A Hydrophilic Channel Is Involved in Oxidative Inactivation of a [NiFeSe] Hydrogenase
- Sónia ZacariasSónia ZacariasInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, PortugalMore by Sónia Zacarias,
- Adriana TemporãoAdriana TemporãoInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, PortugalMore by Adriana Temporão,
- Melisa del BarrioMelisa del BarrioAix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, FranceMore by Melisa del Barrio,
- Vincent FourmondVincent FourmondAix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, FranceMore by Vincent Fourmond,
- Christophe LégerChristophe LégerAix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, FranceMore by Christophe Léger,
- Pedro M. Matias*Pedro M. Matias*E-mail for P.M.M.: [email protected]Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, PortugaliBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, PortugalMore by Pedro M. Matias, and
- Inês A. C. Pereira*Inês A. C. Pereira*E-mail for I.A.C.P.: [email protected]Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, PortugalMore by Inês A. C. Pereira
Copyright © 2019 American Chemical Society
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Abstract
Hydrogenases are metalloenzymes that catalyze the redox conversion between H2 and protons. The so-called [NiFeSe] hydrogenases are highly active for both H2 production and oxidation, but like all hydrogenases, they are inhibited by O2. In the [NiFeSe] enzyme from Desulfovibrio vulgaris Hildenborough this inhibition results from the oxidation of an active site cysteine ligand. We designed mutations that constrict a hydrophilic channel which connects the protein surface to this active site cysteine. Two of the variants show markedly increased tolerance to O2 inactivation, while they retain high catalytic activities in both directions of the reaction, and structural studies confirm that these mutations prevent the oxidation of the cysteine. Our results indicate that the diffusion of O2 or ROS to the active site can occur through a hydrophilic water channel, in contrast to the widely held assumption that only hydrophobic channels are involved in active site inactivation. This provides an original strategy for optimizing the enzyme by protein engineering.
Introduction
ARTICLE SECTIONS
Hydrogenases are extremely efficient biocatalysts for H2 production and oxidation, which use only earth-abundant transition metals as cofactors. The study of these enzymes and their catalytic mechanism may lead to important insights in the efforts to develop an H2-based economy.(1) However, most hydrogenases are sensitive to O2, which hampers their use in large-scale applications. Thus, understanding and preventing the inhibition of hydrogenases by O2 is of major interest, as it could crucially influence the applicability of these enzymes as efficient catalysts in an H2-fueled economy.
The most common hydrogenases are the [NiFe] and the [FeFe] hydrogenases, so named on the basis of the active site metal content.(1) In both classes, this active site is deeply buried inside the protein and a network of hydrophobic channels guides the diffusion of substrate H2 and inhibitors, such as O2 and CO, between the solvent and the active site.(2−10) Most [FeFe] hydrogenases are slowly but irreversibly damaged by O2.(10) The inactivation of the so-called “standard” [NiFe] hydrogenases involves the formation of a mixture of two EPR-detectable inactive states where an oxygen species bridges the active site metal ions; the two inactive states can be reactivated by reduction, albeit at very different rates.(11−13) The [NiFeSe] hydrogenases are a subclass of the [NiFe] hydrogenases, where a selenocysteine (Sec) replaces cysteine as one of the nickel terminal ligands (Figure S1).(14,15) Two [NiFeSe] hydrogenases have been studied in detail, those from Desulfomicrobium baculatum(16) and Desulfovibrio vulgaris Hildenborough.(17) Both exhibit very high catalytic activities,(17−19) and the Dm. baculatum enzyme was shown to be less inhibited by H2 and O2 than the [NiFe] counterparts, which results in the ability to produce H2 under low levels of O2.(19−21) The active site Sec ligand of the D. vulgaris Hildenborough [NiFeSe] hydrogenase was observed to play a key role in its high activity and oxygen tolerance,(18) but other features are also relevant. These include a “cage effect” observed through experimental data and predicted by theoretical calculations, enabling a high H2 density near the active site,(22−24) and differences in the proton transfer pathways and H2 channels.(22) [NiFeSe] hydrogenases are inactivated in the presence of high O2 concentrations, but they can be quickly reactivated by reduction,(19,25) probably because of the reversible reactivity of Sec with O2.(26) An exogenous sulfur atom bound to Ni, observed in the structure of D. vulgaris Hildenborough [NiFeSe] hydrogenase (Figure S1), may also play a role by preventing the formation of inactive species where an oxide bridges the two metal ions.(18,27) Nevertheless, oxygen damage has been observed in the crystal structures of both [NiFeSe] hydrogenases. In the aerobically isolated enzyme from D. vulgaris Hildenborough the proximal iron–sulfur cluster is reversibly modified by the binding of two oxygen atoms, and the terminal Ni ligand, Cys75 (large subunit), is irreversibly oxidized to sulfinate.(27) In addition, the sulfur species at the active site causes the side chain of Sec489 to be present in three possible conformations, named I, II, and III (Figure S1), where conformer III corresponds to that found in the active reduced form of the [NiFe] and [NiFeSe] hydrogenases.(27,28) In the [NiFeSe] hydrogenase from Dm. baculatum, the crystal structure of the enzyme purified aerobically displayed a complex mixture of oxidized states.(29) Some of these could be resolved only when the enzyme was purified anaerobically, showing a mixture of three oxidized states, including seleninate and selenate species and an additional oxygen or sulfur atom bridging the Ni and Fe ions. Despite the structural similarity between the two [NiFeSe] hydrogenases,(28,30) the available data indicate that the effects of O2 inactivation are quite enzyme specific.(27,29) Nevertheless, it should be noted that the experimental conditions used to expose both enzymes to O2 were different.
Recently, we developed a system for recombinant expression of a soluble form of the [NiFeSe] hydrogenase from D. vulgaris Hildenborough,(18) allowing the production of stable and abundant protein with high H2 production and oxidation activities.(18,31) The recombinant enzyme has been applied in optimized biofuel cells, which show benchmark open circuit voltages and current densities for H2 oxidation.(32−34) Its high H2 production activity makes this enzyme attractive for photochemical H2 production(35) and other applications such as electrochemical ATP synthesis.(36)
The engineering of a [NiFeSe] hydrogenase to improve its O2 tolerance has not been reported before. Here, we used rational design to prevent the irreversible oxidation of Cys75. The mechanism of Cys75 oxidation probably involves a reaction with reactive oxygen species (ROS),(37) which may be formed close to the active site by reduction of O2. However, Cys75 is not directly accessible via the hydrophobic channel through which O2 molecules are proposed to diffuse.(2−9) It is possible that the oxidation of Cys75 may arise due to its location at the end of a water channel leading to the protein surface, which is only found in the crystal structures of [NiFeSe] hydrogenases (Figure 1). However, this channel is hydrophilic and, as a nonpolar molecule, O2 is expected to diffuse instead through the hydrophobic channels;(2) therefore, evidence for O2 diffusion through this water channel would be unprecedented for hydrogenases. We examined the effects of replacing two residues that line this hydrophilic channel, Gly50, located near the protein surface, and Gly491, which is near Cys75. We observed that mutations G491A and G491S prevent, or considerably slow, the oxidation of Cys75, resulting in more oxygen tolerant variants and demonstrating the role of this solvent channel in providing access of O2 or ROS to the active site in the D. vulgaris Hildenborough [NiFeSe] hydrogenase.
Figure 1
Figure 1. Solvent channel in the 0.95 Å resolution structure of the anaerobically crystallized wild-type [NiFeSe] hydrogenase from D. vulgaris Hildenborough (PDB 5JSH). The protein Cα backbone is shown in light cyan for the small subunit and gray for the large subunit, the water molecules in the solvent channel are shown as red spheres, the active site and the iron–sulfur clusters are depicted in ball-and-stick representation with atom colors gray for carbon, blue for nitrogen, red for oxygen, gold for sulfur, and brown for iron, and the G50 and G491 residues are highlighted in orange.
Results
ARTICLE SECTIONS
Rational Design of [NiFeSe] Hydrogenase Variants
On the basis of a structural alignment between the D. vulgaris Hildenborough [NiFeSe] hydrogenase and other members of the [NiFe] hydrogenase family (Figure S2) we selected two residues in the large subunit of the [NiFeSe] enzyme, which are in a solvent channel linking Cys75 to the protein surface (Figure 1). Gly50 is near the protein surface at ca. 15 Å from the Ni atom in the active site, whereas Gly491 is very close to the active site at a distance of ca. 6 Å from the same atom. In [NiFe] hydrogenases, Gly50 and Gly491 are replaced with threonine or glutamine and alanine or serine, respectively; thus, a similar channel does not exist. With the aim of blocking the putative solvent channel leading directly to Cys75 and trying to prevent its oxidation, we generated the G50T and G491A variants, which reproduce the sequence of standard [NiFe] hydrogenases. We also produced the G491S, G491T, and G491V variants, to test the effects of side chain polarity and size at position 491.
Kinetic and Structural Characterization of the [NiFeSe] Hydrogenase Variants
Variants G50T, G491A, and G491S were successfully purified and characterized. However, no stable protein could be obtained for the G491T and G491V variants. Although threonine and valine are not bulky residues, the additional carbon atom at position 491 may induce a steric disruption. The G50T, G491A, and G491S mutations moderately decreased the H2 production and oxidation activities of the enzyme (Table 1), but the turnover frequencies remained near or above 3000 s–1, which is high in the context of [NiFe] hydrogenases. All variants retain a catalytic bias toward H2 production in the solution assays.
Table 1. Turnover Rate (s–1) for D. vulgaris Hildenborough [NiFeSe] Hydrogenase and Variantsa
| WT | G491A | G491S | G50T | |
|---|---|---|---|---|
| H2 evolution | 8270 ± 380b | 6020 ± 100 | 3510 ± 140 | 3820 ± 210 |
| H2 uptake | 4850 ± 260 | 4080 ± 80 | 2810 ± 150 | nd |
a
Turnover rate for H2 production at 37 °C in 50 mM Tris-HCl buffer at pH 7 with 1 mM MV reduced with 15 mM sodium dithionite. Turnover rate for H2 oxidation under 0.5 bar H2, at 30 °C, in 50 mM Tris/HCl pH 8 containing 2 mM of MV. nd = not determined.
b
From ref (18).
Crystal structures of the G50T, G491A, and G491S variants were obtained under aerobic conditions at resolutions of 1.10, 1.36, and 1.20 Å, respectively (Tables S1 and S2). The previously reported 1.30 Å structure of the recombinant WT [NiFeSe] hydrogenase in the as-isolated, oxidized form (PDB 5JSH) is used for comparison.(18) No changes are observed in the overall protein structures, relative to the WT. The structural analysis of the variants will focus on the oxidative modification of the active site (Table S3), namely in the relative abundance of the three different Sec conformations (Figure S1) and degree of oxidation of Cys75 to sulfenate and sulfinate.
In the G50T crystal structure, Sec489 displays conformers I, II, and III with occupancies of 46%, 20%, and 34% (Figure S3A,B), similar to those previously reported for the WT structure (52%, 20%, and 29% respectively). In the G491A structure only Sec489 conformers II and III are observed with occupancies of 23% and 77%, respectively, indicating that this residue is mainly in the active, reduced conformation (Figure 2). Finally in the G491S structure, conformers I, II, and III are present with occupancies of 29%, 18%, and 53%, respectively, with Sec489 still predominantly in the active conformation (Figure S3C,D). In the G50T structure, the terminal Cys75 Ni ligand is extensively oxidized to the sulfenate (20%) and sulfinate (54%) states, similarly to the WT (38% and 62%, respectively). Thus, the G50T mutation has no significant effect on the oxidized state of the active site. In contrast, in the G491A structure, Cys75 is not oxidized at all (Figure 2), and in the G491S structure it is only slightly oxidized to the sulfinate state (ca. 20%) (Figure S3C,D). These results are a clear indication that the G491 mutations protect Cys75 from oxidative modification. Furthermore, the G491S variant shows reduced oxygenation of the proximal cluster (Table S3), raising the possibility that this mutation may also reduce access of ROS/O2 to this cluster.
Figure 2
Figure 2. Active site surroundings in the crystal structure of the aerobically purified and crystallized G491A [NiFeSe] hydrogenase variant and its corresponding 2|Fo| – |Fc| (gray mesh, 1.5 map rms) and |Fo| – |Fc| (green mesh, 3.5 map rms) maps. No negative peaks are visible at −3.5 rms in the |Fo| – |Fc| map. Atoms are color-coded as follows: brown, Fe; green, Ni; gold, S; red, O; light blue, C; blue, N; orange, Se. H atoms are omitted for clarity.
Electrochemical Studies of O2 and CO Inhibition
The (in)activation of NiFeSe hydrogenase in response to changes in O2 concentration or redox conditions has been studied by electrochemistry,(19,25) in experiments where electron transfer between the enzyme and the electrode is direct and the turnover frequency is measured as a current.(38,39) In particular, we have shown that D. vulgaris Hildenborough [NiFeSe] hydrogenase is inactivated either in the presence of O2 or at high electrode potential to inactive states that can be reduced when the electrode potential is swept down. The potential where reactivation occurs in voltammetric experiments strongly depends on the scan rate, because the rate of reactivation increases as the electrode potential decreases,(25) as occurs with all [NiFe] and [FeFe] hydrogenases characterized so far. Here we focus on the kinetics of inactivation by O2. The experiment consists in measuring the hydrogen oxidation current (which is proportional to turnover rate) and examining the effect of injecting a small volume of air-saturated buffer solution (containing 240 μM O2) into the electrochemical cell, to reach 0.5 μM O2. The O2 concentration increases instantly at the time of injection and then decreases exponentially over time, as the dissolved O2 is flushed out of the cell by the stream of H2. We perform these experiments at high electrode potential to prevent the direct reduction of O2 on the electrode, which would contribute to the current, decrease the concentration of O2 that the enzyme experiences, and produce reactive oxygen species.(40) The changes in H2 oxidation current obtained for the WT enzyme and variants are shown in Figure 3. When O2 is introduced (at 175 s in the figure), the activity of the WT and G50T variant quickly vanishes, whereas, all things being equal, the G491S and G491A variants are slowly and only partially inactivated. The initial decrease in current, before O2 addition, is due to enzyme desorption and anaerobic oxidative inactivation(41) and was corrected by assuming that the loss of current follows first-order kinetics. The inactivation rate constants were determined by fitting to the data a model that assumes that the enzyme inactivates in an oxygen-dependent reaction (second-order rate constant kinO2), followed by either reactivation (first-order rate constant ka) or irreversible inactivation (k3):(42)
(1)In this scheme, A represents the active species, I the inactive species, and D the dead-end species formed by irreversible inactivation.
(1)In this scheme, A represents the active species, I the inactive species, and D the dead-end species formed by irreversible inactivation.Figure 3
Figure 3. Effect of O2 on the H2 oxidation current of WT [NiFeSe] hydrogenase and variants adsorbed onto a graphite rotating electrode. The gray dashed lines are the best fits of the kinetic model in eq 1. Experimental conditions: [O2] = 0.5 μM, E = 0.14 V vs SHE, 1 bar H2, pH 7, T = 40 °C, electrode rotation rate 3000 rpm.
The best values of the inactivation and reactivation rate constants are given in Table 2. At the potential used, we observed a very slow reactivation (ka ≈ 10–4 s–1) for all four enzymes. The k3 rate constants were small (possibly zero) and could not be determined accurately. The values of kinO2 in Table 2 demonstrate that the two G491 mutations decrease the rate of inactivation by O2 about 7-fold.
Table 2. | Kinetic Properties of the D. vulgaris Hildenborough [NiFeSe] Hydrogenase WT and Variants
| kinO2 (mM–1 s–1)a | koutO2 (s–1)a | appkinCO (mM–1 s–1)b | koutCO (s–1)b | |
|---|---|---|---|---|
| WT | 527 ± 10 | (3 ± 1) × 10–4 | 290 ± 50 | 0.3 ± 0.1 |
| G50T | 629 ± 260 | (3 ± 0.1) × 10–4 | 410 ± 220 | 0.8 ± 0.5 |
| G491A | 77 ± 15 | (6 ± 2) × 10–4 | 380 ± 80 | 0.9 ± 0.4 |
| G491S | 86 ± 15 | (8 ± 3) × 10–4 | 400 ± 170 | 1.0 ± 0.1 |
a
Conditions: E = 0.14 V vs SHE, 1 bar H2, pH 7, T = 40 °C, [O2] = 0.5 μM.
b
Conditions: E = −0.06 V vs SHE, 1 bar H2, pH 7, T = 40 °C, [CO] = 1, 2, 4 μM.
In [NiFe] hydrogenases, it is believed that both CO and O2 access the active site through the same gate at the end of a hydrophobic gas channel, and it was found that the rate of CO inhibition is a good proxy for the rate of intramolecular diffusion of other gases to the active site.(7) We evaluated the rates of inhibition by CO (kinCO) and the rate of CO release (koutCO) for the WT and the variants by monitoring the H2 oxidation current after exposure to CO at −60 mV and 40 °C (Figure S6). In all cases, the current decreases upon CO injection and then recovers its initial value as CO is flushed away from the solution, showing that the enzymes are reversibly inhibited by CO. To determine the constants of CO inhibition, we fitted to the data a model that assumes bimolecular CO binding to the active and first-order kinetics for CO release:(5)
(2)
(2)Since CO inhibition of [NiFe] hydrogenases is competitive(40) the measured rate constant of CO binding is not the true rate but is multiplied by Km/(Km + [H2]).(7,43) This does not matter here for comparison of the rates, since we have not observed any effect of the mutations on the Michaelis constant for H2. In contrast to what we observed with O2, all four enzymes react with CO at about the same rate.
Effect of O2 Exposure on H2 Uptake Activity in Solution
To study the effect of prolonged air exposure and the reversibility of oxidative damage, we first activated each enzyme with H2 and then exposed it to air for 1 h, 4 h, or overnight (ca. 16 h) at room temperature. After exposure to air, no activity was detected in either the WT or variants, consistent with previous evidence that reactivation requires that the enzyme be reduced.(19,25) The inactive samples were then incubated under H2 for 10, 30, or 90 min, before measuring how much activity remained or was irreversibly lost. The G50T variant was not tested in this experiment, since the structural and electrochemical data indicated no significant difference from the WT.
For the WT enzyme we observed that, after 1 h exposure to air and reactivation with H2, the enzyme loses 30% of the initial activity. The loss in activity increases with exposure time, reaching 50% after overnight exposure to air (Figure 4). In contrast, the G491S variant is more tolerant to O2, since even after overnight air exposure (followed by reduction), the activity was not significantly affected. The G491A variant is less sensitive than the WT during the first hours, but overnight air exposure results in a similar loss of activity, by about 50%. The duration of the incubation under H2 made no difference (Figure S4), with 10 min being enough to reactivate all the proteins. The different behavior between the G491S and G491A variants and the WT suggests that this mutation prevents, or at least considerably delays, the formation of inactive species that do not reactivate. This experiment also emphasizes the robust catalytic behavior of the [NiFeSe] hydrogenase, since the turnovers for WT and variants are all above 1000 s–1 even after overnight air exposure and reactivation, which is a very high activity in comparison to most [NiFe] hydrogenases.
Figure 4
Figure 4. H2 uptake activity of WT and variants after 1 h (green), 4 h (blue), and 16 h (gray) exposure to air, followed by a 30 min reactivation under 0.5 atm of H2. The activities were normalized by the corresponding maximum activity of each protein (dark blue), reported in Table 1. Each experiment was performed three times (technical replicates), and the error bars show the corresponding standard deviations.
Structural Analysis of the [NiFeSe] Hydrogenase Channels
We used the software CAVER(44) to analyze and compare the shapes of the channels in the WT D. vulgaris Hildenborough [NiFeSe] hydrogenase,(18) in the variants obtained in this work, in the Dm. baculatum [NiFeSe] hydrogenase,(29) and in the standard [NiFe] hydrogenases given in Figure S2. Hydrogen atoms were added to the analyzed structures, whenever necessary, using the PHENIX phenix.ready_set tool.(45) The D. vulgaris Myiazaki F [NiFe] hydrogenase(46) is shown as representative of the results obtained. As illustrated in Figure 5 and previously described,(2) both [NiFe] and [NiFeSe] hydrogenases contain a branched hydrophobic channel system through which H2 and other small gas molecules diffuse to/from the active site.(8,9) A second channel system is present in both [NiFeSe] hydrogenases, linking the Ni terminal ligands Sec489 and Cys75 (D. vulgaris Hildenborough numbering) to the protein surface (Figure 5B,C). CAVER calculations revealed that this channel is not present in the [NiFe] hydrogenases analyzed (Figure 5 and Table S4), here represented by the D. vulgaris Myiazaki F [NiFe] hydrogenase (Figure 5A). In the D. vulgaris Hildenborough [NiFeSe] hydrogenase, this second channel has a hydrophilic character, starts from the active site, and then divides into two branches (Figures 5B and 6A); the first branch continues the initial hydrophilic segment, while the second is lined by hydrophobic residues and is absent in the [NiFeSe] hydrogenase from Dm. baculatum (Figure 5C).
Figure 5
Figure 5. Channels in the high-resolution structures of [NiFe] hydrogenase from D. vulgaris Myiazaki F (A, 0.86 Å, PDB 489H), [NiFeSe] hydrogenases from D. vulgaris Hildenborough (B, 0.95 Å, PDB 5JSK, crystallized anaerobically), and Dm. baculatum (C, 1.4 Å, PDB 4KN9), calculated with CAVER. The hydrophobic channel system allowing H2 exchange with the active site is shown in light magenta, and the channels connecting Sec and Cys75 Sγ atoms with the enzyme exterior are displayed in blue. The protein Cα backbones are shown as gray tubes; the active site and the iron–sulfur clusters are shown in ball-and-stick representation with atom colors gray for carbon, blue for nitrogen, red for oxygen, gold for sulfur, and brown for iron; the G50 and G491 residues in D. vulgaris Hildenborough and their structurally equivalent Thr56 and Ala548 residues in D. vulgaris Myiazaki F and G45 and G494 in Dm. baculatum are displayed in ball-and-stick representation with carbon atoms colored yellow. For clarity, only the side chains of the protein residues are shown.
Figure 6
Figure 6. Close-up view of the channels in the structure of the D. vulgaris Hildenborough [NiFeSe] hydrogenase and variants, calculated with CAVER: (A) wild-type (PDB 5JSK); (B) G50T variant; (C) G491A variant; (D) G491S variant. The channels connecting the enzyme exterior with the Sec and the Cys75 Sγ atoms are displayed as meshes. The hydrophilic branch of the channel is shown in blue and the hydrophobic branch in green. The water molecules enclosed by the channels are represented as red spheres.
The G50T mutation is located after the branching point, near the end of the hydrophilic branch closer to the surface, while the G491A and G491S mutations are in the first segment of the hydrophilic channel, before the branching point. Figure 6 shows that the G50T mutation obstructs the hydrophilic branch (Figure 6B), while the G491A and G491S substitutions block channel access to Cys75 (Figure 6C,D).
Discussion
ARTICLE SECTIONS
In [NiFe] hydrogenases, the importance of hydrophobic gas channels was emphasized when experiments on O2-tolerant regulatory hydrogenases (RH) revealed that bulky residues at the end of the hydrophobic channel narrow the access of gas molecules to the active site. This was initially proposed as the possible reason for the O2 tolerance of these enzymes.(3,47,48) These observations prompted studies on the standard O2-sensitive [NiFe] hydrogenase from D. fructosovorans, where bulky residues were introduced in the hydrophobic channel to restrict the access of inhibitory gases O2 and CO. The effect of modifying the gas channel in the D. fructosovorans hydrogenase was to slow O2 access to the active site(7) and also to increase the reactivation rates.(49) These mutations also resulted in strongly decreased H2 production rates, since H2 also diffuses through the same channel, in a step that limits the rate of H2 production in these mutants.(49)
[NiFeSe] hydrogenases are powerful biocatalysts, which we aim to improve further by decreasing their O2 sensitivity. Here, we tested mutations aiming to prevent the irreversible oxidation of Cys75. This residue is not directly accessible via the hydrophobic channel through which H2 and O2 are believed to diffuse; it is positioned at the end of a hydrophilic solvent channel that connects the active site of [NiFeSe] hydrogenases to the surface. This solvent channel seems to be present only in [NiFeSe] hydrogenases, since it is not seen in any of the O2-resistant or O2-sensitive [NiFe] hydrogenases that have been crystallized (Table S4). We tested the hypothesis that the hydrophilic channel leading to Cys75 also guides O2 or ROS toward this residue, by examining how replacing amino acids whose side chains line this tunnel affects Cys75 oxidation and oxygen sensitivity. We observed that the G50T mutation has no significant effect, but both the G491A and G491S mutations significantly improve resistance against Cys75 irreversible oxidation. In protein film voltammetry experiments, the G491A and G491S variants are more slowly inactivated by O2 than the WT, with 7-fold lower rate constants for O2 inhibition. In addition, the results of solution assays show that the G491S variant is much more tolerant to prolonged air exposure, recovering nearly full activity even after overnight exposure to air. Therefore, our kinetic data reveal that the mutation prevents the formation of irreversibly oxidized species. From a molecular point of view, the X-ray structures of the variants show that the G491 mutations prevent the oxidation of Cys75. Channel prediction with CAVER explains why the G50T mutation has no effect on Cys75 oxidation: this residue is positioned at the entrance of the hydrophilic water channel, but a branching point in this channel still allows access of oxygen species to the active site in the G50T variant. In contrast, the G491 residue is close to the active site, and its substitution does prevent (or strongly delay) the access of oxidizing species to Cys75. In support of our hypothesis, we note that the oxidation of this terminal Ni ligand has never been observed in any [NiFe] hydrogenase where this tunnel is absent. Moreover, the rate of inhibition of the WT [NiFeSe] hydrogenase by O2 (530 mM–1 s–1 at 40 °C) is well above the typical range observed with [NiFe] hydrogenases (5–20 mM–1 s–1, from ref (42)), whereas the G491 mutations decrease the rate of inhibition to a less remarkable value of around 80 mM–1 s–1, which likely corresponds to O2 diffusion through the hydrophobic channel as observed in standard [NiFe] hydrogenases.(42) This supports the idea that the mutations render ineffective the hydrophilic channel that is not present in [NiFe] hydrogenases.
Interestingly, the G491 mutations slow O2 inhibition but have no effect on CO inhibition. Carbon monoxide is a competitive inhibitor of [NiFe] hydrogenases, and in the case of D. fructosovorans [NiFe] hydrogenase, the rate of inhibition by CO is limited by the rate of CO access to the active site.(5,7) Here we observed no correlation between changes in kinO2 and kinCO, suggesting that, in D. vulgaris Hildenborough [NiFeSe] hydrogenase, the CO molecules diffuse preferentially through the hydrophobic channel system, whereas O2/ROS diffuse more quickly through the hydrophilic channel system, as revealed by the inhibition kinetics.
Overall, our structural, biochemical, and electrochemical results provide strong support to the hypothesis that O2 or ROS can reach the active site of D. vulgaris Hildenborough [NiFeSe] hydrogenase through a hydrophilic water channel. This argues against the general idea that aerobic inactivation occurs only through O2 diffusion through hydrophobic channels. A few previous studies have also suggested that O2 diffusion can occur through hydrophilic channels: in the enzyme nitrogenase it has been observed that a hydrophilic channel is used for transport of the nonpolar substrate N2.(50,51) In catalase, MD simulations indicate that the same channel is used by substrates and products, H2O2, H2O, and O2.(52) Interestingly, in O2-tolerant hydrogenases it has been proposed that hydrophilic channels are involved in removal of the water molecules produced during H2 oxidation in the presence of O2.(53)
All D. vulgaris [NiFeSe] hydrogenase variants studied here are slightly less active than the WT but still more active than any [NiFe] hydrogenase. Residue 491 is close to the highly conserved Glu28 (Figure 6C,D), which in [NiFe] hydrogenases is part of the proton transfer pathway.(54) A plausible explanation for the mutation-induced decrease in activity is that the bulkier side chains of the G491 variants may place some steric hindrance in the side-chain motion of Glu28 and thus interfere with proton transfer. This could be especially relevant in the case of the G491S variant, where in the crystal structure Ser491 forms a hydrogen bond with Glu28 that could further hinder the proton transfer action of Glu28. Indeed, as shown in Table 1, the H2 evolution activities of the variants are more affected than those of H2 oxidation, and particularly so in the case of G491S. In the G50T variant, the threonine side chain breaks the chain of water molecules in the hydrophilic channel, possibly creating a barrier to proton transfer and thus lowering the enzyme activity. Proton transfer is proposed to be the rate-limiting step of H2 production in the WT form of D. fructosovorans [NiFe] hydrogenase.(55) If proton transfer also limits the rate of H2 production by D. vulgaris Hildenborough [NiFeSe] hydrogenase, then mutations that interfere with the proton transfer pathway should slow H2 evolution more than H2 uptake, as indeed observed in our experiments (Table 1). The oxidation of Cys75 to the sulfinate state is also expected to lower the activity of the G491S variant. This modification is apparently formed during prolonged air exposure (long purification and crystallization), since we observe reduced levels of Cys75 sulfinate in aerobically crystallized samples after starting to work with recombinant protein (∼60% vs 100% previously), for which affinity purification is much faster.(18) We have previously measured the decrease in activity of the WT enzyme under the crystallization conditions(56) and observed a gradual loss of activity, reaching about 10% after 10 days. This indicates that formation of Cys75 sulfinate leads to activity reduction, but whether the modified form retains any activity or not is difficult to ascertain. The observation that the reduced form (conformer III) could still be obtained by H2 reduction of aerobic crystals,(27) where Cys75 sulfinate was already present in most molecules, may suggest that some activity is retained. On the other hand, the precision of the crystallographic assignments for the sulfinate state is about ±10%; thus, we cannot discard that there were some active enzyme molecules left without the sulfinate modification, which were sufficient to generate enough electrons inside the crystal leading to reduction of the sulfinate-modified molecules (also obtained with dithionite(27)).
Concluding Remarks
ARTICLE SECTIONS
In conclusion, we show that a single mutation (G491A or G491S) can improve the O2 tolerance of a [NiFeSe] hydrogenase, while it retains high activity in both directions of H2 catalysis. Both variants exhibit a lower rate of inactivation by O2 and a faster rate of reactivation after O2 exposure. These results emphasize the fact that the O2 sensitivity of hydrogenases from different families may be determined by specific structural features. Furthermore, our results also reveal that O2 and/or ROS can access the active site of [NiFeSe] hydrogenase through a hydrophilic channel, in contrast with the general assumption that O2 molecules are guided only by hydrophobic channels.
Methods
ARTICLE SECTIONS
Variant Expression and Purification
The D. vulgaris Hildenborough [NiFeSe] hydrogenase variants were created by site-directed mutagenesis with an NZYMutagenesis kit (Nzytech) using the expression vector pMOIP03 carrying a hysAStrephysB fragment as a template.(18,31) The vectors were electroporated into the IPAR01 deletion strain,(18) using the Gene Pulser XCell apparatus (Biorad) at 1250 V, 250 Ω, and 25 μF.(57) Cells were grown at 37 °C in standard lactate–sulfate medium supplement with 1 μM NiCl2·6H2O and 1 μM NaSeO3·5H2O. Cells were collected and disrupted in the French press at 6.9 MPa. The expression of the protein variants was assessed by Western blot against native D. vulgaris Hildenborough [NiFeSe] hydrogenase and against the Strep-Tactin AP (IBA Lifesciences). For purification the soluble fraction was first applied in a Q-Sepharose HP column (XK 26/10, GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.6 and a stepwise NaCl gradient of 50 mM/step was performed. Fractions with [NiFeSe] hydrogenase activity were affinity-purified using a gravity column containing Strep-Tactin resin (IBA Lifesciences) equilibrated with 100 mM Tris-HCl pH 8.0 and 150 mM NaCl (buffer W); the recombinant protein was eluted with buffer W plus 2.5 mM desthiobiotin. The purity of [NiFeSe] hydrogenase WT and variants was analyzed by SDS-PAGE stained with Comassie Blue.
Hydrogenase Activities
H2-oxidation activity was routinely determined spectrophotometrically by following the H2-dependent reduction of methylviologen (MV) at 604 nm (ε = 13.6 mM–1 cm–1) at 30 °C inside a Coy anaerobic chamber (98% N2, 2% H2). The assay solution contained 2 mM MV in H2-saturated 50 mM Tris-HCl buffer at pH 8, and the as-isolated enzyme was previously activated with 0.5 bar H2 for 30 min. One unit of enzyme is defined as the amount of hydrogenase reducing 2 μmol of MV per minute, which is equivalent to 1 μmol of H2 oxidized per minute.
For H2 production the enzyme was reduced with 1 mM methylviologen (MV) reduced by 15 mM sodium dithionite and the reaction mixture was incubated in a shaker at 37 °C for 10 min. A headspace sample was measured, every 4 min, in a Trace GC Ultra gas chromatograph (Thermo Scientific) equipped with a thermal conductivity detector and a MolSieve 5A 80/100 column (Althech) with N2 as a carrier gas.
Electrochemistry
All electrochemical experiments were carried out in a glovebox (Jacomex) filled with N2, with the electrochemical setup and equipment as previously described.(40) We used a pyrolytic graphite edge (PGE) rotating disk working electrode (area ∼3 mm2), a platinum wire as a counter electrode, and a saturated calomel electrode (SCE), located in a Luggin side arm of the electrochemical cell and immersed in 0.1 M NaCl, as a reference. The enzyme was coadsorbed with neomycin(58) on the working electrode. The electrode surface was first polished with an aqueous alumina slurry (1 μm) and sonicated. Then it was covered with 0.5 μL of neomycin (200 mg/mL in water) and 1 μL of the D. vulgaris Hildenborough [NiFeSe] hydrogenase (50 μg/mL in pH 7 mixed buffer consisting of 5 mM MES, 5 mM CHES, 5 mM HEPES, 5 mM TAPS, 5 mM sodium acetate, and 100 mM NaCl). The protein film was allowed to dry and then rinsed with water. The electrochemical cell contained pH 7 mixed buffer and was continuously flushed with pure H2. The temperature was regulated at 40 °C by circulating water in the double jacket of the cell. The data were analyzed using QSoas,(59) an open source program available at http://www.qsoas.org.
O2 Tolerance Studies in Solution
Samples of the as-isolated enzymes (0.25 μM) were activated for 1 h in 0.5 bar H2, 50 mM Tris-HCl pH 8, and 1 mM MV and were kept under an H2 atmosphere overnight. The H2 oxidation activity was measured and taken as corresponding to the maximum activity of each protein. Next, the enzyme solutions were put in contact with air and diluted to 0.125 μM in an aerobic buffer of 50 mM Tris-HCl pH 8. Three samples of each enzyme solution were kept under aerobic conditions for 1 h, 4 h, or overnight (∼16 h) at room temperature, after which no H2 oxidation activity was detected in any of the samples. Following this air exposure, oxidized MV was added to each sample up to a concentration of 1 mM, and the enzymes were reactivated again under 0.5 bar H2 for 10, 30, or 90 min, following which H2 oxidation activities were measured.
Crystallization and X-ray Diffraction Data Collection
Crystals of the three D. vulgaris [NiFeSe] hydrogenase variants were obtained at 20 °C by the sitting drop variant of the vapor diffusion method. Drops of 1 or 2 μL of pure protein (80–114 μM) were mixed with an equal volume of reservoir solution containing 20% PEG 1500 (w/v) and 100 mM Tris-HCl pH 7 and equilibrated against 500 μL of the reservoir solution.
Crystals appeared after about 1 week and were harvested, briefly dipped in a cryoprotecting solution with the same composition as the reservoir solution and adjusted to include 10% (v/v) glycerol, flash-cooled in liquid nitrogen, and shipped to a synchrotron beamline for data collection. The G50T and G491S data sets were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at beamlines ID29 and ID30A-3, respectively, and the G491A data set was measured at the beamline I03 of the Diamond Light Source (DLS, Didcot, U.K.). The data collection and processing statistics are given in Table S1.
Structure Determination and Refinement
The crystal structures were determined by the molecular replacement method with PHASER(65) via the CCP4 Graphics User Interface.(66) The coordinates of the protein chains of the large and small subunits of the previously published crystal structure of D. vulgaris [NiFeSe] hydrogenase (PDB 5JSH)(18) were used as search models, after removal of all nonprotein atoms. Following a quick initial refinement with REFMAC,(67) COOT(68) was used to perform model corrections and addition of the active site metals and Fe–S clusters. Refinement was continued with PHENIX,(45) consisting of five macrocycles with refinement of positional coordinates, individual isotropic atomic displacement parameters for all non-hydrogen atoms, and anomalous dispersion parameters for the Se atom. Model inspection and editing was done with COOT against σA-weighted 2|Fo| – |Fc| and |Fo| – |Fc| electron density maps. Water molecules were added with PHENIX and checked with COOT. Hydrogen atoms in calculated positions were added to the structural models and included in the refinement in riding positions. In the final refinement stages, individual anisotropic atomic displacement parameters for all protein non-hydrogen atoms were refined for structures G50T and G491S. For structure G491A, TLS (translation-libration-screw) rigid body refinement of atomic displacement parameters was carried out instead, followed by refinement of individual isotropic B factors. Six and seven TLS groups were used respectively for the small subunit (chain A) and the large subunit (chain B). These groups were adapted from the list determined with the find_tls_groups tool in PHENIX from a previous full isotropic refinement to also include the active site and the iron–sulfur clusters.
MOLPROBITY(69) was used to investigate the model geometry in combination with the validation tools provided in COOT: for all three crystal structures reported here, the number of Ramachandran outliers did not exceed 0.13% of the total number of nonproline and nonglycine residues and the clash score did not exceed 2.5. The refinement statistics are included in Table S2.
Structure figures were created using the PyMOL Molecular Graphics System, Version 2.1.0 Open Source (Schrödinger, LLC). CAVER calculations were done using the PyMOL plug-in with default parameters (minimum probe radius of 0.9 Å, shell depth of 4 Å, shell radius of 3 Å, a clustering threshold of 3.5, and a starting point optimization with 3 Å maximum distance and a desired radius of 5 Å) and atomic coordinates including hydrogen atoms in calculated positions. The final atomic coordinates and experimental structure factors were deposited in the Worldwide Protein Data Bank(70) with accession codes 6RTP, 6RU9, and 6RUC for the G50T, G491A, and G491S structures, respectively.
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.9b02347.
View of the active site conformations in the as-isolated D. vulgaris Hildenborough [NiFeSe] hydrogenase, structure-based sequence alignments used to select the D. vulgaris Hildenborough [NiFeSe] hydrogenase mutants, X-ray data collection, processing, and refinement statistics, structural details at the active site and proximal [Fe4S4] cluster, views of the active site of the aerobically purified and crystallized [NiFeSe] hydrogenase G50T and G491S variants, H2 uptake activity of WT and variants, channels predicted by CAVER in [NiFe] and [NiFeSe] hydrogenase structures, detailed views of the hydrophilic channel and hydrophobic side channel, and graph showing the effect of CO on the H2 oxidation current of WT [NiFeSe] hydrogenase and variants adsorbed onto a graphite rotating electrode (PDF)
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Author Information
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- Pedro M. Matias - Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal;
http://orcid.org/0000-0001-6170-451X;
Email: [email protected]
- Melisa del Barrio - Aix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, France;http://orcid.org/0000-0002-6947-6686
- Vincent Fourmond - Aix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, France;http://orcid.org/0000-0001-9837-6214
- Christophe Léger - Aix Marseille Univ., CNRS, Bioénergétique et Ingénierie des Protéines, UMR 7281 Marseille, France;http://orcid.org/0000-0002-8871-6059
The authors declare no competing financial interest.
Acknowledgments
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This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) through fellowship SFRH/BD/100314/2014 (to S.Z.), grant PTDC/BBB-BEP/2885/2014 (to P.M.M. and I.A.C.P.), and R&D units UID/Multi/04551/2013 (Green-IT) and LISBOA-01-0145-FEDER-007660 (MostMicro) cofunded by FCT/MCTES and FEDER funds through COMPETE2020/POCI. The work of M. del B., V. F., and C. L. was supported by CNRS, Aix Marseille Université, Agence Nationale de la Recherche (ANR-14-CE05-0010), and the Excellence Initiative of AixMarseille University - A*MIDEX, a French “Investissements d’Avenir” programme (ANR-11-IDEX-0001-02). Support from the ESRF and the beamline staff of ID29 and ID30A-3 for the G50T and G491S data collections is acknowledged, and we also thank Diamond Light Source for access to beamline I02 under proposal number MX10515 for the G491A data collection. Funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 810856 is also acknowledged, and the French authors are part of the French bioinorganic chemistry research network (www.frenchbic.cnrs.fr).
References
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This article references 70 other publications.
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- 7Liebgott, P.; Leroux, F.; Burlat, B.; Dementin, S.; Baffert, C.; Lautier, T.; Fourmond, V.; Ceccaldi, P.; Cavazza, C.; Meynial-Salles, I.; Soucaille, P.; Fontecilla-Camps, J. C.; Guigliarelli, B.; Bertrand, P.; Rousset, M.; Léger, C. Relating Diffusion along the Substrate Tunnel and Oxygen Sensitivity in Hydrogenase. Nat. Chem. Biol. 2010, 6, 63– 70, DOI: 10.1038/nchembio.276[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFWksb7M&md5=39c97ff63b8b0d0c1b3136c3bb22859bRelating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenaseLiebgott, Pierre-Pol; Leroux, Fanny; Burlat, Benedicte; Dementin, Sebastien; Baffert, Carole; Lautier, Thomas; Fourmond, Vincent; Ceccaldi, Pierre; Cavazza, Christine; Meynial-Salles, Isabelle; Soucaille, Philippe; Fontecilla-Camps, Juan Carlos; Guigliarelli, Bruno; Bertrand, Patrick; Rousset, Marc; Leger, ChristopheNature Chemical Biology (2010), 6 (1), 63-70CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a mol. channel whose structure may det. the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallog. and mol. dynamics, but kinetic data are scarce. Using protein film voltammetry, we detd. and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quant. define the relations between diffusion, the Michaelis const. for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.
- 8Kalms, J.; Schmidt, A.; Frielingsdorf, S.; Van Der Linden, P.; Von Stetten, D.; Lenz, O.; Carpentier, P.; Scheerer, P. Krypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport. Angew. Chem., Int. Ed. 2016, 55, 5586– 5590, DOI: 10.1002/anie.201508976[Crossref], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtlKgsLo%253D&md5=b2e5d335671bc7226fc1233a2b89903bKrypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas TransportKalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, PatrickAngewandte Chemie, International Edition (2016), 55 (18), 5586-5590CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2-tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calcns. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2-tolerant and O2-sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.
- 9Kalms, J.; Schmidt, A.; Frielingsdorf, S.; Utesch, T.; Gotthard, G.; von Stetten, D.; van der Linden, P.; Royant, A.; Mroginski, M. A.; Carpentier, P.; Lenz, O.; Scheerer, P. Tracking the Route of Molecular Oxygen in O2-Tolerant Membrane-Bound [NiFe] Hydrogenase. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), E2229– E2237, DOI: 10.1073/pnas.1712267115[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1WjsbbJ&md5=871c5a1671f144663859bfcc1a0aa404Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenaseKalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; Utesch, Tillmann; Gotthard, Guillaume; von Stetten, David; van der Linden, Peter; Royant, Antoine; Mroginski, Maria Andrea; Carpentier, Philippe; Lenz, Oliver; Scheerer, PatrickProceedings of the National Academy of Sciences of the United States of America (2018), 115 (10), E2229-E2237CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas mols. through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic redn. of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed exptl. using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallog. and computational studies. This combined approach revealed several sites of O2 mols. within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concns. used for MBH crystal derivatization. The examn. of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme.
- 10Kubas, A.; Orain, C.; De Sancho, D.; Saujet, L.; Sensi, M.; Gauquelin, C.; Meynial-Salles, I.; Soucaille, P.; Bottin, H.; Baffert, C.; Fourmond, V.; Best, R.; Blumberger, J.; Léger, C. Mechanism of O2 Diffusion and Reduction in FeFe Hydrogenases. Nat. Chem. 2017, 9, 88– 95, DOI: 10.1038/nchem.2592[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVSrsLzJ&md5=d4de66253d7c17f395ad5edae4910449Mechanism of O2 diffusion and reduction in FeFe hydrogenasesKubas, Adam; Orain, Christophe; De Sancho, David; Saujet, Laure; Sensi, Matteo; Gauquelin, Charles; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Herve; Baffert, Carole; Fourmond, Vincent; Best, Robert B.; Blumberger, Jochen; Leger, ChristopheNature Chemistry (2017), 9 (1), 88-95CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)[FeFe]-hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnol. devices. Here, the authors combined electrochem., site-directed mutagenesis, mol. dynamics simulations, and quantum chem. calcns. to uncover the mol. mechanism of O2 diffusion within the enzyme and its reactions at the active site. The authors proposed that the partial reversibility of the reaction with O2 resulted from the 4-electron redn. of O2 to water. The 3rd electron/proton transfer step was the bottleneck for water prodn., competing with formation of a highly reactive OH radical and hydroxylated Cys residue. The rapid delivery of electrons and protons to the active site was therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
- 11Fernández, V. M.; Hatchikian, E. C.; Cammack, R. Properties and Reactivation of Two Different Deactivated Forms of Desulfovibrio Gigas Hydrogenase. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 832, 69– 79, DOI: 10.1016/0167-4838(85)90175-X[Crossref], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXmtV2ktrc%253D&md5=d3650cd7715ef57a004a2d6755f80b6aProperties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenaseFernandez, Victor M.; Hatchikian, E. Claude; Cammack, RichardBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1985), 832 (1), 69-79CODEN: BBAEDZ; ISSN:0167-4838.It was previously shown that D. gigas hydrogenase, as isolated, has a relatively low activity in the H2-Me viologen reductase assay, and that the activity is slowly stimulated ≤10-fold by H2 or strong reductants. The enzyme, before reductive activation, is also totally inactive in 1H-3H exchange and H2-dichloroindophenol (DCIP) reductase assays. The behavior of the enzyme in various states of activation is discussed in terms of 3 different states: the active state, which is active in all assays; the unready state, which is totally inactive; and the ready state, which does not react with H2, but which is rapidly activated by strong reductants. The conditions for the slow activation of the unready state of D. gigas hydrogenase were investigated. The rate of activation was independent of enzyme concn. over a wide range, which rules out mechanisms involving intermol. electron exchange. The rate was only slightly affected by pH in the range 6-9, but was strongly temp. dependent, with an activation energy of 88 kJ/mol. The enzyme could be activated by dithiothreitol + the mediator dye indigo tetrasulfonate, but not by dithiothreitol alone. No effects were seen during treatments with weaker reductants, thioredoxin, Fe2+, sulfide, or Ni2+. The activation does not involve conversions of a metal center or the cleavage of an accessible SS bridge. Presumably, it involves an intramol. change, possibly in the redox state or coordination of a metal center. The active form of D. gigas hydrogenase was rapidly activated by O, producing mostly the unready state, which could be reactivated only slowly. By contrast, anaerobic reoxidn. by DCIP was able to convert the enzyme mostly to the ready state. This was identified as being inactive in 1H-3H exchange and H2-DCIP reductase assays but rapidly activated in the H2-Me viologen reductase assay (DCIP prevents this). A similar oxidn. of the active enzyme may take place in the cell as a protection against O.
- 12Lamle, S. E.; Albracht, S. P. J.; Armstrong, F. A. Electrochemical Potential-Step Investigations of the Aerobic Interconversions of [NiFe]-Hydrogenase from Allochromatium Vinosum: Insights into the Puzzling Difference between Unready and Ready Oxidized Inactive States. J. Am. Chem. Soc. 2004, 126, 14899– 14909, DOI: 10.1021/ja047939v[ACS Full Text], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXovVKgtL8%253D&md5=57d6b9248e8610a2aca59dfb0c48e341Electrochemical Potential-Step Investigations of the Aerobic Interconversions of [NiFe]-Hydrogenase from Allochromatium vinosum: Insights into the Puzzling Difference between Unready and Ready Oxidized Inactive StatesLamle, Sophie E.; Albracht, Simon P. J.; Armstrong, Fraser A.Journal of the American Chemical Society (2004), 126 (45), 14899-14909CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Dynamic electrochem. studies, incorporating catalytic voltammetry and detailed potential-step manipulations, provide compelling evidence that the oxidized inactive state of [NiFe]-hydrogenases termed Unready (or Ni-A) contains a product of partial redn. of O2 that is trapped in the active site.
- 13Abou Hamdan, A.; Burlat, B.; Gutiérrez-Sanz, Ó.; Liebgott, P.; Baffert, C.; De Lacey, A. L.; Rousset, M.; Guigliarelli, B.; Léger, C.; Dementin, S. O2-Independent Formation of the Inactive States of NiFe Hydrogenase. Nat. Chem. Biol. 2013, 9, 15– 17, DOI: 10.1038/nchembio.1110[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s7isFWrsw%253D%253D&md5=86fca973f7f6abad383486947d4d3279O2-independent formation of the inactive states of NiFe hydrogenaseAbou Hamdan Abbas; Burlat Benedicte; Gutierrez-Sanz Oscar; Liebgott Pierre-Pol; Baffert Carole; De Lacey Antonio L; Rousset Marc; Guigliarelli Bruno; Leger Christophe; Dementin SebastienNature chemical biology (2013), 9 (1), 15-7 ISSN:.We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.
- 14Baltazar, C.; Marques, M.; Soares, C. M.; De Lacey, A.; Pereira, I. A. C.; Matias, P. M. Nickel-Iron-Selenium Hydrogenases - An Overview. Eur. J. Inorg. Chem. 2011, 2011, 948– 962, DOI: 10.1002/ejic.201001127
- 15Wombwell, C.; Caputo, C. A.; Reisner, E. [NiFeSe]-Hydrogenase Chemistry. Acc. Chem. Res. 2015, 48, 2858– 2865, DOI: 10.1021/acs.accounts.5b00326[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1yltrzE&md5=bef139745dc865d1978624c5eecb06ca[NiFeSe]-Hydrogenase ChemistryWombwell, Claire; Caputo, Christine A.; Reisner, ErwinAccounts of Chemical Research (2015), 48 (11), 2858-2865CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The development of technol. for the inexpensive generation of the renewable energy vector H2 through water splitting is of immediate economic, ecol., and humanitarian interest. Recent interest in hydrogenases has been fueled by their exceptionally high catalytic rates for H2 prodn. at a marginal overpotential, which is presently only matched by the nonscalable noble metal platinum. The mechanistic understanding of hydrogenase function guides the design of synthetic catalysts, and selection of a suitable hydrogenase enables direct applications in electro- and photocatalysis. [FeFe]-hydrogenases display excellent H2 evolution activity, but they are irreversibly damaged upon exposure to O2, which currently prevents their use in full water splitting systems. O2-tolerant [NiFe]-hydrogenases are known, but they are typically strongly biased toward H2 oxidn., while H2 prodn. by [NiFe]-hydrogenases is often product (H2) inhibited. [NiFeSe]-hydrogenases are a subclass of [NiFe]-hydrogenases with a selenocysteine residue coordinated to the active site nickel center in place of a cysteine. They exhibit a combination of unique properties that are highly advantageous for applications in water splitting compared with other hydrogenases. They display a high H2 evolution rate with marginal inhibition by H2 and tolerance to O2. [NiFeSe]-hydrogenases are therefore one of the most active mol. H2 evolution catalysts applicable in water splitting. Herein, we summarize our recent progress in exploring the unique chem. of [NiFeSe]-hydrogenases through biomimetic model chem. and the chem. with [NiFeSe]-hydrogenases in semi-artificial photosynthetic systems. We gain perspective from the structural, spectroscopic, and electrochem. properties of the [NiFeSe]-hydrogenases and compare them with the chem. of synthetic models of this hydrogenase active site. Our synthetic models give insight into the effects on the electronic properties and reactivity of the active site upon the introduction of selenium. We have utilized the exceptional properties of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum in a no. of photocatalytic H2 prodn. schemes, which are benchmark systems in terms of single site activity, tolerance toward O2, and in vitro water splitting with biol. mols. Each system comprises a light-harvesting component, which allows for light-driven electron transfer to the hydrogenase in order for it to catalyze H2 prodn. A system with [NiFeSe]-hydrogenase on a dye-sensitized TiO2 nanoparticle gives an enzyme-semiconductor hybrid for visible light-driven generation of H2 with an enzyme-based turnover frequency of 50 s-1. A stable and inexpensive polymeric carbon nitride as a photosensitizer in combination with the [NiFeSe]-hydrogenase shows good activity for more than 2 days. Light-driven H2 evolution with the enzyme and an org. dye under high O2 levels demonstrates the excellent robustness and feasibility of water splitting with a hydrogenase-based scheme. This has led, most recently, to the development of a light-driven full water splitting system with a [NiFeSe]-hydrogenase wired to the water oxidn. enzyme photosystem II in a photoelectrochem. cell. In contrast to the other systems, this photoelectrochem. system does not rely on a sacrificial electron donor and allowed us to establish the long sought after light-driven water splitting with an isolated hydrogenase.
- 16Teixeira, M.; Fauque, G.; Moura, I.; Lespinat, P. A.; Berlier, Y.; Prickril, B.; Peck, H.; Xavier, A. V.; Gall, J. Le; Moura, J. J. G. Nickel-[Iron-Sulfur]-Selenium-Containing Hydrogenases from Desulfovibrio Baculatus (DSM 1743). Eur. J. Biochem. 1987, 167, 47– 58, DOI: 10.1111/j.1432-1033.1987.tb13302.x[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXlt12lu7w%253D&md5=88ebdaadc996173e66e129417a7d4c5aNickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic propertiesTeixeira, Miguel; Fauque, Guy; Moura, Isabel; Lespinat, Paul A.; Berlier, Yves; Prickril, Ben; Peck, Harry D., Jr.; Xavier, Antonio V.; Le Gall, Jean; Moura, Jose J. G.European Journal of Biochemistry (1987), 167 (1), 47-58CODEN: EJBCAI; ISSN:0014-2956.Hydrogenase from D. baculatus (DSM 1743) was purified from each of 3 different fractions: sol. periplasmic (wash), sol. cytoplasmic (cell disruption), and membrane-bound (detergent solubilization). Plasma-emission metal anal. detected in all 3 fractions the presence of Fe plus Ni and Se in equimol. amts. These hydrogenases were composed of 2 nonidentical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g = ∼2.0 when obsd. at low temp. (<20 K). The periplasmic and membrane-bound enzymes also presented addnl. EPR signals, observable up to 77 K, with g > 2.0, and assigned to Ni(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06, and 2.0. The signals obsd. in the membrane-bound prepns. could be decompd. into 2 sets with g = 2.34, 2.16, and ∼2.0 (component I) and g = 2.33, 2.24, and ∼2.0 (component II). In the reduced state, after exposure to a H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temps. and microwave powers, and in samples partially and fully reduced (under H2 or dithionite), allowed the identification of 2 different Fe-S centers: center I (g = 2.03, 1.89 and 1.86) detectable at <10 K, and center II (g = 2.06, 1.95, and 1.88) which was easily satd. at low temps. Addnl. EPR signals due to transient Ni species were detected with g = >2.0, and a rhombic EPR signal at 77 K developed at g = 2.20, 2.16, and 2.0. This EPR signal is reminiscent of the Ni signal C (g = 2.19, 2.14 and 2.02) obsd. in intermediate redox states of the well-characterized Desulfovibrio gigas hydrogenase. During the course of a redox titrn. at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity at approx. -320 mV. Low-temp. studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g = 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The sol. hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such step to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymic activity. The catalytic properties were also followed by the 1H-2H exchange reaction. The isolated hydrogenases produced H2/HD ratios higher than those obsd. for non-Se-contg. hydrogenases.
- 17Valente, F.; Oliveira, S.; Gnadt, N.; Pacheco, I.; Coelho, A. V.; Xavier, A. V.; Teixeira, M.; Soares, C. M.; Pereira, I. A. C. Hydrogenases in Desulfovibrio Vulgaris Hildenborough: Structural and Physiologic Characterisation of the Membrane-Bound [NiFeSe] Hydrogenase. JBIC, J. Biol. Inorg. Chem. 2005, 10, 667– 682, DOI: 10.1007/s00775-005-0022-4[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1CqtLvO&md5=6ea253f7b728ec043dcec87ee8d390aaHydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenaseValente, Filipa M. A.; Oliveira, A. Sofia F.; Gnadt, Nicole; Pacheco, Isabel; Coelho, Ana V.; Xavier, Antonio V.; Teixeira, Miguel; Soares, Claudio M.; Pereira, Ines A. C.JBIC, Journal of Biological Inorganic Chemistry (2005), 10 (6), 667-682CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulfate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase assocd. with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H2 prodn. activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidn. state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane assocn. Cleavage of this group originates a sol., less active form of the enzyme. Sequence anal. shows that [NiFeSe] Hases from Desulfovibrionacae form a sep. family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S]2+/1+ cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe]1 Hase, with the Type I cytochrome c 3 as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centers were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.
- 18Marques, M.; Tapia, C.; Gutiérrez-Sanz, Ó.; Ramos, A. R.; Keller, K. L.; Wall, J. D.; De Lacey, A. L.; Matias, P. M.; Pereira, I. A. C. The Direct Role of Selenocysteine in [NiFeSe] Hydrogenase Maturation and Catalysis. Nat. Chem. Biol. 2017, 13, 544– 550, DOI: 10.1038/nchembio.2335[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvFahtb8%253D&md5=e46b54a3255f5195c93026c15ffe192eThe direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysisMarques, Marta C.; Tapia, Cristina; Gutierrez-Sanz, Oscar; Ramos, Ana Raquel; Keller, Kimberly L.; Wall, Judy D.; De Lacey, Antonio L.; Matias, Pedro M.; Pereira, Ines A. C.Nature Chemical Biology (2017), 13 (5), 544-550CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Hydrogenases are highly active enzymes for hydrogen prodn. and oxidn. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 prodn. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for prodn. of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.
- 19Parkin, A.; Goldet, G.; Cavazza, C.; Fontecilla-Camps, J. C.; Armstrong, F. A. The Difference a Se Makes? Oxygen-Tolerant Hydrogen Production by the [NiFeSe]-Hydrogenase from Desulfomicrobium Baculatum. J. Am. Chem. Soc. 2008, 130, 13410– 13416, DOI: 10.1021/ja803657d[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFSjtrvI&md5=082d232b1fd5fd6e8fdf80b3c185b466The Difference a Se Makes? Oxygen-Tolerant Hydrogen Production by the [NiFeSe]-Hydrogenase from Desulfomicrobium baculatumParkin, Alison; Goldet, Gabrielle; Cavazza, Christine; Fontecilla-Camps, Juan C.; Armstrong, Fraser A.Journal of the American Chemical Society (2008), 130 (40), 13410-13416CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein film voltammetry (PFV) studies of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum show it to be a highly efficient H2 cycling catalyst. In the presence of 100% H2, the ratio of H2 prodn. to H2 oxidn. activity is higher than for any conventional [NiFe]-hydrogenases (lacking a selenocysteine ligand) that have been investigated to date. Although traces of O2 (« 1%) rapidly and completely remove H2 oxidn. activity, the enzyme sustains partial activity for H2 prodn. even in the presence of 1% O2 in the atm. That H2 prodn. should be partly allowed, whereas H2 oxidn. is not, is explained because the inactive product of O2 attack is reductively reactivated very rapidly, but this requires a potential that is almost as neg. as the thermodn. potential for the 2H+/H2 couple. The study provides further encouragement and clues regarding the feasibility of microbial/enzymic H2 prodn. free from restrictions of anaerobicity.
- 20Reisner, E.; Powell, D. J.; Cavazza, C.; Fontecilla-Camps, J. C.; Armstrong, F. A. Visible Light-Driven H2 Production by Hydrogenases Attached to Dye-Sensitized TiO2 Nanoparticles. J. Am. Chem. Soc. 2009, 131, 18457– 18466, DOI: 10.1021/ja907923r[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVKhsL%252FJ&md5=be81b7fbe9a198b0599afacfb4c58eeaVisible Light-Driven H2 Production by Hydrogenases Attached to Dye-Sensitized TiO2 NanoparticlesReisner, Erwin; Powell, Daniel J.; Cavazza, Christine; Fontecilla-Camps, Juan C.; Armstrong, Fraser A.Journal of the American Chemical Society (2009), 131 (51), 18457-18466CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A study of hybrid, enzyme-modified nanoparticles able to produce H2 using visible light as the energy source has been carried out to establish per-site performance stds. for H2 prodn. catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO2, with triethanolamine as a sacrificial electron donor, produces H2 at a turnover frequency of approx. 50 (mol H2) s-1 (mol total hydrogenase)-1 at pH 7 and 25 °C, even under the typical solar irradn. of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for bench-top applications.
- 21Wakerley, D. W.; Reisner, E. Oxygen-Tolerant Proton Reduction Catalysis: Much O2 about Nothing?. Energy Environ. Sci. 2015, 8, 2283– 2295, DOI: 10.1039/C5EE01167A[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1Sltbg%253D&md5=c077a781550e753ea5bae966ac9219e4Oxygen-tolerant proton reduction catalysis: much O2 about nothing?Wakerley, David W.; Reisner, ErwinEnergy & Environmental Science (2015), 8 (8), 2283-2295CODEN: EESNBY; ISSN:1754-5706. (Royal Society of Chemistry)Proton redn. catalysts are an integral component of artificial photosynthetic systems for the prodn. of H2. This perspective covers such catalysts with respect to their tolerance towards the potential catalyst inhibitor O2. O2 is abundant in our atm. and generated as a byproduct during the water splitting process, therefore maintaining proton redn. activity in the presence of O2 is important for the widespread prodn. of H2. This perspective article summarizes viable strategies for avoiding the adverse effects of aerobic environments to encourage their adoption and improvement in future research. H2-evolving enzymic systems, mol. synthetic catalysts and catalytic surfaces are discussed with respect to their interaction with O2 and anal. techniques through which O2-tolerant catalysts can be studied are described.
- 22Baltazar, C.; Teixeira, V. H.; Soares, C. M. Structural Features of [NiFeSe] and [NiFe] Hydrogenases Determining Their Different Properties: A Computational Approach. JBIC, J. Biol. Inorg. Chem. 2012, 17, 543– 555, DOI: 10.1007/s00775-012-0875-2[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlOmt70%253D&md5=d10cf43da4d06895abea888ae0c4c0f6Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approachBaltazar, Carla S. A.; Teixeira, Vitor H.; Soares, Claudio M.JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (4), 543-555CODEN: JJBCFA; ISSN:0949-8257. (Springer)Hydrogenases are metalloenzymes that catalyze the reversible reaction H2 ↹ 2H+ + 2e-, being potentially useful in H2 prodn. or oxidn. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O2 inhibition and produce more H2 than std. [NiFe] hydrogenases. However, the mol. determinants responsible for these properties remain unknown. Hydrophobic pathways for H2 diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H2 and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a std. [NiFe] hydrogenase. We performed mol. dynamics simulations of H2 diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas. The comparison showed that H2 d. near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H2 to the active site. We have also detd. a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas. The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H2 and proton pathways.
- 23Gutiérrez-Sanz, Ó.; Marques, M.; Baltazar, C.; Fernández, V. M.; Soares, C. M.; Pereira, I. A. C.; De Lacey, A. L. Influence of the Protein Structure Surrounding the Active Site on the Catalytic Activity of [NiFeSe] Hydrogenases. JBIC, J. Biol. Inorg. Chem. 2013, 18, 419– 427, DOI: 10.1007/s00775-013-0986-4[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjsV2jsL0%253D&md5=ba933361c2265bbaf85f477319c5cf6fInfluence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenasesGutierrez-Sanz, Oscar; Marques, Marta C.; Baltazar, Carla S. A.; Fernandez, Victor M.; Soares, Claudio M.; Pereira, Ines A. C.; De Lacey, Antonio L.JBIC, Journal of Biological Inorganic Chemistry (2013), 18 (4), 419-427CODEN: JJBCFA; ISSN:0949-8257. (Springer)A combined exptl. and theor. study of the catalytic activity of Desulfovibrio vulgaris [NiFeSe] hydrogenase was performed by H/D exchange mass spectrometry and mol. dynamics (MD) simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or prodn. of H2. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the Ni atom in the active site instead of cysteine. The aim of this research was to det. how much the specific catalytic properties of this hydrogenase were influenced by the replacement of a S atom by a Se atom in the coordination of the bimetallic active site vs. the changes in the protein structure surrounding the active site. The pH dependence of the D2/H+ exchange activity and the high isotope effect obsd. in the Km for the H2 substrate and in the single exchange/double exchange ratio suggest that a "cage effect" due to the protein structure surrounding the active site was modulating the enzymic catalysis. This "cage effect" was supported by MD simulations of the diffusion of H2 and D2 from the outside to the inside of the protein, which showed different accumulation of these substrates in a cavity next to the active site.
- 24Tamura, T.; Tsunekawa, N.; Nemoto, M.; Inagaki, K.; Hirano, T.; Sato, F. Molecular Evolution of Gas Cavity in [NiFeSe] Hydrogenases Resurrected in Silico. Sci. Rep. 2016, 6, 19742, DOI: 10.1038/srep19742[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs12jtbg%253D&md5=6c0f6dd0646de41a7d15e2940b0cb735Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silicoTamura, Takashi; Tsunekawa, Naoki; Nemoto, Michiko; Inagaki, Kenji; Hirano, Toshiyuki; Sato, FumitoshiScientific Reports (2016), 6 (), 19742CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Oxygen tolerance of selenium-contg. [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulfate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homol. modeling and mol. dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under abs. anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.
- 25Ceccaldi, P.; Marques, M.; Fourmond, V.; Pereira, I. A. C.; Léger, C. Oxidative Inactivation of NiFeSe Hydrogenase. Chem. Commun. 2015, 51, 14223– 14226, DOI: 10.1039/C5CC05930E[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmtrjE&md5=3cc06940ad564a04949151ff00fe7dceOxidative inactivation of NiFeSe hydrogenaseCeccaldi, Pierre; Marques, Marta C.; Fourmond, Vincent; Pereira, Ines Cardoso; Leger, ChristopheChemical Communications (Cambridge, United Kingdom) (2015), 51 (75), 14223-14226CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The authors propose a resoln. to the paradox that spectroscopic studies of NiFeSe hydrogenase have not revealed any major signal attributable to NiIII states formed upon reaction with O2, despite the fact that 2 inactive states are formed upon either aerobic or anaerobic oxidn.
- 26Maroney, M. J.; Hondal, R. J. Selenium versus Sulfur: Reversibility of Chemical Reactions and Resistance to Permanent Oxidation in Proteins and Nucleic Acids. Free Radical Biol. Med. 2018, 127, 228– 237, DOI: 10.1016/j.freeradbiomed.2018.03.035[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntFajt7g%253D&md5=16adde751b6085fe8451e6bbaa0eec8fSelenium, sulfur, and the Reversibility of chemical reactions and resistance to permanent oxidation in proteins and nucleic acidsMaroney, Michael J.; Hondal, Robert J.Free Radical Biology & Medicine (2018), 127 (), 228-237CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)A review. This review highlights the contributions of Jean Chaudiere to the field of selenium biochem. Chaudiere was the first to recognize that one of the main reasons that selenium in the form of selenocysteine is used in proteins is due to the fact that it strongly resists permanent oxidn. The foundations for this important concept was laid down by Al Tappel in the 1960's and even before by others. The concept of oxygen tolerance first recognized in the study of glutathione peroxidase was further advanced and refined by those studying [NiFeSe]-hydrogenases, selenosubtilisin, and thioredoxin reductase. After 200 years of selenium research, work by Marcus Conrad and coworkers studying glutathione peroxidase-4 has provided definitive evidence for Chaudiere's original hypothesis (Ingold et al., 2018) [36]. While the reaction of selenium with oxygen is readily reversible, there are many other examples of this phenomenon of reversibility. Many reactions of selenium can be described as "easy in - easy out". This is due to the strong nucleophilic character of selenium to attack electrophiles, but then this reaction can be reversed due to the strong electrophilic character of selenium and the weakness of the selenium-carbon bond. Several examples of this are described.
- 27Marques, M.; Coelho, R.; Pereira, I. A. C.; Matias, P. M. Redox State-Dependent Changes in the Crystal Structure of [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough. Int. J. Hydrogen Energy 2013, 38, 8664– 8682, DOI: 10.1016/j.ijhydene.2013.04.132[Crossref], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosFertbo%253D&md5=9fd85def4bd733acddec965ae314aa3aRedox state-dependent changes in the crystal structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris HildenboroughMarques, Marta C.; Coelho, Ricardo; Pereira, Ines A. C.; Matias, Pedro M.International Journal of Hydrogen Energy (2013), 38 (21), 8664-8682CODEN: IJHEDX; ISSN:0360-3199. (Elsevier Ltd.)Hydrogenases are enzymes that can potentially be used in bioelec. devices or for biol. hydrogen prodn., the most studied of which are the [NiFe] type. Most [NiFe] hydrogenases are inactivated by oxygen and the few known O2-tolerant enzymes are hydrogen-uptake enzymes, unsuitable for hydrogen prodn., due to strong product inhibition. In contrast, the [NiFeSe] hydrogenases, where a selenocysteine is bound to the nickel, are very attractive alternatives because of their high hydrogen prodn. activity and fast reactivation after O2 exposure. Here we report five high-resoln. crystallog. 3D structures of the sol. form of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough in three different redox states (oxidized as-isolated, H2-reduced and air re-oxidized), which revealed the structural changes that take place at the active site during enzyme redn. and re-oxidn. The results provide new insights into the pathways of O2 inactivation in [NiFe], and in particular [NiFeSe], hydrogenases. In addn., they suggest that different enzymes may display different oxidized states upon exposure to O2, which are probably detd. by the protein structure.
- 28Marques, M.; Coelho, R.; De Lacey, A. L.; Pereira, I. A. C.; Matias, P. M. The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State. J. Mol. Biol. 2010, 396, 893– 907, DOI: 10.1016/j.jmb.2009.12.013[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitValtr8%253D&md5=fa4d04e98e831a42830404f607fb44d8The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: A hydrogenase without a bridging ligand in the active site in its oxidized, "as-isolated" stateMarques, Marta C.; Coelho, Ricardo; De Lacey, Antonio L.; Pereira, Ines A. C.; Matias, Pedro M.Journal of Molecular Biology (2010), 396 (4), 893-907CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)H2 is a good energy vector, and its prodn. from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biol. prodn. of H2 because they are capable of high prodn. rates even in the presence of moderate amts. of O2, lessening the requirements for anaerobic conditions. Here, the 3-dimensional structure of [NiFeSe] Hase of D. vulgaris Hildenborough was detd. in its oxidized "as-isolated" form at 2.04-Å resoln. Remarkably, this is the 1st structure of an oxidized Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra S atom was obsd. binding Ni and Se, leading to a SeCys conformation that shielded the NiFe site from contact with O2. This structure provided several insights that may explain the fast activation and O2 tolerance of these enzymes.
- 29Volbeda, A.; Amara, P.; Iannello, M.; De Lacey, A. L.; Cavazza, C.; Fontecilla-Camps, J. C. Structural Foundations for the O2 Resistance of Desulfomicrobium Baculatum [NiFeSe]-Hydrogenase. Chem. Commun. (Cambridge, U. K.) 2013, 49, 7061– 7063, DOI: 10.1039/c3cc43619e[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFSit7jN&md5=01da88066da9970ae41fc16d27a66465Structural foundations for the O2 resistance of Desulfomicrobium baculatum [NiFeSe]-hydrogenaseVolbeda, Anne; Amara, Patricia; Iannello, Marina; De Lacey, Antonio L.; Cavazza, Christine; Fontecilla-Camps, Juan CarlosChemical Communications (Cambridge, United Kingdom) (2013), 49 (63), 7061-7063CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The authors show how the NiFeSe site of anaerobically purified O2-resistant hydrogenase of D. baculatum reacts with air to give a seleninate as the 1st product. Less oxidized states of the active site were readily reduced in the presence of x-rays. Reductive enzyme activation required an efficient pathway for water escape. The crystal structure of the enzyme is reported. The crystal space group was P212121 with cell dimensions a = 106.2, b = 108.7, and c = 136.5 Å.
- 30Garcin, E.; Vernede, X.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J. C. The Crystal Structure of a Reduced [NiFeSe] Hydrogenase Provides an Image of the Activated Catalytic Center. Structure 1999, 7, 557– 566, DOI: 10.1016/S0969-2126(99)80072-0[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVSitrw%253D&md5=881c8e351d7fc1dbe6050bec883b20f4The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic centerGarcin, E.; Vernede, X.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J. C.Structure (London) (1999), 7 (5), 557-566CODEN: STRUE6; ISSN:0969-2126. (Current Biology Publications)[NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2 ↔ 2H+ + 2e-. They are generally heterodimeric, contain 3 Fe-S clusters in their small subunit and a Ni-Fe-contg. active site in their large subunit that includes a selenocysteine (SeCys) ligand. Here, the authors report the x-ray crystal structure at 2.15 Å resoln. of periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of oxidized, as-prepd., Desulfovibrio gigas and the reduced D. baculatum hydrogenases showed that in the reduced enzyme the Ni-Fe distance was 0.4 Å shorter than in the oxidized enzyme. In addn., the putative oxo ligand, detected in the as-prepd. D. gigas enzyme, was absent from the D. baculatum hydrogenase. The authors also obsd. higher-than-av. temp. factors for both the active site Ni-selenocysteine ligand and the neighboring Glu-18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the mol. surface. Other differences between [NiFeSe] and [NiFe] hydrogenases were the presence of a 3rd [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative Fe center that substitutes the Mg2+ ion that has already been described at the C-terminus of the large subunit of 2 [NiFe] hydrogenases. The heterolytic cleavage of H2 seems to be mediated by the Ni center and the selenocysteine residue. In addn. to modifying the catalytic properties of the enzyme, the Se ligand might protect the Ni atom from oxidn. It was concluded that the putative oxo ligand is a signature of inactive "unready" [NiFe] hydrogenases.
- 31Zacarias, S.; Vélez, M.; Pita, M.; De Lacey, A. L.; Matias, P. M.; Pereira, I. A. C. Methods in Enzymology 2018, 613, 169, DOI: 10.1016/bs.mie.2018.10.003[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3crotVynug%253D%253D&md5=63af0b1c66a5c68e7034e56922866798Characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris HildenboroughZacarias Sonia; Velez Marisela; Pita Marcos; De Lacey Antonio L; Matias Pedro M; Pereira Ines A CMethods in enzymology (2018), 613 (), 169-201 ISSN:.The [NiFeSe] hydrogenases are a subgroup of the well-characterized family of [NiFe] hydrogenases, in which a selenocysteine is a ligand to the nickel atom in the binuclear NiFe active site instead of cysteine. These enzymes display very interesting catalytic properties for biological hydrogen production and bioelectrochemical applications: high H2 production activity, bias for H2 evolution, low H2 inhibition, and some degree of O2 tolerance. Here we describe the methodologies employed to study the [NiFeSe] hydrogenase isolated from the sulfate-reducing bacteria D. vulgaris Hildenborough and the creation of a homologous expression system for production of variant forms of the enzyme.
- 32Ruff, A.; Szczesny, J.; Zacarias, S.; Pereira, I. A. C.; Plumeré, N.; Schuhmann, W. Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough under Oxidative Conditions. ACS Energy Lett. 2017, 2, 964– 968, DOI: 10.1021/acsenergylett.7b00167[ACS Full Text], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVelsrw%253D&md5=93d67508e80d067b63c190b60f124459Protection and reactivation of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough under oxidative conditionsRuff, Adrian; Szczesny, Julian; Zacarias, Sonia; Pereira, Ines A. C.; Plumere, Nicolas; Schuhmann, WolfgangACS Energy Letters (2017), 2 (5), 964-968CODEN: AELCCP; ISSN:2380-8195. (American Chemical Society)We report on the fabrication of bioanodes for H2 oxidn. based on [NiFeSe] hydrogenase. The enzyme was elec. wired by means of a specifically designed low-potential viologen-modified polymer, which delivers benchmark H2 oxidizing currents even under deactivating conditions owing to efficient protection against O2 combined with a viologen-induced reactivation of the O2-inhibited enzyme. Moreover, the viologen-modified polymer allows for electrochem. co-deposition of polymer and biocatalyst and, by this, for control of the film thickness. Protection and reactivation of the enzyme was demonstrated in thick and thin reaction layers.
- 33Ruff, A.; Szczesny, J.; Marković, N.; Conzuelo, F.; Zacarias, S.; Pereira, I. A. C.; Lubitz, W.; Schuhmann, W. A Fully Protected Hydrogenase/Polymer-Based Bioanode for High-Performance Hydrogen/Glucose Biofuel Cells. Nat. Commun. 2018, 9, 3675, DOI: 10.1038/s41467-018-06106-3[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3nsV2ruw%253D%253D&md5=14b1677590384718edbc3d1b051df0daA fully protected hydrogenase/polymer-based bioanode for high-performance hydrogen/glucose biofuel cellsRuff Adrian; Szczesny Julian; Markovic Nikola; Conzuelo Felipe; Schuhmann Wolfgang; Zacarias Sonia; Pereira Ines A C; Lubitz WolfgangNature communications (2018), 9 (1), 3675 ISSN:.Hydrogenases with Ni- and/or Fe-based active sites are highly active hydrogen oxidation catalysts with activities similar to those of noble metal catalysts. However, the activity is connected to a sensitivity towards high-potential deactivation and oxygen damage. Here we report a fully protected polymer multilayer/hydrogenase-based bioanode in which the sensitive hydrogen oxidation catalyst is protected from high-potential deactivation and from oxygen damage by using a polymer multilayer architecture. The active catalyst is embedded in a low-potential polymer (protection from high-potential deactivation) and covered with a polymer-supported bienzymatic oxygen removal system. In contrast to previously reported polymer-based protection systems, the proposed strategy fully decouples the hydrogenase reaction form the protection process. Incorporation of the bioanode into a hydrogen/glucose biofuel cell provides a benchmark open circuit voltage of 1.15 V and power densities of up to 530 μW cm(-2) at 0.85 V.
- 34Szczesny, J.; Marković, N.; Conzuelo, F.; Zacarias, S.; Pereira, I. A. C.; Lubitz, W.; Plumeré, N.; Schuhmann, W.; Ruff, A. A Gas Breathing Hydrogen/Air Biofuel Cell Comprising a Redox Polymer/Hydrogenase-Based Bioanode. Nat. Commun. 2018, 9, 4715, DOI: 10.1038/s41467-018-07137-6[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cvosFGrug%253D%253D&md5=c57ae509fb6b30179d48ba64f2ec4677A gas breathing hydrogen/air biofuel cell comprising a redox polymer/hydrogenase-based bioanodeSzczesny Julian; Markovic Nikola; Conzuelo Felipe; Schuhmann Wolfgang; Ruff Adrian; Zacarias Sonia; Pereira Ines A C; Lubitz Wolfgang; Plumere NicolasNature communications (2018), 9 (1), 4715 ISSN:.Hydrogen is one of the most promising alternatives for fossil fuels. However, the power output of hydrogen/oxygen fuel cells is often restricted by mass transport limitations of the substrate. Here, we present a dual-gas breathing H2/air biofuel cell that overcomes these limitations. The cell is equipped with a hydrogen-oxidizing redox polymer/hydrogenase gas-breathing bioanode and an oxygen-reducing bilirubin oxidase gas-breathing biocathode (operated in a direct electron transfer regime). The bioanode consists of a two layer system with a redox polymer-based adhesion layer and an active, redox polymer/hydrogenase top layer. The redox polymers protect the biocatalyst from high potentials and oxygen damage. The bioanodes show remarkable current densities of up to 8 mA cm(-2). A maximum power density of 3.6 mW cm(-2) at 0.7 V and an open circuit voltage of up to 1.13 V were achieved in biofuel cell tests, representing outstanding values for a device that is based on a redox polymer-based hydrogenase bioanode.
- 35Tapia, C.; Zacarias, S.; Pereira, I. A. C.; Conesa, J. C.; Pita, M.; De Lacey, A. L. In Situ Determination of Photobioproduction of H2 by In2S3-[NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough Using Only Visible Light. ACS Catal. 2016, 6, 5691– 5698, DOI: 10.1021/acscatal.6b01512[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1aju7vI&md5=e0a75db3466993373a2d25a9174bce54In Situ Determination of Photobioproduction of H2 by In2S3-[NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough Using Only Visible LightTapia, Cristina; Zacarias, Sonia; Pereira, Ines A. C.; Conesa, Jose C.; Pita, Marcos; De Lacey, Antonio L.ACS Catalysis (2016), 6 (9), 5691-5698CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)An interesting strategy for photocatalytic prodn. of hydrogen from water and sunlight is the formation of a hybrid photocatalyst that combines an inorg. semiconductor able to absorb in the visible light spectral range with an enzymic catalyst for reducing protons. How to optimize the interfacing of In2S3 particles with the sol. form of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough by means of its initial H2 photoprodn. rate is studied. The kinetics of the photocatalytic process was studied by membrane-inlet mass spectrometry, in order to optimize the interaction between both components of the hybrid photocatalyst. Membrane-inlet mass spectrometry allows measuring in the same expt., for comparison, the rate of H2 prodn. by the photocatalyst hybrid directly in the aq. soln. in real time and the result of a std. assay of the hydrogenase activity. An incubation period of 6 h with mild stirring of hydrogenase with In2S3 particles was necessary for optimal interaction of the enzyme mols. with the porous surface of the semiconductor. A turnover frequency of the NiFeSe hydrogenase (TOFHase) for H2 photobioprodn. of 986 s-1 was measured under the optimized conditions. This means that the immobilized hydrogenase has a photocatalytic efficiency for H2 generation which is 94% of that obtained in the std. specific activity test of H2 prodn. using reduced Me viologen as an electron donor.
- 36Gutiérrez-Sanz, Ó.; Natale, P.; Márquez, I.; Marques, M.; Zacarias, S.; Pita, M.; Pereira, I. A. C.; López-Montero, I.; De Lacey, A. L.; Vélez, M. H2-Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration. Angew. Chem., Int. Ed. 2016, 55, 6216– 6220, DOI: 10.1002/anie.201600752[Crossref], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlKmur4%253D&md5=866777a35d90c2cdea5cc096d6d1a4f5H2-Fueled ATP Synthesis on an Electrode: Mimicking Cellular RespirationGutierrez-Sanz, Oscar; Natale, Paolo; Marquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Ines A. C.; Lopez-Montero, Ivan; De Lacey, Antonio L.; Velez, MariselaAngewandte Chemie, International Edition (2016), 55 (21), 6216-6220CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)ATP, the mol. used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0-ATPase in a lipid membrane can couple the electrochem. oxidn. of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochem. fuel locally when required in biomedical devices or enzymic synthesis of valuable products.
- 37Paulsen, C. E.; Carroll, K. S. Cysteine-Mediated Redox Signaling: Chemistry, Biology, and Tools for Discovery. Chem. Rev. 2013, 113, 4633– 4679, DOI: 10.1021/cr300163e[ACS Full Text], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlert7Y%253D&md5=a071177f7815ccefe7a58abe02d24906Cysteine-mediated redox signaling: Chemistry, biology, and tools for discoveryPaulsen, Candice E.; Carroll, Kate S.Chemical Reviews (Washington, DC, United States) (2013), 113 (7), 4633-4679CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The different oxidative post-translational modifications of protein Cys residue SH groups are reviewed, with particular emphasis on those chem. properties that differentiate one modification from another. Recent progress in using chem. approaches to develop probes that enable selective and direct detection of individual modifications within their native cellular environment are also reviewed. The discussion is complemented with examples from the literature that highlight ways in which Cys oxidn. can be used to control protein function and cell signaling pathways.
- 38Sensi, M.; del Barrio, M.; Baffert, C.; Fourmond, V.; Léger, C. New Perspectives in Hydrogenase Direct Electrochemistry. Curr. Opin. Electrochem. 2017, 5, 135– 145, DOI: 10.1016/j.coelec.2017.08.005[Crossref], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVGgsbzM&md5=3a1aef526f6859e16c65e91f8b05faaaNew perspectives in hydrogenase direct electrochemistrySensi, Matteo; del Barrio, Melisa; Baffert, Carole; Fourmond, Vincent; Leger, ChristopheCurrent Opinion in Electrochemistry (2017), 5 (1), 135-145CODEN: COEUCY; ISSN:2451-9111. (Elsevier B.V.)Electrochem. studies of hydrogenases, the biol. catalysts of H2 oxidn. and prodn., have proven wrong the old saying that enzymes do not easily transfer electrons to electrodes in the absence of mediators. Many distinct hydrogenases have actually been directly connected to electrodes or particles, for studying their catalytic mechanism or for designing solar-fuels catalysts. In this review, we list the electrodes that have proved successful for direct electron transfer to hydrogenases, and we discuss recent results which illustrate new directions in this research field: the study of the biosynthesis of FeFe hydrogenase, the electrochem. characterization of non-std. NiFe or FeFe hydrogenases, the general discussion of what makes a catalyst better in one particular direction of the reaction, and the elucidation of the mol. mechanisms of hydrogenase catalysis by combining electrochem. and theor. chem., spectroscopy or photochem. The electrochem. methods described herein will probably prove useful for studying or using other redox enzymes.
- 39del Barrio, M.; Sensi, M.; Orain, C.; Baffert, C.; Dementin, S.; Fourmond, V.; Léger, C. Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels. Acc. Chem. Res. 2018, 51, 769– 777, DOI: 10.1021/acs.accounts.7b00622[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFagu7s%253D&md5=c6d2e2edab275cf3cea9837d9d58b320Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuelsdel Barrio, Melisa; Sensi, Matteo; Orain, Christophe; Baffert, Carole; Dementin, Sebastien; Fourmond, Vincent; Leger, ChristopheAccounts of Chemical Research (2018), 51 (3), 769-777CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Many enzymes that produce or transform small mols. such as O2, H2, and CO2 embed inorg. cofactors based on transition metals. Their active site, where the chem. reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water mols. transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which det. the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochem., structural, spectroscopic, theor., and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extg. quant. information from the comparison of data and kinetic models and by exploring the combination of exptl. kinetics and theor. chem. In studies of these catalytic mechanisms, direct electrochem., the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an elec. current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chem. and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochem. can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramol. diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodn. of intramol. electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equil. potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examg. the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochem. investigations and MD, DFT, and TD-DFT calcns. is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O2, and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and detg. their structure using spectroscopic or structural methods.
- 40Léger, C.; Dementin, S.; Bertrand, P.; Rousset, M.; Guigliarelli, B. Inhibition and Aerobic Inactivation Kinetics of Desulfovibrio Fructosovorans NiFe Hydrogenase Studied by Protein Film Voltammetry. J. Am. Chem. Soc. 2004, 126, 12162– 12172, DOI: 10.1021/ja046548d[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntFGhs74%253D&md5=919d588f69d5af2f75a716b826cf0781Inhibition and Aerobic Inactivation Kinetics of Desulfovibrio fructosovorans NiFe Hydrogenase Studied by Protein Film VoltammetryLeger, Christophe; Dementin, Sebastien; Bertrand, Patrick; Rousset, Marc; Guigliarelli, BrunoJournal of the American Chemical Society (2004), 126 (38), 12162-12172CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addn. in the electrochem. cell of H2, CO, or O2 allow simple and virtually instantaneous detns. of the Michaelis const., inhibition const., or rate of inactivation, resp., thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we det. and discuss how its affinity for the active site changes as the driving force for the H+/H2 reaction is continuously varied. Inactivation by O2 is a slow, bimol. process (with pH-independent rate const. ≈ 3×104 s-1 M-1 at 40°, under one atm of H2) that leads to a mixt. of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H2. This exptl. approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.
- 41Fourmond, V.; Lautier, T.; Baffert, C.; Leroux, F.; Liebgott, P.; Dementin, S.; Rousset, M.; Arnoux, P.; Pignol, D.; Meynial-Salles, I.; Soucaille, P.; Bertrand, P.; Léger, C. Correcting for Electrocatalyst Desorption and Inactivation in Chronoamperometry Experiments. Anal. Chem. 2009, 81, 2962, DOI: 10.1021/ac8025702[ACS Full Text], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtl2qsr0%253D&md5=4c664f44beca6f3a84d7b55d49e8c0fdCorrecting for Electrocatalyst Desorption and Inactivation in Chronoamperometry ExperimentsFourmond, Vincent; Lautier, Thomas; Baffert, Carole; Leroux, Fanny; Liebgott, Pierre-Pol; Dementin, Sebastien; Rousset, Marc; Arnoux, Pascal; Pignol, David; Meynial-Salles, Isabelle; Soucaille, Phillippe; Bertrand, Patrick; Leger, ChristopheAnalytical Chemistry (Washington, DC, United States) (2009), 81 (8), 2962-2968CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Chronoamperometric expts. with adsorbed electrocatalysts are commonly performed either for anal. purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concn. is being increased to det. a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such expts., the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. The authors propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric expts. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control expt. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. The authors describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case the authors discuss the trustfulness and the benefit of the correction. This method also applies to expts. where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concn. of catalyst.
- 42del Barrio, M.; Guendon, C.; Kpebe, A.; Baffert, C.; Fourmond, V.; Brugna, M.; Léger, C. A Valine-to-Cysteine Mutation Further Increases the Oxygen Tolerance of Escherichia Coli NiFe Hydrogenase Hyd-1. ACS Catal. 2019, 9, 4084– 4088, DOI: 10.1021/acscatal.9b00543[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtFGnurc%253D&md5=37545f094f547cdcb0b1b91884744c33Valine-to-cysteine mutation further increases the oxygen tolerance of Escherichia coli NiFe hydrogenase Hyd-1del Barrio, Melisa; Guendon, Chloe; Kpebe, Arlette; Baffert, Carole; Fourmond, Vincent; Brugna, Myriam; Leger, ChristopheACS Catalysis (2019), 9 (5), 4084-4088CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Some NiFe hydrogenases are particularly resistant to O2 as a result of either the natural presence of a particular FeS cluster or the artificial replacement of a conserved valine residue near the Ni site. We show that the two protective effects can be combined in a single enzyme by constructing and characterizing the V78C variant of the naturally O2-tolerant Escherichia coli NiFe hydrogenase Hyd-1. We elucidated the effect of the mutation by comparing the kinetics of inhibition by CO and O2 of a no. of wild-type forms and valine-to-cysteine variants of NiFe hydrogenases.
- 43Almeida, M. G.; Silveira, C. M.; Guigliarelli, B.; Bertrand, P.; Moura, J. J. G.; Moura, I.; Léger, C. A Needle in a Haystack: The Active Site of the Membrane-Bound Complex Cytochrome c Nitrite Reductase. FEBS Lett. 2007, 581, 284– 288, DOI: 10.1016/j.febslet.2006.12.023[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslyqtQ%253D%253D&md5=27acbb3f784d63ef94913955b213507fA needle in a haystack: The active site of the membrane-bound complex cytochrome c nitrite reductaseAlmeida, M. Gabriela; Silveira, Celia M.; Guigliarelli, Bruno; Bertrand, Patrick; Moura, Jose J. G.; Moura, Isabel; Leger, ChristopheFEBS Letters (2007), 581 (2), 284-288CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron redn. of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The no. of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously det. the redn. potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.
- 44Chovancova, E.; Pavelka, A.; Benes, P.; Strnad, O.; Brezovsky, J.; Kozlikova, B.; Gora, A.; Sustr, V.; Klvana, M.; Medek, P.; Biedermannova, L.; Sochor, J.; Damborsky, J. CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures. PLoS Comput. Biol. 2012, 8, e1002708 DOI: 10.1371/journal.pcbi.1002708[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1ansbfI&md5=aff24be751fef33d531b446cf6ab86c5CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structuresChovancova, Eva; Pavelka, Antonin; Benes, Petr; Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, JiriPLoS Computational Biology (2012), 8 (10), e1002708CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)Tunnels and channels facilitate the transport of small mols., ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chem. properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromol. structures. Herein we present a new version of CAVER enabling automatic anal. of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calcn. and clustering of pathways. A trajectory from a mol. dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the anal. of mol. dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estd. the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that anal. of mol. dynamics simulation is essential for the estn. of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochem. phenomena in the area of mol. transport, mol. recognition and enzymic catalysis. The software is freely available as a multiplatform command-line application online.
- 45Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A.; Moriarty, N.; Oeffner, R.; Read, R.; Richardson, D.; Richardson, J.; Terwilliger, T.; Zwart, P. PHENIX: A Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221, DOI: 10.1107/S0907444909052925[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisbc%253D&md5=67b439ff4bd61c659cae37ca4209b7bcPHENIX: a comprehensive Python-based system for macromolecular structure solutionAdams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
- 46Ogata, H.; Nishikawa, K.; Lubitz, W. Hydrogens Detected by Subatomic Resolution Protein Crystallography in a [NiFe] Hydrogenase. Nature 2015, 520, 571– 574, DOI: 10.1038/nature14110[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MvpvVanuw%253D%253D&md5=0bfe17f42242357775645917812a4f5bHydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenaseOgata Hideaki; Nishikawa Koji; Lubitz WolfgangNature (2015), 520 (7548), 571-4 ISSN:.The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-angstrom resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 ÅA resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 ÅA and 1.78ÅA, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
- 47Buhrke, T.; Lenz, O.; Krauss, N.; Friedrich, B. Oxygen Tolerance of the H2-Sensing [NiFe] Hydrogenase from Ralstonia Eutropha H16 Is Based on Limited Access of Oxygen to the Active Site. J. Biol. Chem. 2005, 280, 23791– 23796, DOI: 10.1074/jbc.M503260200[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXltlKhsrc%253D&md5=557d20410fde7e57e3820aa1240014b3Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active SiteBuhrke, Thorsten; Lenz, Oliver; Krauss, Norbert; Friedrich, BaerbelJournal of Biological Chemistry (2005), 280 (25), 23791-23796CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton redn. Hydrogen metab. predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. The authors have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the mol. background of its oxygen tolerance. Evidence is presented that the shape and size of the intramol. hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivs. that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of std. [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnol. application.
- 48Duché, O.; Elsen, S.; Cournac, L.; Colbeau, A. Enlarging the Gas Access Channel to the Active Site Renders the Regulatory Hydrogenase HupUV of Rhodobacter Capsulatus O2 Sensitive without Affecting Its Transductory Activity. FEBS J. 2005, 272, 3899– 3908, DOI: 10.1111/j.1742-4658.2005.04806.x[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MzoslOjsA%253D%253D&md5=27d9bdabfeb56608dc294b43f50cc19dEnlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activityDuche Ophelie; Elsen Sylvie; Cournac Laurent; Colbeau AnnetteThe FEBS journal (2005), 272 (15), 3899-908 ISSN:1742-464X.In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.
- 49Abou Hamdan, A.; Liebgott, P.; Fourmond, V.; Gutiérrez-Sanz, Ó.; De Lacey, A. L.; Infossi, P.; Rousset, M.; Dementin, S.; Léger, C. Relation between Anaerobic Inactivation and Oxygen Tolerance in a Large Series of NiFe Hydrogenase Mutants. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19916– 19921, DOI: 10.1073/pnas.1212258109[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslKhtA%253D%253D&md5=5a7fb740150648d2b1c4b67bef976f14Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutantsAbou Hamdan, Abbas; Liebgott, Pierre-Pol; Fourmond, Vincent; Gutierrez-Sanz, Oscar; De Lacey, Antonio L.; Infossi, Pascale; Rousset, Marc; Dementin, Sebastien; Leger, ChristopheProceedings of the National Academy of Sciences of the United States of America (2012), 109 (49), 19916-19921, S19916/1-S19916/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Ni-contg. hydrogenases, the biol. catalysts of H2 oxidn. and prodn., reversibly inactivate under anaerobic, oxidizing conditions. Here, the authors aimed at understanding the mechanism of (in)activation and what dets. its kinetics, because there is a correlation between fast reductive reactivation and O2 tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnol. Direct electrochem. is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here, the authors describe simple anal. models that were used to characterize and compare 16 mutants, obtained by substituting the position-74 Val residue of O2-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans. The authors obsd. that this substitution could accelerate reactivation up to 1000-fold, depending on the polarity of the position 74 residue side-chain. In terms of kinetics of anaerobic (in)activation and O2 tolerance, the Val-to-His (V74H) mutation had the most spectacular effect: The V74H mutant compared favorably with the O2-tolerant hydrogenase from Aquifex aeolicus, which was used here as a benchmark.
- 50Durrant, M. C. Controlled Protonation of Iron–Molybdenum Cofactor by Nitrogenase: A Structural and Theoretical Analysis. Biochem. J. 2001, 355, 569– 576, DOI: 10.1042/bj3550569[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjslCntb0%253D&md5=4138f63f02aa70c2d582c2f80ce9dda4Controlled protonation of iron-molybdenum cofactor by nitrogenase: a structural and theoretical analysisDurrant, Marcus C.Biochemical Journal (2001), 355 (3), 569-576CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Qual. mol. modeling has been used to identify possible routes for transfer of protons from the surface of the nitrogenase protein to the iron-molybdenum cofactor (FeMoco) and to substrates during catalysis. Three proton-transfer routes have been identified; a water-filled channel running from the protein exterior to the homocitrate ligand of FeMoco, and two hydrogen-bonded chains to specific FeMoco sulfur atoms. It is suggested that the water channel is used for multiple proton deliveries to the substrate, as well as in diffusion of products and substrates between FeMoco and the bulk solvent, whereas the two hydrogen-bonded chains each allow a single proton to be added to, and subsequently depart from, FeMoco during the catalytic cycle. Possible functional differences in the proton-transfer channels are discussed in terms of assessment of the protein environment and specific hydrogen-bonding effects. The implications of these observations are discussed in terms of the suppression of wasteful prodn. of dihydrogen by nitrogenase and the Lowe-Thorneley scheme for dinitrogen redn.
- 51Barney, B. M.; Yurth, M. G.; Santos, P. C.; Dean, D. R.; Seefeldt, C.; Carolina, N. A Substrate Channel in the Nitrogenase MoFe Protein. JBIC, J. Biol. Inorg. Chem. 2009, 14, 1015– 1022, DOI: 10.1007/s00775-009-0544-2[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmt1SktLo%253D&md5=99d668f2f9e91f56817ba27891399497A substrate channel in the nitrogenase MoFe proteinBarney, Brett M.; Yurth, Michael G.; Dos Santos, Patricia C.; Dean, Dennis R.; Seefeldt, Lance C.JBIC, Journal of Biological Inorganic Chemistry (2009), 14 (7), 1015-1022CODEN: JJBCFA; ISSN:0949-8257. (Springer)Nitrogenase catalyzes the six electron/six proton redn. of N2 to two ammonia mols. at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the α-subunit of the MoFe protein, with no obvious access for substrates. Examn. of high-resoln. X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate redn. kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the redn. of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, Vmax values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the Km values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.
- 52Amara, P.; Andreoletti, P.; Jouve, H. M.; Field, M. J. Ligand Diffusion in the Catalase from Proteus Mirabilis: A Molecular Dynamics Study. Protein Sci. 2001, 10, 1927– 1935, DOI: 10.1110/ps.14201[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXntFeqsLo%253D&md5=c5b9a7949c64211261c4e5dcf985e936Ligand diffusion in the catalase from Proteus mirabilis: a molecular dynamics studyAmara, Patricia; Andreoletti, Pierre; Jouve, Helene Marie; Field, Martin J.Protein Science (2001), 10 (10), 1927-1935CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)The role of the channels and cavities present in the catalase from Proteus mirabilis (PMC) was investigated using mol. dynamics (MD) simulations. The reactant and products of the reaction, H2O2 → 1/2 O2 + H2O, catalyzed by the enzyme were allowed to diffuse to and from the active site. Dynamic fluctuations in the structure are found necessary for the opening of the major channel, identified in the X-ray model, which allows access to the active site. This channel is the only pathway to the active site obsd. during the dynamics, and both the products and reactant use it. H2O and O2 are also detected in a cavity defined by the heme and Ser196, which could play an important role during the reaction. Free energy profiles of the ligands diffusing through the major channel indicate that the barriers to ligand diffusion are less than 20 kJ mol-1 for each of the species. It is not clear from our study that minor channels play a role for access to the protein active site or to the protein surface.
- 53Fritsch, J.; Scheerer, P.; Frielingsdorf, S.; Kroschinsky, S.; Friedrich, B.; Lenz, O.; Spahn, C. M. T. The Crystal Structure of an Oxygen-Tolerant Hydrogenase Uncovers a Novel Iron-Sulphur Centre. Nature 2011, 479, 249– 252, DOI: 10.1038/nature10505[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlWksr7L&md5=0baaeed344d6182959296d5dec1627c6The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulfur centerFritsch, Johannes; Scheerer, Patrick; Frielingsdorf, Stefan; Kroschinsky, Sebastian; Friedrich, Baerbel; Lenz, Oliver; Spahn, Christian M. T.Nature (London, United Kingdom) (2011), 479 (7372), 249-252CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Hydrogenases are abundant enzymes that catalyze the reversible interconversion of H2 into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O2 are considered to be central to H2-based technologies, such as enzymic fuel cells and for light-driven H2 prodn. Despite comprehensive genetic, biochem., electrochem., and spectroscopic investigations, the mol. background allowing a structural interpretation of how the catalytic center is protected from irreversible inactivation by O2 has remained unclear. Here, the authors present the crystal structure of an O2-tolerant [NiFe]-hydrogenase from the aerobic H2 oxidizer, Ralstonia eutropha H16 at 1.5 Å resoln. The heterodimeric enzyme consisted of a large subunit harboring the catalytic center in the H2-reduced state and a small subunit contg. an electron relay consisting of 3 different Fe-S clusters. The cluster proximal to the active site displayed an unprecedented [4Fe-3S] structure and was coordinated by 6 Cys residues. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas mol. approaching the active site. It serves as an electron acceptor in the course of H2 oxidn. and as an electron-delivering device upon O2 attack at the active site. This dual function was supported by the capability of the novel Fe-S cluster to adopt 3 redox states at physiol. redox potentials. The 2nd structural feature was a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biol. and chem. H2-converting catalysts that are capable of cycling H2 in air.
- 54Dementin, S.; Burlat, B.; De Lacey, A. L.; Pardo, A.; Adryanczyk-Perrier, G.; Guigliarelli, B.; Fernández, V. M.; Rousset, M. A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] Hydrogenase. J. Biol. Chem. 2004, 279, 10508– 10513, DOI: 10.1074/jbc.M312716200[Crossref], [PubMed], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvFChs7c%253D&md5=38c266f2161f170bf8acbf3ccad891b3A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] HydrogenaseDementin, Sebastien; Burlat, Benedicte; De Lacey, Antonio L.; Pardo, Alejandro; Adryanczyk-Perrier, Geraldine; Guigliarelli, Bruno; Fernandez, Victor M.; Rousset, MarcJournal of Biological Chemistry (2004), 279 (11), 10508-10513CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Kinetic, EPR, and Fourier transform IR spectroscopic anal. of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H2/ortho-H2 conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover nos. for the oxidn. of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H2 oxidn. and hydrogen isotope exchange activities, but did not change the para-H2/ortho-H2 conversion activity. The spectroscopic properties of this mutant were totally different, esp. in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.
- 55Bertrand, P.; Dole, F.; Asso, M.; Guigliarelli, B. Is There a Rate-Limiting Step in the Catalytic Cycle of [Ni-Fe] Hydrogenases?. JBIC, J. Biol. Inorg. Chem. 2000, 5, 682– 691, DOI: 10.1007/s007750000152[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXovV2hsL4%253D&md5=dd2371710d29fd6647321fbb8a9237d6Is there a rate-limiting step in the catalytic cycle of [Ni-Fe] hydrogenases?Bertrand, Patrick; Dole, Francois; Asso, Marcel; Guigliarelli, BrunoJBIC, Journal of Biological Inorganic Chemistry (2000), 5 (6), 682-691CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopersicina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, resp. This proposal is consistent with the data available from other Ni-Fe enzymes.
- 56Marques, M. F. C. Ph.D. Thesis, Structural and Functional Studies of a High Activity NiFeSe Hydrogenase; Universidade Nova de Lisboa: Oeiras, 2014.Google ScholarThere is no corresponding record for this reference.
- 57Keller, K. L.; Wall, J. D.; Chhabra, S.; Voigt, C. Methods Enzymol. 2011, 497, 503, DOI: 10.1016/B978-0-12-385075-1.00022-6[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpsVGksr4%253D&md5=352517f8a4ab96546bb12c6fcccc3794Methods for engineering sulfate reducing bacteria of the genus DesulfovibrioKeller, Kimberly L.; Wall, Judy D.; Chhabra, SwapnilMethods in Enzymology (2011), 497 (Synthetic Biology, Part A), 503-517CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. Sulfate reducing bacteria (SRB) are physiol. important given their nearly ubiquitous presence and have important applications in the areas of bioremediation and bioenergy. This chapter provides details on the steps used for homologous-recombination mediated chromosomal manipulation of Desulfovibrio vulgaris Hildenborough, a well-studied sulfate reducer. More specifically, we focus on the implementation of a "parts" based approach for suicide vector assembly, important aspects of anaerobic culturing, choices for antibiotic selection, electroporation-based DNA transformation, as well as tools for screening and verifying genetically modified constructs. These methods, which in principle may be extended to other SRB, are applicable for functional genomics investigations, as well as metabolic engineering manipulations.
- 58Bertrand, P.; Frangioni, B.; Dementin, S.; Sabaty, M.; Arnoux, P.; Guigliarelli, B.; Pignol, D.; Léger, C. Effects of Slow Substrate Binding and Release in Redox Enzymes: Theory and Application to Periplasmic Nitrate Reductase. J. Phys. Chem. B 2007, 111, 10300– 10311, DOI: 10.1021/jp074340j[ACS Full Text], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos1Sks7c%253D&md5=4741b536281eb2ebaede3caa699fd39fEffects of Slow Substrate Binding and Release in Redox Enzymes: Theory and Application to Periplasmic Nitrate ReductaseBertrand, Patrick; Frangioni, Bettina; Dementin, Sebastien; Sabaty, Monique; Arnoux, Pascal; Guigliarelli, Bruno; Pignol, David; Leger, ChristopheJournal of Physical Chemistry B (2007), 111 (34), 10300-10311CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)For redox enzymes, the technique of protein film voltammetry (PFV) makes it possible to det. the entire profile of activity against driving force by allowing the enzyme to directly exchange electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chem. steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully anal. treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quant. conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concn. dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the redn. potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional expts.; the position of the wave (i.e., the driving force that is required to elicit catalysis) departs from the redn. potential of the active site even at the lowest substrate concn., and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramol. electron transfer from the proximal [4Fe4S]+ cluster to the active site MoV.
- 59Fourmond, V. QSoas: A Versatile Software for Data Analysis. Anal. Chem. 2016, 88, 5050– 5052, DOI: 10.1021/acs.analchem.6b00224[ACS Full Text], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsVKksrk%253D&md5=d22e37a8f55f5b5e579f5711f0f713ebQSoas: A Versatile Software for Data AnalysisFourmond, VincentAnalytical Chemistry (Washington, DC, United States) (2016), 88 (10), 5050-5052CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Undoubtedly, the most natural way to confirm a model is to quant. verify its predictions. However, this is not done systematically, and one of the reasons for that is the lack of appropriate tools for analyzing data, because the existing tools do not implement the required models or they lack the flexibility required to perform data anal. in a reasonable time. We present QSoas, an open-source, cross-platform data anal. program written to overcome these problems. In addn. to std. data anal. procedures and full automation using scripts, QSoas features a very powerful data fitting interface with support for arbitrary functions, differential equation and kinetic system integration, and flexible global fits. QSoas is available from http://www.qoas.org.
- 60Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132, DOI: 10.1107/S0907444909047337[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1SisLc%253D&md5=1aa9a38aeb3ce95af4ffb7d8b8a142bdSoftware XDS for image rotation, recognition and crystal symmetry assignmentKabsch, WolfgangActa Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
- 61Vonrhein, C.; Flensburg, C.; Keller, P.; Sharff, A.; Smart, O.; Paciorek, W.; Womack, T.; Bricogne, G. Data Processing and Analysis with the AutoPROC Toolbox. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 293– 302, DOI: 10.1107/S0907444911007773[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtLs%253D&md5=a0a3579f6b371b6312819883864aa80dData processing and analysis with the autoPROC toolboxVonrhein, Clemens; Flensburg, Claus; Keller, Peter; Sharff, Andrew; Smart, Oliver; Paciorek, Wlodek; Womack, Thomas; Bricogne, GerardActa Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 293-302CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A typical diffraction expt. will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and nos. that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concg. on difficulties that may often show up during the first steps along the path of turning the expt. (i.e. data collection) into a model (i.e. interpreted electron d.). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analyzing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the expt. is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
- 62Evans, P. Scaling and Assessment of Data Quality. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72– 82, DOI: 10.1107/S0907444905036693[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7jM&md5=293d3876e534c0c57813990515bb3c76Scaling and assessment of data qualityEvans, PhilipActa Crystallographica, Section D: Biological Crystallography (2006), D62 (1), 72-82CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)The various phys. factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analyzed to set the real resoln. of the data set, to detect bad regions (e.g. bad images), to analyze radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation anal. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be detd. from scores such as correlation coeffs. between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.
- 63Tickle, I. J.; Flensburg, C.; Keller, P.; Paciorek, W.; Sharff, A.; Vonrhein, C.; Bricogne, G. STARANISO; Global Phasing: Cambridge, United Kingdom, 2018.Google ScholarThere is no corresponding record for this reference.
- 64Evans, P. R.; Murshudov, G. N. How Good Are My Data and What Is the Resolution?. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1204– 1214, DOI: 10.1107/S0907444913000061[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVejsL8%253D&md5=3f649426c21e9d423f94ef8d7c568233How good are my data and what is the resolution?Evans, Philip R.; Murshudov, Garib N.Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1204-1214CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Following integration of the obsd. diffraction spots, the process of data redn.' initially aims to det. the point-group symmetry of the data and the likely space group. This can be performed with the program POINTLESS. The scaling program then puts all the measurements on a common scale, avs. measurements of symmetry-related reflections (using the symmetry detd. previously) and produces many statistics that provide the first important measures of data quality. A new scaling program, AIMLESS, implements scaling models similar to those in SCALA but adds some addnl. analyses. From the analyses, a no. of decisions can be made about the quality of the data and whether some measurements should be discarded. The effective resoln.' of a data set is a difficult and possibly contentious question (particularly with referees of papers) and this is discussed in the light of tests comparing the data-processing statistics with trials of refinement against obsd. and simulated data, and automated model-building and comparison of maps calcd. with different resoln. limits. These trials show that adding weak high-resoln. data beyond the commonly used limits may make some improvement and does no harm.
- 65McCoy, A. J. Solving Structures of Protein Complexes by Molecular Replacement with Phaser. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 32– 41, DOI: 10.1107/S0907444906045975[Crossref], [PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlSqu77F&md5=323a5297bdf622ebaf398a87c39dc794Solving structures of protein complexes by molecular replacement with PhaserMcCoy, Airlie J.Acta Crystallographica, Section D: Biological Crystallography (2007), 63 (1), 32-41CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Mol. replacement (MR) generally becomes more difficult as the no. of components in the asym. unit requiring sep. MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asym. units to be built up from individual components with a 'tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a no. of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large no. of copies of the same component in the asym. unit or where the components of the asym. unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the std. 'automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.
- 66Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A Graphical User Interface to the CCP4 Program Suite Research Papers A Graphical User Interface to the CCP 4 Program Suite. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1131– 1137, DOI: 10.1107/S0907444903008126[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvFSksLs%253D&md5=25a0168dd7ea9f930036453a829e1e3dA graphical user interface to the CCP4 program suitePotterton, Elizabeth; Briggs, Peter; Turkenburg, Maria; Dodson, EleanorActa Crystallographica, Section D: Biological Crystallography (2003), D59 (7), 1131-1137CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)CCP4i is a graphical user interface that makes running programs from the CCP4 suite simpler and quicker. It is particularly directed at inexperienced users and tightly linked to introductory and scientific documentation. It also provides a simple project-management system and visualization tools. The system is readily extensible and not specific to CCP4 software.
- 67Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the Refinement of Macromolecular Crystal Structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355– 367, DOI: 10.1107/S0907444911001314[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtbk%253D&md5=f8f3202d246908500057ad7c71015b7bREFMAC5 for the refinement of macromolecular crystal structuresMurshudov, Garib N.; Skubak, Pavol; Lebedev, Andrey A.; Pannu, Navraj S.; Steiner, Roberto A.; Nicholls, Robert A.; Winn, Martyn D.; Long, Fei; Vagin, Alexei A.Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 355-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)This paper describes various components of the macromol. crystallog. refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS exptl. diffraction data. To ensure chem. and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolns. at least as low as 4 Å can be achieved thanks to low-resoln. refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, 'jelly-body' restraints and the use of novel long-range restraints on at. displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 addnl. offers TLS parameterization and, when high-resoln. data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resoln. spectrum encountered in macromol. crystallog.
- 68Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501, DOI: 10.1107/S0907444910007493[Crossref], [PubMed], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
- 69Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C.; Richardson, D. C. MolProbity: All-Atom Structure Validation for Macromolecular Crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 12– 21, DOI: 10.1107/S0907444909042073[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktg%253D%253D&md5=b5fc7574f43f01dd6e43c3663ca4f779MolProbity: all-atom structure validation for macromolecular crystallographyChen, Vincent B.; Arendall, W. Bryan, III; Headd, Jeffrey J.; Keedy, Daniel A.; Immormino, Robert M.; Kapral, Gary J.; Murray, Laura W.; Richardson, Jane S.; Richardson, David C.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 12-21CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact anal., complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallog. provides a wealth of biol. important mol. data in the form of at. three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystn. to data collection to phasing to model building to refinement, have made solving a structure using crystallog. easier than ever. However, despite these improvements, local errors that can affect biol. interpretation are widespread at low resoln. and even high-resoln. structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is crit. both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
- 70Berman, H.; Henrick, K.; Nakamura, H. Announcing the Worldwide Protein Data Bank. Nat. Struct. Mol. Biol. 2003, 10, 980– 980, DOI: 10.1038/nsb1203-980[Crossref], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXptFOmsbY%253D&md5=8146ff5fc67f64e31b2f8c475473879cAnnouncing the worldwide Protein Data BankBerman, Helen; Henrick, Kim; Nakamura, HarukiNature Structural Biology (2003), 10 (12), 980CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Group)There is no expanded citation for this reference.
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Abstract
Figure 1
Figure 1. Solvent channel in the 0.95 Å resolution structure of the anaerobically crystallized wild-type [NiFeSe] hydrogenase from D. vulgaris Hildenborough (PDB 5JSH). The protein Cα backbone is shown in light cyan for the small subunit and gray for the large subunit, the water molecules in the solvent channel are shown as red spheres, the active site and the iron–sulfur clusters are depicted in ball-and-stick representation with atom colors gray for carbon, blue for nitrogen, red for oxygen, gold for sulfur, and brown for iron, and the G50 and G491 residues are highlighted in orange.
Figure 2
Figure 2. Active site surroundings in the crystal structure of the aerobically purified and crystallized G491A [NiFeSe] hydrogenase variant and its corresponding 2|Fo| – |Fc| (gray mesh, 1.5 map rms) and |Fo| – |Fc| (green mesh, 3.5 map rms) maps. No negative peaks are visible at −3.5 rms in the |Fo| – |Fc| map. Atoms are color-coded as follows: brown, Fe; green, Ni; gold, S; red, O; light blue, C; blue, N; orange, Se. H atoms are omitted for clarity.
Figure 3
Figure 3. Effect of O2 on the H2 oxidation current of WT [NiFeSe] hydrogenase and variants adsorbed onto a graphite rotating electrode. The gray dashed lines are the best fits of the kinetic model in eq 1. Experimental conditions: [O2] = 0.5 μM, E = 0.14 V vs SHE, 1 bar H2, pH 7, T = 40 °C, electrode rotation rate 3000 rpm.
Figure 4
Figure 4. H2 uptake activity of WT and variants after 1 h (green), 4 h (blue), and 16 h (gray) exposure to air, followed by a 30 min reactivation under 0.5 atm of H2. The activities were normalized by the corresponding maximum activity of each protein (dark blue), reported in Table 1. Each experiment was performed three times (technical replicates), and the error bars show the corresponding standard deviations.
Figure 5
Figure 5. Channels in the high-resolution structures of [NiFe] hydrogenase from D. vulgaris Myiazaki F (A, 0.86 Å, PDB 489H), [NiFeSe] hydrogenases from D. vulgaris Hildenborough (B, 0.95 Å, PDB 5JSK, crystallized anaerobically), and Dm. baculatum (C, 1.4 Å, PDB 4KN9), calculated with CAVER. The hydrophobic channel system allowing H2 exchange with the active site is shown in light magenta, and the channels connecting Sec and Cys75 Sγ atoms with the enzyme exterior are displayed in blue. The protein Cα backbones are shown as gray tubes; the active site and the iron–sulfur clusters are shown in ball-and-stick representation with atom colors gray for carbon, blue for nitrogen, red for oxygen, gold for sulfur, and brown for iron; the G50 and G491 residues in D. vulgaris Hildenborough and their structurally equivalent Thr56 and Ala548 residues in D. vulgaris Myiazaki F and G45 and G494 in Dm. baculatum are displayed in ball-and-stick representation with carbon atoms colored yellow. For clarity, only the side chains of the protein residues are shown.
Figure 6
Figure 6. Close-up view of the channels in the structure of the D. vulgaris Hildenborough [NiFeSe] hydrogenase and variants, calculated with CAVER: (A) wild-type (PDB 5JSK); (B) G50T variant; (C) G491A variant; (D) G491S variant. The channels connecting the enzyme exterior with the Sec and the Cys75 Sγ atoms are displayed as meshes. The hydrophilic branch of the channel is shown in blue and the hydrophobic branch in green. The water molecules enclosed by the channels are represented as red spheres.
References
ARTICLE SECTIONSThis article references 70 other publications.
- 1Lubitz, W.; Ogata, H.; Rudiger, O.; Reijerse, E. Hydrogenases. Chem. Rev. 2014, 114, 4081– 4148, DOI: 10.1021/cr4005814[ACS Full Text], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXks1Sisrs%253D&md5=36a052b8100bfabd655a0798c17d14d0HydrogenasesLubitz, Wolfgang; Ogata, Hideaki; Ruediger, Olaf; Reijerse, EdwardChemical Reviews (Washington, DC, United States) (2014), 114 (8), 4081-4148CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The current state of knowledge on hydrogenases, esp. recent advances made in understanding the detailed structure and function of these important enzymes. The authors provide an overview of important previous achievements with the main focus on [NiFe] and [FeFe] hydrogenases, and in part also on [Fe] hydrogenases. Recent progress on biomimetic model systems for hydrogenases and devices using hydrogenases both in fuel cells and for H2 prodn. are presented with emphasis on functional aspects. The great progress made in synthesizing model systems for hydrogenases that are functionally active is promising for the future employment of such catalysts in hydrogen technologies.
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- 3Volbeda, A.; Montet, Y.; Vernâ, X.; Hatchikian, E. C.; Fontecilla-camps, J. C. High-Resolution Crystallographic Analysis of Desulfovibrio Fructosovorans [NiFe] Hydrogenase. Int. J. Hydrogen Energy 2002, 27, 1449– 1461, DOI: 10.1016/S0360-3199(02)00072-1[Crossref], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xnt1Kit7Y%253D&md5=5c98724806ecd5509d4ed72a9d360644High-resolution crystallographic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenaseVolbeda, Anne; Montet, Yael; Vernede, Xavier; Hatchikian, E. Claude; Fontecilla-Camps, Juan C.International Journal of Hydrogen Energy (2002), 27 (11-12), 1449-1461CODEN: IJHEDX; ISSN:0360-3199. (Elsevier Science Ltd.)Two 1.8 Å resoln. crystal structures of an oxidized form of the [NiFe] hydrogenase of Desulfovibrio (D.) fructosovorans are reported. The high data quality allows for a detailed anal. of the active site geometry, confirming asym. bridging of the Ni and Fe ion by the two cysteine sulfur atoms and one oxygen atom as previously obsd. in the D. gigas enzyme. The CO ligand is now clearly distinguishable from the two CN- ligands, as it refines to a significantly shorter distance to the Fe. The refined structures confirm the presence of long, mainly hydrophobic cavities that most probably provide pathways for H2 diffusion between the mol. surface and the deeply buried active site. Amino acid sequence comparisons suggest that these cavities are significantly narrower in the so-called sensor hydrogenases, which may explain why this special class of enzymes is insensitive to O2.
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- 5Leroux, F.; Dementin, S.; Burlat, B.; Cournac, L.; Volbeda, A.; Champ, S.; Martin, L.; Guigliarelli, B.; Bertrand, P.; Fontecilla-Camps, J. C.; Rousset, M.; Léger, C. Experimental Approaches to Kinetics of Gas Diffusion in Hydrogenase. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11188– 11193, DOI: 10.1073/pnas.0803689105[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVShtbvJ&md5=db422c03a9fc8ea7cc3b63ee06e1a6a4Experimental approaches to kinetics of gas diffusion in hydrogenaseLeroux, Fanny; Dementin, Sebastien; Burlat, Benedicte; Cournac, Laurent; Volbeda, Anne; Champ, Stephanie; Martin, Lydie; Guigliarelli, Bruno; Bertrand, Patrick; Fontecilla-Camps, Juan; Rousset, Marc; Leger, ChristopheProceedings of the National Academy of Sciences of the United States of America (2008), 105 (32), 11188-11193CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Hydrogenases, which catalyze H2 to H+ conversion as part of the bioenergetic metab. of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallog. and mol. dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quant. methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the X-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in detg. these.
- 6Fontecilla-Camps, J. C.; Amara, P.; Cavazza, C.; Nicolet, Y.; Volbeda, A. Structure-Function Relationships of Anaerobic Gas-Processing Metalloenzymes. Nature 2009, 460, 814– 822, DOI: 10.1038/nature08299[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXpvFCrt70%253D&md5=c57814b40ec4560a317f88764b80b7bdStructure-function relationships of anaerobic gas-processing metalloenzymesFontecilla-Camps, Juan C.; Amara, Patricia; Cavazza, Christine; Nicolet, Yvain; Volbeda, AnneNature (London, United Kingdom) (2009), 460 (7257), 814-822CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)A review. R. Reactions involving H2, N2, CO, CO2 and CH4 are likely to have been central to the origin of life. This is indicated by the active site structures of the enzymes involved, which are often reminiscent of minerals. Through the combined efforts of protein crystallog., various types of spectroscopy, theor. calcns., and model chem., it has been possible to put forward plausible mechanisms for gas-based metab. by extant microorganisms. Although the reactions are based on metal centers, the protein matrix regulates reactivity and substrate and product trafficking through internal pathways, specific ligation, and dielectricity.
- 7Liebgott, P.; Leroux, F.; Burlat, B.; Dementin, S.; Baffert, C.; Lautier, T.; Fourmond, V.; Ceccaldi, P.; Cavazza, C.; Meynial-Salles, I.; Soucaille, P.; Fontecilla-Camps, J. C.; Guigliarelli, B.; Bertrand, P.; Rousset, M.; Léger, C. Relating Diffusion along the Substrate Tunnel and Oxygen Sensitivity in Hydrogenase. Nat. Chem. Biol. 2010, 6, 63– 70, DOI: 10.1038/nchembio.276[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFWksb7M&md5=39c97ff63b8b0d0c1b3136c3bb22859bRelating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenaseLiebgott, Pierre-Pol; Leroux, Fanny; Burlat, Benedicte; Dementin, Sebastien; Baffert, Carole; Lautier, Thomas; Fourmond, Vincent; Ceccaldi, Pierre; Cavazza, Christine; Meynial-Salles, Isabelle; Soucaille, Philippe; Fontecilla-Camps, Juan Carlos; Guigliarelli, Bruno; Bertrand, Patrick; Rousset, Marc; Leger, ChristopheNature Chemical Biology (2010), 6 (1), 63-70CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a mol. channel whose structure may det. the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallog. and mol. dynamics, but kinetic data are scarce. Using protein film voltammetry, we detd. and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quant. define the relations between diffusion, the Michaelis const. for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.
- 8Kalms, J.; Schmidt, A.; Frielingsdorf, S.; Van Der Linden, P.; Von Stetten, D.; Lenz, O.; Carpentier, P.; Scheerer, P. Krypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport. Angew. Chem., Int. Ed. 2016, 55, 5586– 5590, DOI: 10.1002/anie.201508976[Crossref], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtlKgsLo%253D&md5=b2e5d335671bc7226fc1233a2b89903bKrypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas TransportKalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, PatrickAngewandte Chemie, International Edition (2016), 55 (18), 5586-5590CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2-tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calcns. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2-tolerant and O2-sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.
- 9Kalms, J.; Schmidt, A.; Frielingsdorf, S.; Utesch, T.; Gotthard, G.; von Stetten, D.; van der Linden, P.; Royant, A.; Mroginski, M. A.; Carpentier, P.; Lenz, O.; Scheerer, P. Tracking the Route of Molecular Oxygen in O2-Tolerant Membrane-Bound [NiFe] Hydrogenase. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), E2229– E2237, DOI: 10.1073/pnas.1712267115[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1WjsbbJ&md5=871c5a1671f144663859bfcc1a0aa404Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenaseKalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; Utesch, Tillmann; Gotthard, Guillaume; von Stetten, David; van der Linden, Peter; Royant, Antoine; Mroginski, Maria Andrea; Carpentier, Philippe; Lenz, Oliver; Scheerer, PatrickProceedings of the National Academy of Sciences of the United States of America (2018), 115 (10), E2229-E2237CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas mols. through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic redn. of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed exptl. using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallog. and computational studies. This combined approach revealed several sites of O2 mols. within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concns. used for MBH crystal derivatization. The examn. of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme.
- 10Kubas, A.; Orain, C.; De Sancho, D.; Saujet, L.; Sensi, M.; Gauquelin, C.; Meynial-Salles, I.; Soucaille, P.; Bottin, H.; Baffert, C.; Fourmond, V.; Best, R.; Blumberger, J.; Léger, C. Mechanism of O2 Diffusion and Reduction in FeFe Hydrogenases. Nat. Chem. 2017, 9, 88– 95, DOI: 10.1038/nchem.2592[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVSrsLzJ&md5=d4de66253d7c17f395ad5edae4910449Mechanism of O2 diffusion and reduction in FeFe hydrogenasesKubas, Adam; Orain, Christophe; De Sancho, David; Saujet, Laure; Sensi, Matteo; Gauquelin, Charles; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Herve; Baffert, Carole; Fourmond, Vincent; Best, Robert B.; Blumberger, Jochen; Leger, ChristopheNature Chemistry (2017), 9 (1), 88-95CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)[FeFe]-hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnol. devices. Here, the authors combined electrochem., site-directed mutagenesis, mol. dynamics simulations, and quantum chem. calcns. to uncover the mol. mechanism of O2 diffusion within the enzyme and its reactions at the active site. The authors proposed that the partial reversibility of the reaction with O2 resulted from the 4-electron redn. of O2 to water. The 3rd electron/proton transfer step was the bottleneck for water prodn., competing with formation of a highly reactive OH radical and hydroxylated Cys residue. The rapid delivery of electrons and protons to the active site was therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
- 11Fernández, V. M.; Hatchikian, E. C.; Cammack, R. Properties and Reactivation of Two Different Deactivated Forms of Desulfovibrio Gigas Hydrogenase. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 832, 69– 79, DOI: 10.1016/0167-4838(85)90175-X[Crossref], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXmtV2ktrc%253D&md5=d3650cd7715ef57a004a2d6755f80b6aProperties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenaseFernandez, Victor M.; Hatchikian, E. Claude; Cammack, RichardBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1985), 832 (1), 69-79CODEN: BBAEDZ; ISSN:0167-4838.It was previously shown that D. gigas hydrogenase, as isolated, has a relatively low activity in the H2-Me viologen reductase assay, and that the activity is slowly stimulated ≤10-fold by H2 or strong reductants. The enzyme, before reductive activation, is also totally inactive in 1H-3H exchange and H2-dichloroindophenol (DCIP) reductase assays. The behavior of the enzyme in various states of activation is discussed in terms of 3 different states: the active state, which is active in all assays; the unready state, which is totally inactive; and the ready state, which does not react with H2, but which is rapidly activated by strong reductants. The conditions for the slow activation of the unready state of D. gigas hydrogenase were investigated. The rate of activation was independent of enzyme concn. over a wide range, which rules out mechanisms involving intermol. electron exchange. The rate was only slightly affected by pH in the range 6-9, but was strongly temp. dependent, with an activation energy of 88 kJ/mol. The enzyme could be activated by dithiothreitol + the mediator dye indigo tetrasulfonate, but not by dithiothreitol alone. No effects were seen during treatments with weaker reductants, thioredoxin, Fe2+, sulfide, or Ni2+. The activation does not involve conversions of a metal center or the cleavage of an accessible SS bridge. Presumably, it involves an intramol. change, possibly in the redox state or coordination of a metal center. The active form of D. gigas hydrogenase was rapidly activated by O, producing mostly the unready state, which could be reactivated only slowly. By contrast, anaerobic reoxidn. by DCIP was able to convert the enzyme mostly to the ready state. This was identified as being inactive in 1H-3H exchange and H2-DCIP reductase assays but rapidly activated in the H2-Me viologen reductase assay (DCIP prevents this). A similar oxidn. of the active enzyme may take place in the cell as a protection against O.
- 12Lamle, S. E.; Albracht, S. P. J.; Armstrong, F. A. Electrochemical Potential-Step Investigations of the Aerobic Interconversions of [NiFe]-Hydrogenase from Allochromatium Vinosum: Insights into the Puzzling Difference between Unready and Ready Oxidized Inactive States. J. Am. Chem. Soc. 2004, 126, 14899– 14909, DOI: 10.1021/ja047939v[ACS Full Text], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXovVKgtL8%253D&md5=57d6b9248e8610a2aca59dfb0c48e341Electrochemical Potential-Step Investigations of the Aerobic Interconversions of [NiFe]-Hydrogenase from Allochromatium vinosum: Insights into the Puzzling Difference between Unready and Ready Oxidized Inactive StatesLamle, Sophie E.; Albracht, Simon P. J.; Armstrong, Fraser A.Journal of the American Chemical Society (2004), 126 (45), 14899-14909CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Dynamic electrochem. studies, incorporating catalytic voltammetry and detailed potential-step manipulations, provide compelling evidence that the oxidized inactive state of [NiFe]-hydrogenases termed Unready (or Ni-A) contains a product of partial redn. of O2 that is trapped in the active site.
- 13Abou Hamdan, A.; Burlat, B.; Gutiérrez-Sanz, Ó.; Liebgott, P.; Baffert, C.; De Lacey, A. L.; Rousset, M.; Guigliarelli, B.; Léger, C.; Dementin, S. O2-Independent Formation of the Inactive States of NiFe Hydrogenase. Nat. Chem. Biol. 2013, 9, 15– 17, DOI: 10.1038/nchembio.1110[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s7isFWrsw%253D%253D&md5=86fca973f7f6abad383486947d4d3279O2-independent formation of the inactive states of NiFe hydrogenaseAbou Hamdan Abbas; Burlat Benedicte; Gutierrez-Sanz Oscar; Liebgott Pierre-Pol; Baffert Carole; De Lacey Antonio L; Rousset Marc; Guigliarelli Bruno; Leger Christophe; Dementin SebastienNature chemical biology (2013), 9 (1), 15-7 ISSN:.We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.
- 14Baltazar, C.; Marques, M.; Soares, C. M.; De Lacey, A.; Pereira, I. A. C.; Matias, P. M. Nickel-Iron-Selenium Hydrogenases - An Overview. Eur. J. Inorg. Chem. 2011, 2011, 948– 962, DOI: 10.1002/ejic.201001127
- 15Wombwell, C.; Caputo, C. A.; Reisner, E. [NiFeSe]-Hydrogenase Chemistry. Acc. Chem. Res. 2015, 48, 2858– 2865, DOI: 10.1021/acs.accounts.5b00326[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1yltrzE&md5=bef139745dc865d1978624c5eecb06ca[NiFeSe]-Hydrogenase ChemistryWombwell, Claire; Caputo, Christine A.; Reisner, ErwinAccounts of Chemical Research (2015), 48 (11), 2858-2865CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The development of technol. for the inexpensive generation of the renewable energy vector H2 through water splitting is of immediate economic, ecol., and humanitarian interest. Recent interest in hydrogenases has been fueled by their exceptionally high catalytic rates for H2 prodn. at a marginal overpotential, which is presently only matched by the nonscalable noble metal platinum. The mechanistic understanding of hydrogenase function guides the design of synthetic catalysts, and selection of a suitable hydrogenase enables direct applications in electro- and photocatalysis. [FeFe]-hydrogenases display excellent H2 evolution activity, but they are irreversibly damaged upon exposure to O2, which currently prevents their use in full water splitting systems. O2-tolerant [NiFe]-hydrogenases are known, but they are typically strongly biased toward H2 oxidn., while H2 prodn. by [NiFe]-hydrogenases is often product (H2) inhibited. [NiFeSe]-hydrogenases are a subclass of [NiFe]-hydrogenases with a selenocysteine residue coordinated to the active site nickel center in place of a cysteine. They exhibit a combination of unique properties that are highly advantageous for applications in water splitting compared with other hydrogenases. They display a high H2 evolution rate with marginal inhibition by H2 and tolerance to O2. [NiFeSe]-hydrogenases are therefore one of the most active mol. H2 evolution catalysts applicable in water splitting. Herein, we summarize our recent progress in exploring the unique chem. of [NiFeSe]-hydrogenases through biomimetic model chem. and the chem. with [NiFeSe]-hydrogenases in semi-artificial photosynthetic systems. We gain perspective from the structural, spectroscopic, and electrochem. properties of the [NiFeSe]-hydrogenases and compare them with the chem. of synthetic models of this hydrogenase active site. Our synthetic models give insight into the effects on the electronic properties and reactivity of the active site upon the introduction of selenium. We have utilized the exceptional properties of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum in a no. of photocatalytic H2 prodn. schemes, which are benchmark systems in terms of single site activity, tolerance toward O2, and in vitro water splitting with biol. mols. Each system comprises a light-harvesting component, which allows for light-driven electron transfer to the hydrogenase in order for it to catalyze H2 prodn. A system with [NiFeSe]-hydrogenase on a dye-sensitized TiO2 nanoparticle gives an enzyme-semiconductor hybrid for visible light-driven generation of H2 with an enzyme-based turnover frequency of 50 s-1. A stable and inexpensive polymeric carbon nitride as a photosensitizer in combination with the [NiFeSe]-hydrogenase shows good activity for more than 2 days. Light-driven H2 evolution with the enzyme and an org. dye under high O2 levels demonstrates the excellent robustness and feasibility of water splitting with a hydrogenase-based scheme. This has led, most recently, to the development of a light-driven full water splitting system with a [NiFeSe]-hydrogenase wired to the water oxidn. enzyme photosystem II in a photoelectrochem. cell. In contrast to the other systems, this photoelectrochem. system does not rely on a sacrificial electron donor and allowed us to establish the long sought after light-driven water splitting with an isolated hydrogenase.
- 16Teixeira, M.; Fauque, G.; Moura, I.; Lespinat, P. A.; Berlier, Y.; Prickril, B.; Peck, H.; Xavier, A. V.; Gall, J. Le; Moura, J. J. G. Nickel-[Iron-Sulfur]-Selenium-Containing Hydrogenases from Desulfovibrio Baculatus (DSM 1743). Eur. J. Biochem. 1987, 167, 47– 58, DOI: 10.1111/j.1432-1033.1987.tb13302.x[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXlt12lu7w%253D&md5=88ebdaadc996173e66e129417a7d4c5aNickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic propertiesTeixeira, Miguel; Fauque, Guy; Moura, Isabel; Lespinat, Paul A.; Berlier, Yves; Prickril, Ben; Peck, Harry D., Jr.; Xavier, Antonio V.; Le Gall, Jean; Moura, Jose J. G.European Journal of Biochemistry (1987), 167 (1), 47-58CODEN: EJBCAI; ISSN:0014-2956.Hydrogenase from D. baculatus (DSM 1743) was purified from each of 3 different fractions: sol. periplasmic (wash), sol. cytoplasmic (cell disruption), and membrane-bound (detergent solubilization). Plasma-emission metal anal. detected in all 3 fractions the presence of Fe plus Ni and Se in equimol. amts. These hydrogenases were composed of 2 nonidentical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g = ∼2.0 when obsd. at low temp. (<20 K). The periplasmic and membrane-bound enzymes also presented addnl. EPR signals, observable up to 77 K, with g > 2.0, and assigned to Ni(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06, and 2.0. The signals obsd. in the membrane-bound prepns. could be decompd. into 2 sets with g = 2.34, 2.16, and ∼2.0 (component I) and g = 2.33, 2.24, and ∼2.0 (component II). In the reduced state, after exposure to a H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temps. and microwave powers, and in samples partially and fully reduced (under H2 or dithionite), allowed the identification of 2 different Fe-S centers: center I (g = 2.03, 1.89 and 1.86) detectable at <10 K, and center II (g = 2.06, 1.95, and 1.88) which was easily satd. at low temps. Addnl. EPR signals due to transient Ni species were detected with g = >2.0, and a rhombic EPR signal at 77 K developed at g = 2.20, 2.16, and 2.0. This EPR signal is reminiscent of the Ni signal C (g = 2.19, 2.14 and 2.02) obsd. in intermediate redox states of the well-characterized Desulfovibrio gigas hydrogenase. During the course of a redox titrn. at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity at approx. -320 mV. Low-temp. studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g = 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The sol. hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such step to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymic activity. The catalytic properties were also followed by the 1H-2H exchange reaction. The isolated hydrogenases produced H2/HD ratios higher than those obsd. for non-Se-contg. hydrogenases.
- 17Valente, F.; Oliveira, S.; Gnadt, N.; Pacheco, I.; Coelho, A. V.; Xavier, A. V.; Teixeira, M.; Soares, C. M.; Pereira, I. A. C. Hydrogenases in Desulfovibrio Vulgaris Hildenborough: Structural and Physiologic Characterisation of the Membrane-Bound [NiFeSe] Hydrogenase. JBIC, J. Biol. Inorg. Chem. 2005, 10, 667– 682, DOI: 10.1007/s00775-005-0022-4[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1CqtLvO&md5=6ea253f7b728ec043dcec87ee8d390aaHydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenaseValente, Filipa M. A.; Oliveira, A. Sofia F.; Gnadt, Nicole; Pacheco, Isabel; Coelho, Ana V.; Xavier, Antonio V.; Teixeira, Miguel; Soares, Claudio M.; Pereira, Ines A. C.JBIC, Journal of Biological Inorganic Chemistry (2005), 10 (6), 667-682CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulfate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase assocd. with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H2 prodn. activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidn. state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane assocn. Cleavage of this group originates a sol., less active form of the enzyme. Sequence anal. shows that [NiFeSe] Hases from Desulfovibrionacae form a sep. family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S]2+/1+ cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe]1 Hase, with the Type I cytochrome c 3 as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centers were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.
- 18Marques, M.; Tapia, C.; Gutiérrez-Sanz, Ó.; Ramos, A. R.; Keller, K. L.; Wall, J. D.; De Lacey, A. L.; Matias, P. M.; Pereira, I. A. C. The Direct Role of Selenocysteine in [NiFeSe] Hydrogenase Maturation and Catalysis. Nat. Chem. Biol. 2017, 13, 544– 550, DOI: 10.1038/nchembio.2335[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvFahtb8%253D&md5=e46b54a3255f5195c93026c15ffe192eThe direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysisMarques, Marta C.; Tapia, Cristina; Gutierrez-Sanz, Oscar; Ramos, Ana Raquel; Keller, Kimberly L.; Wall, Judy D.; De Lacey, Antonio L.; Matias, Pedro M.; Pereira, Ines A. C.Nature Chemical Biology (2017), 13 (5), 544-550CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Hydrogenases are highly active enzymes for hydrogen prodn. and oxidn. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 prodn. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for prodn. of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.
- 19Parkin, A.; Goldet, G.; Cavazza, C.; Fontecilla-Camps, J. C.; Armstrong, F. A. The Difference a Se Makes? Oxygen-Tolerant Hydrogen Production by the [NiFeSe]-Hydrogenase from Desulfomicrobium Baculatum. J. Am. Chem. Soc. 2008, 130, 13410– 13416, DOI: 10.1021/ja803657d[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFSjtrvI&md5=082d232b1fd5fd6e8fdf80b3c185b466The Difference a Se Makes? Oxygen-Tolerant Hydrogen Production by the [NiFeSe]-Hydrogenase from Desulfomicrobium baculatumParkin, Alison; Goldet, Gabrielle; Cavazza, Christine; Fontecilla-Camps, Juan C.; Armstrong, Fraser A.Journal of the American Chemical Society (2008), 130 (40), 13410-13416CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein film voltammetry (PFV) studies of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum show it to be a highly efficient H2 cycling catalyst. In the presence of 100% H2, the ratio of H2 prodn. to H2 oxidn. activity is higher than for any conventional [NiFe]-hydrogenases (lacking a selenocysteine ligand) that have been investigated to date. Although traces of O2 (« 1%) rapidly and completely remove H2 oxidn. activity, the enzyme sustains partial activity for H2 prodn. even in the presence of 1% O2 in the atm. That H2 prodn. should be partly allowed, whereas H2 oxidn. is not, is explained because the inactive product of O2 attack is reductively reactivated very rapidly, but this requires a potential that is almost as neg. as the thermodn. potential for the 2H+/H2 couple. The study provides further encouragement and clues regarding the feasibility of microbial/enzymic H2 prodn. free from restrictions of anaerobicity.
- 20Reisner, E.; Powell, D. J.; Cavazza, C.; Fontecilla-Camps, J. C.; Armstrong, F. A. Visible Light-Driven H2 Production by Hydrogenases Attached to Dye-Sensitized TiO2 Nanoparticles. J. Am. Chem. Soc. 2009, 131, 18457– 18466, DOI: 10.1021/ja907923r[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVKhsL%252FJ&md5=be81b7fbe9a198b0599afacfb4c58eeaVisible Light-Driven H2 Production by Hydrogenases Attached to Dye-Sensitized TiO2 NanoparticlesReisner, Erwin; Powell, Daniel J.; Cavazza, Christine; Fontecilla-Camps, Juan C.; Armstrong, Fraser A.Journal of the American Chemical Society (2009), 131 (51), 18457-18466CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A study of hybrid, enzyme-modified nanoparticles able to produce H2 using visible light as the energy source has been carried out to establish per-site performance stds. for H2 prodn. catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO2, with triethanolamine as a sacrificial electron donor, produces H2 at a turnover frequency of approx. 50 (mol H2) s-1 (mol total hydrogenase)-1 at pH 7 and 25 °C, even under the typical solar irradn. of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for bench-top applications.
- 21Wakerley, D. W.; Reisner, E. Oxygen-Tolerant Proton Reduction Catalysis: Much O2 about Nothing?. Energy Environ. Sci. 2015, 8, 2283– 2295, DOI: 10.1039/C5EE01167A[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpt1Sltbg%253D&md5=c077a781550e753ea5bae966ac9219e4Oxygen-tolerant proton reduction catalysis: much O2 about nothing?Wakerley, David W.; Reisner, ErwinEnergy & Environmental Science (2015), 8 (8), 2283-2295CODEN: EESNBY; ISSN:1754-5706. (Royal Society of Chemistry)Proton redn. catalysts are an integral component of artificial photosynthetic systems for the prodn. of H2. This perspective covers such catalysts with respect to their tolerance towards the potential catalyst inhibitor O2. O2 is abundant in our atm. and generated as a byproduct during the water splitting process, therefore maintaining proton redn. activity in the presence of O2 is important for the widespread prodn. of H2. This perspective article summarizes viable strategies for avoiding the adverse effects of aerobic environments to encourage their adoption and improvement in future research. H2-evolving enzymic systems, mol. synthetic catalysts and catalytic surfaces are discussed with respect to their interaction with O2 and anal. techniques through which O2-tolerant catalysts can be studied are described.
- 22Baltazar, C.; Teixeira, V. H.; Soares, C. M. Structural Features of [NiFeSe] and [NiFe] Hydrogenases Determining Their Different Properties: A Computational Approach. JBIC, J. Biol. Inorg. Chem. 2012, 17, 543– 555, DOI: 10.1007/s00775-012-0875-2[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlOmt70%253D&md5=d10cf43da4d06895abea888ae0c4c0f6Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approachBaltazar, Carla S. A.; Teixeira, Vitor H.; Soares, Claudio M.JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (4), 543-555CODEN: JJBCFA; ISSN:0949-8257. (Springer)Hydrogenases are metalloenzymes that catalyze the reversible reaction H2 ↹ 2H+ + 2e-, being potentially useful in H2 prodn. or oxidn. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O2 inhibition and produce more H2 than std. [NiFe] hydrogenases. However, the mol. determinants responsible for these properties remain unknown. Hydrophobic pathways for H2 diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H2 and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a std. [NiFe] hydrogenase. We performed mol. dynamics simulations of H2 diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas. The comparison showed that H2 d. near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H2 to the active site. We have also detd. a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas. The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H2 and proton pathways.
- 23Gutiérrez-Sanz, Ó.; Marques, M.; Baltazar, C.; Fernández, V. M.; Soares, C. M.; Pereira, I. A. C.; De Lacey, A. L. Influence of the Protein Structure Surrounding the Active Site on the Catalytic Activity of [NiFeSe] Hydrogenases. JBIC, J. Biol. Inorg. Chem. 2013, 18, 419– 427, DOI: 10.1007/s00775-013-0986-4[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjsV2jsL0%253D&md5=ba933361c2265bbaf85f477319c5cf6fInfluence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenasesGutierrez-Sanz, Oscar; Marques, Marta C.; Baltazar, Carla S. A.; Fernandez, Victor M.; Soares, Claudio M.; Pereira, Ines A. C.; De Lacey, Antonio L.JBIC, Journal of Biological Inorganic Chemistry (2013), 18 (4), 419-427CODEN: JJBCFA; ISSN:0949-8257. (Springer)A combined exptl. and theor. study of the catalytic activity of Desulfovibrio vulgaris [NiFeSe] hydrogenase was performed by H/D exchange mass spectrometry and mol. dynamics (MD) simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or prodn. of H2. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the Ni atom in the active site instead of cysteine. The aim of this research was to det. how much the specific catalytic properties of this hydrogenase were influenced by the replacement of a S atom by a Se atom in the coordination of the bimetallic active site vs. the changes in the protein structure surrounding the active site. The pH dependence of the D2/H+ exchange activity and the high isotope effect obsd. in the Km for the H2 substrate and in the single exchange/double exchange ratio suggest that a "cage effect" due to the protein structure surrounding the active site was modulating the enzymic catalysis. This "cage effect" was supported by MD simulations of the diffusion of H2 and D2 from the outside to the inside of the protein, which showed different accumulation of these substrates in a cavity next to the active site.
- 24Tamura, T.; Tsunekawa, N.; Nemoto, M.; Inagaki, K.; Hirano, T.; Sato, F. Molecular Evolution of Gas Cavity in [NiFeSe] Hydrogenases Resurrected in Silico. Sci. Rep. 2016, 6, 19742, DOI: 10.1038/srep19742[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs12jtbg%253D&md5=6c0f6dd0646de41a7d15e2940b0cb735Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silicoTamura, Takashi; Tsunekawa, Naoki; Nemoto, Michiko; Inagaki, Kenji; Hirano, Toshiyuki; Sato, FumitoshiScientific Reports (2016), 6 (), 19742CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Oxygen tolerance of selenium-contg. [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulfate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homol. modeling and mol. dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under abs. anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.
- 25Ceccaldi, P.; Marques, M.; Fourmond, V.; Pereira, I. A. C.; Léger, C. Oxidative Inactivation of NiFeSe Hydrogenase. Chem. Commun. 2015, 51, 14223– 14226, DOI: 10.1039/C5CC05930E[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmtrjE&md5=3cc06940ad564a04949151ff00fe7dceOxidative inactivation of NiFeSe hydrogenaseCeccaldi, Pierre; Marques, Marta C.; Fourmond, Vincent; Pereira, Ines Cardoso; Leger, ChristopheChemical Communications (Cambridge, United Kingdom) (2015), 51 (75), 14223-14226CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The authors propose a resoln. to the paradox that spectroscopic studies of NiFeSe hydrogenase have not revealed any major signal attributable to NiIII states formed upon reaction with O2, despite the fact that 2 inactive states are formed upon either aerobic or anaerobic oxidn.
- 26Maroney, M. J.; Hondal, R. J. Selenium versus Sulfur: Reversibility of Chemical Reactions and Resistance to Permanent Oxidation in Proteins and Nucleic Acids. Free Radical Biol. Med. 2018, 127, 228– 237, DOI: 10.1016/j.freeradbiomed.2018.03.035[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntFajt7g%253D&md5=16adde751b6085fe8451e6bbaa0eec8fSelenium, sulfur, and the Reversibility of chemical reactions and resistance to permanent oxidation in proteins and nucleic acidsMaroney, Michael J.; Hondal, Robert J.Free Radical Biology & Medicine (2018), 127 (), 228-237CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)A review. This review highlights the contributions of Jean Chaudiere to the field of selenium biochem. Chaudiere was the first to recognize that one of the main reasons that selenium in the form of selenocysteine is used in proteins is due to the fact that it strongly resists permanent oxidn. The foundations for this important concept was laid down by Al Tappel in the 1960's and even before by others. The concept of oxygen tolerance first recognized in the study of glutathione peroxidase was further advanced and refined by those studying [NiFeSe]-hydrogenases, selenosubtilisin, and thioredoxin reductase. After 200 years of selenium research, work by Marcus Conrad and coworkers studying glutathione peroxidase-4 has provided definitive evidence for Chaudiere's original hypothesis (Ingold et al., 2018) [36]. While the reaction of selenium with oxygen is readily reversible, there are many other examples of this phenomenon of reversibility. Many reactions of selenium can be described as "easy in - easy out". This is due to the strong nucleophilic character of selenium to attack electrophiles, but then this reaction can be reversed due to the strong electrophilic character of selenium and the weakness of the selenium-carbon bond. Several examples of this are described.
- 27Marques, M.; Coelho, R.; Pereira, I. A. C.; Matias, P. M. Redox State-Dependent Changes in the Crystal Structure of [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough. Int. J. Hydrogen Energy 2013, 38, 8664– 8682, DOI: 10.1016/j.ijhydene.2013.04.132[Crossref], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosFertbo%253D&md5=9fd85def4bd733acddec965ae314aa3aRedox state-dependent changes in the crystal structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris HildenboroughMarques, Marta C.; Coelho, Ricardo; Pereira, Ines A. C.; Matias, Pedro M.International Journal of Hydrogen Energy (2013), 38 (21), 8664-8682CODEN: IJHEDX; ISSN:0360-3199. (Elsevier Ltd.)Hydrogenases are enzymes that can potentially be used in bioelec. devices or for biol. hydrogen prodn., the most studied of which are the [NiFe] type. Most [NiFe] hydrogenases are inactivated by oxygen and the few known O2-tolerant enzymes are hydrogen-uptake enzymes, unsuitable for hydrogen prodn., due to strong product inhibition. In contrast, the [NiFeSe] hydrogenases, where a selenocysteine is bound to the nickel, are very attractive alternatives because of their high hydrogen prodn. activity and fast reactivation after O2 exposure. Here we report five high-resoln. crystallog. 3D structures of the sol. form of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough in three different redox states (oxidized as-isolated, H2-reduced and air re-oxidized), which revealed the structural changes that take place at the active site during enzyme redn. and re-oxidn. The results provide new insights into the pathways of O2 inactivation in [NiFe], and in particular [NiFeSe], hydrogenases. In addn., they suggest that different enzymes may display different oxidized states upon exposure to O2, which are probably detd. by the protein structure.
- 28Marques, M.; Coelho, R.; De Lacey, A. L.; Pereira, I. A. C.; Matias, P. M. The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State. J. Mol. Biol. 2010, 396, 893– 907, DOI: 10.1016/j.jmb.2009.12.013[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitValtr8%253D&md5=fa4d04e98e831a42830404f607fb44d8The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: A hydrogenase without a bridging ligand in the active site in its oxidized, "as-isolated" stateMarques, Marta C.; Coelho, Ricardo; De Lacey, Antonio L.; Pereira, Ines A. C.; Matias, Pedro M.Journal of Molecular Biology (2010), 396 (4), 893-907CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)H2 is a good energy vector, and its prodn. from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biol. prodn. of H2 because they are capable of high prodn. rates even in the presence of moderate amts. of O2, lessening the requirements for anaerobic conditions. Here, the 3-dimensional structure of [NiFeSe] Hase of D. vulgaris Hildenborough was detd. in its oxidized "as-isolated" form at 2.04-Å resoln. Remarkably, this is the 1st structure of an oxidized Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra S atom was obsd. binding Ni and Se, leading to a SeCys conformation that shielded the NiFe site from contact with O2. This structure provided several insights that may explain the fast activation and O2 tolerance of these enzymes.
- 29Volbeda, A.; Amara, P.; Iannello, M.; De Lacey, A. L.; Cavazza, C.; Fontecilla-Camps, J. C. Structural Foundations for the O2 Resistance of Desulfomicrobium Baculatum [NiFeSe]-Hydrogenase. Chem. Commun. (Cambridge, U. K.) 2013, 49, 7061– 7063, DOI: 10.1039/c3cc43619e[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFSit7jN&md5=01da88066da9970ae41fc16d27a66465Structural foundations for the O2 resistance of Desulfomicrobium baculatum [NiFeSe]-hydrogenaseVolbeda, Anne; Amara, Patricia; Iannello, Marina; De Lacey, Antonio L.; Cavazza, Christine; Fontecilla-Camps, Juan CarlosChemical Communications (Cambridge, United Kingdom) (2013), 49 (63), 7061-7063CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)The authors show how the NiFeSe site of anaerobically purified O2-resistant hydrogenase of D. baculatum reacts with air to give a seleninate as the 1st product. Less oxidized states of the active site were readily reduced in the presence of x-rays. Reductive enzyme activation required an efficient pathway for water escape. The crystal structure of the enzyme is reported. The crystal space group was P212121 with cell dimensions a = 106.2, b = 108.7, and c = 136.5 Å.
- 30Garcin, E.; Vernede, X.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J. C. The Crystal Structure of a Reduced [NiFeSe] Hydrogenase Provides an Image of the Activated Catalytic Center. Structure 1999, 7, 557– 566, DOI: 10.1016/S0969-2126(99)80072-0[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVSitrw%253D&md5=881c8e351d7fc1dbe6050bec883b20f4The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic centerGarcin, E.; Vernede, X.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J. C.Structure (London) (1999), 7 (5), 557-566CODEN: STRUE6; ISSN:0969-2126. (Current Biology Publications)[NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2 ↔ 2H+ + 2e-. They are generally heterodimeric, contain 3 Fe-S clusters in their small subunit and a Ni-Fe-contg. active site in their large subunit that includes a selenocysteine (SeCys) ligand. Here, the authors report the x-ray crystal structure at 2.15 Å resoln. of periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of oxidized, as-prepd., Desulfovibrio gigas and the reduced D. baculatum hydrogenases showed that in the reduced enzyme the Ni-Fe distance was 0.4 Å shorter than in the oxidized enzyme. In addn., the putative oxo ligand, detected in the as-prepd. D. gigas enzyme, was absent from the D. baculatum hydrogenase. The authors also obsd. higher-than-av. temp. factors for both the active site Ni-selenocysteine ligand and the neighboring Glu-18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the mol. surface. Other differences between [NiFeSe] and [NiFe] hydrogenases were the presence of a 3rd [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative Fe center that substitutes the Mg2+ ion that has already been described at the C-terminus of the large subunit of 2 [NiFe] hydrogenases. The heterolytic cleavage of H2 seems to be mediated by the Ni center and the selenocysteine residue. In addn. to modifying the catalytic properties of the enzyme, the Se ligand might protect the Ni atom from oxidn. It was concluded that the putative oxo ligand is a signature of inactive "unready" [NiFe] hydrogenases.
- 31Zacarias, S.; Vélez, M.; Pita, M.; De Lacey, A. L.; Matias, P. M.; Pereira, I. A. C. Methods in Enzymology 2018, 613, 169, DOI: 10.1016/bs.mie.2018.10.003[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3crotVynug%253D%253D&md5=63af0b1c66a5c68e7034e56922866798Characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris HildenboroughZacarias Sonia; Velez Marisela; Pita Marcos; De Lacey Antonio L; Matias Pedro M; Pereira Ines A CMethods in enzymology (2018), 613 (), 169-201 ISSN:.The [NiFeSe] hydrogenases are a subgroup of the well-characterized family of [NiFe] hydrogenases, in which a selenocysteine is a ligand to the nickel atom in the binuclear NiFe active site instead of cysteine. These enzymes display very interesting catalytic properties for biological hydrogen production and bioelectrochemical applications: high H2 production activity, bias for H2 evolution, low H2 inhibition, and some degree of O2 tolerance. Here we describe the methodologies employed to study the [NiFeSe] hydrogenase isolated from the sulfate-reducing bacteria D. vulgaris Hildenborough and the creation of a homologous expression system for production of variant forms of the enzyme.
- 32Ruff, A.; Szczesny, J.; Zacarias, S.; Pereira, I. A. C.; Plumeré, N.; Schuhmann, W. Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough under Oxidative Conditions. ACS Energy Lett. 2017, 2, 964– 968, DOI: 10.1021/acsenergylett.7b00167[ACS Full Text], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsVelsrw%253D&md5=93d67508e80d067b63c190b60f124459Protection and reactivation of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough under oxidative conditionsRuff, Adrian; Szczesny, Julian; Zacarias, Sonia; Pereira, Ines A. C.; Plumere, Nicolas; Schuhmann, WolfgangACS Energy Letters (2017), 2 (5), 964-968CODEN: AELCCP; ISSN:2380-8195. (American Chemical Society)We report on the fabrication of bioanodes for H2 oxidn. based on [NiFeSe] hydrogenase. The enzyme was elec. wired by means of a specifically designed low-potential viologen-modified polymer, which delivers benchmark H2 oxidizing currents even under deactivating conditions owing to efficient protection against O2 combined with a viologen-induced reactivation of the O2-inhibited enzyme. Moreover, the viologen-modified polymer allows for electrochem. co-deposition of polymer and biocatalyst and, by this, for control of the film thickness. Protection and reactivation of the enzyme was demonstrated in thick and thin reaction layers.
- 33Ruff, A.; Szczesny, J.; Marković, N.; Conzuelo, F.; Zacarias, S.; Pereira, I. A. C.; Lubitz, W.; Schuhmann, W. A Fully Protected Hydrogenase/Polymer-Based Bioanode for High-Performance Hydrogen/Glucose Biofuel Cells. Nat. Commun. 2018, 9, 3675, DOI: 10.1038/s41467-018-06106-3[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3nsV2ruw%253D%253D&md5=14b1677590384718edbc3d1b051df0daA fully protected hydrogenase/polymer-based bioanode for high-performance hydrogen/glucose biofuel cellsRuff Adrian; Szczesny Julian; Markovic Nikola; Conzuelo Felipe; Schuhmann Wolfgang; Zacarias Sonia; Pereira Ines A C; Lubitz WolfgangNature communications (2018), 9 (1), 3675 ISSN:.Hydrogenases with Ni- and/or Fe-based active sites are highly active hydrogen oxidation catalysts with activities similar to those of noble metal catalysts. However, the activity is connected to a sensitivity towards high-potential deactivation and oxygen damage. Here we report a fully protected polymer multilayer/hydrogenase-based bioanode in which the sensitive hydrogen oxidation catalyst is protected from high-potential deactivation and from oxygen damage by using a polymer multilayer architecture. The active catalyst is embedded in a low-potential polymer (protection from high-potential deactivation) and covered with a polymer-supported bienzymatic oxygen removal system. In contrast to previously reported polymer-based protection systems, the proposed strategy fully decouples the hydrogenase reaction form the protection process. Incorporation of the bioanode into a hydrogen/glucose biofuel cell provides a benchmark open circuit voltage of 1.15 V and power densities of up to 530 μW cm(-2) at 0.85 V.
- 34Szczesny, J.; Marković, N.; Conzuelo, F.; Zacarias, S.; Pereira, I. A. C.; Lubitz, W.; Plumeré, N.; Schuhmann, W.; Ruff, A. A Gas Breathing Hydrogen/Air Biofuel Cell Comprising a Redox Polymer/Hydrogenase-Based Bioanode. Nat. Commun. 2018, 9, 4715, DOI: 10.1038/s41467-018-07137-6[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cvosFGrug%253D%253D&md5=c57ae509fb6b30179d48ba64f2ec4677A gas breathing hydrogen/air biofuel cell comprising a redox polymer/hydrogenase-based bioanodeSzczesny Julian; Markovic Nikola; Conzuelo Felipe; Schuhmann Wolfgang; Ruff Adrian; Zacarias Sonia; Pereira Ines A C; Lubitz Wolfgang; Plumere NicolasNature communications (2018), 9 (1), 4715 ISSN:.Hydrogen is one of the most promising alternatives for fossil fuels. However, the power output of hydrogen/oxygen fuel cells is often restricted by mass transport limitations of the substrate. Here, we present a dual-gas breathing H2/air biofuel cell that overcomes these limitations. The cell is equipped with a hydrogen-oxidizing redox polymer/hydrogenase gas-breathing bioanode and an oxygen-reducing bilirubin oxidase gas-breathing biocathode (operated in a direct electron transfer regime). The bioanode consists of a two layer system with a redox polymer-based adhesion layer and an active, redox polymer/hydrogenase top layer. The redox polymers protect the biocatalyst from high potentials and oxygen damage. The bioanodes show remarkable current densities of up to 8 mA cm(-2). A maximum power density of 3.6 mW cm(-2) at 0.7 V and an open circuit voltage of up to 1.13 V were achieved in biofuel cell tests, representing outstanding values for a device that is based on a redox polymer-based hydrogenase bioanode.
- 35Tapia, C.; Zacarias, S.; Pereira, I. A. C.; Conesa, J. C.; Pita, M.; De Lacey, A. L. In Situ Determination of Photobioproduction of H2 by In2S3-[NiFeSe] Hydrogenase from Desulfovibrio Vulgaris Hildenborough Using Only Visible Light. ACS Catal. 2016, 6, 5691– 5698, DOI: 10.1021/acscatal.6b01512[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1aju7vI&md5=e0a75db3466993373a2d25a9174bce54In Situ Determination of Photobioproduction of H2 by In2S3-[NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough Using Only Visible LightTapia, Cristina; Zacarias, Sonia; Pereira, Ines A. C.; Conesa, Jose C.; Pita, Marcos; De Lacey, Antonio L.ACS Catalysis (2016), 6 (9), 5691-5698CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)An interesting strategy for photocatalytic prodn. of hydrogen from water and sunlight is the formation of a hybrid photocatalyst that combines an inorg. semiconductor able to absorb in the visible light spectral range with an enzymic catalyst for reducing protons. How to optimize the interfacing of In2S3 particles with the sol. form of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough by means of its initial H2 photoprodn. rate is studied. The kinetics of the photocatalytic process was studied by membrane-inlet mass spectrometry, in order to optimize the interaction between both components of the hybrid photocatalyst. Membrane-inlet mass spectrometry allows measuring in the same expt., for comparison, the rate of H2 prodn. by the photocatalyst hybrid directly in the aq. soln. in real time and the result of a std. assay of the hydrogenase activity. An incubation period of 6 h with mild stirring of hydrogenase with In2S3 particles was necessary for optimal interaction of the enzyme mols. with the porous surface of the semiconductor. A turnover frequency of the NiFeSe hydrogenase (TOFHase) for H2 photobioprodn. of 986 s-1 was measured under the optimized conditions. This means that the immobilized hydrogenase has a photocatalytic efficiency for H2 generation which is 94% of that obtained in the std. specific activity test of H2 prodn. using reduced Me viologen as an electron donor.
- 36Gutiérrez-Sanz, Ó.; Natale, P.; Márquez, I.; Marques, M.; Zacarias, S.; Pita, M.; Pereira, I. A. C.; López-Montero, I.; De Lacey, A. L.; Vélez, M. H2-Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration. Angew. Chem., Int. Ed. 2016, 55, 6216– 6220, DOI: 10.1002/anie.201600752[Crossref], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlKmur4%253D&md5=866777a35d90c2cdea5cc096d6d1a4f5H2-Fueled ATP Synthesis on an Electrode: Mimicking Cellular RespirationGutierrez-Sanz, Oscar; Natale, Paolo; Marquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Ines A. C.; Lopez-Montero, Ivan; De Lacey, Antonio L.; Velez, MariselaAngewandte Chemie, International Edition (2016), 55 (21), 6216-6220CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)ATP, the mol. used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0-ATPase in a lipid membrane can couple the electrochem. oxidn. of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochem. fuel locally when required in biomedical devices or enzymic synthesis of valuable products.
- 37Paulsen, C. E.; Carroll, K. S. Cysteine-Mediated Redox Signaling: Chemistry, Biology, and Tools for Discovery. Chem. Rev. 2013, 113, 4633– 4679, DOI: 10.1021/cr300163e[ACS Full Text], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlert7Y%253D&md5=a071177f7815ccefe7a58abe02d24906Cysteine-mediated redox signaling: Chemistry, biology, and tools for discoveryPaulsen, Candice E.; Carroll, Kate S.Chemical Reviews (Washington, DC, United States) (2013), 113 (7), 4633-4679CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The different oxidative post-translational modifications of protein Cys residue SH groups are reviewed, with particular emphasis on those chem. properties that differentiate one modification from another. Recent progress in using chem. approaches to develop probes that enable selective and direct detection of individual modifications within their native cellular environment are also reviewed. The discussion is complemented with examples from the literature that highlight ways in which Cys oxidn. can be used to control protein function and cell signaling pathways.
- 38Sensi, M.; del Barrio, M.; Baffert, C.; Fourmond, V.; Léger, C. New Perspectives in Hydrogenase Direct Electrochemistry. Curr. Opin. Electrochem. 2017, 5, 135– 145, DOI: 10.1016/j.coelec.2017.08.005[Crossref], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVGgsbzM&md5=3a1aef526f6859e16c65e91f8b05faaaNew perspectives in hydrogenase direct electrochemistrySensi, Matteo; del Barrio, Melisa; Baffert, Carole; Fourmond, Vincent; Leger, ChristopheCurrent Opinion in Electrochemistry (2017), 5 (1), 135-145CODEN: COEUCY; ISSN:2451-9111. (Elsevier B.V.)Electrochem. studies of hydrogenases, the biol. catalysts of H2 oxidn. and prodn., have proven wrong the old saying that enzymes do not easily transfer electrons to electrodes in the absence of mediators. Many distinct hydrogenases have actually been directly connected to electrodes or particles, for studying their catalytic mechanism or for designing solar-fuels catalysts. In this review, we list the electrodes that have proved successful for direct electron transfer to hydrogenases, and we discuss recent results which illustrate new directions in this research field: the study of the biosynthesis of FeFe hydrogenase, the electrochem. characterization of non-std. NiFe or FeFe hydrogenases, the general discussion of what makes a catalyst better in one particular direction of the reaction, and the elucidation of the mol. mechanisms of hydrogenase catalysis by combining electrochem. and theor. chem., spectroscopy or photochem. The electrochem. methods described herein will probably prove useful for studying or using other redox enzymes.
- 39del Barrio, M.; Sensi, M.; Orain, C.; Baffert, C.; Dementin, S.; Fourmond, V.; Léger, C. Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels. Acc. Chem. Res. 2018, 51, 769– 777, DOI: 10.1021/acs.accounts.7b00622[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFagu7s%253D&md5=c6d2e2edab275cf3cea9837d9d58b320Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuelsdel Barrio, Melisa; Sensi, Matteo; Orain, Christophe; Baffert, Carole; Dementin, Sebastien; Fourmond, Vincent; Leger, ChristopheAccounts of Chemical Research (2018), 51 (3), 769-777CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Many enzymes that produce or transform small mols. such as O2, H2, and CO2 embed inorg. cofactors based on transition metals. Their active site, where the chem. reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water mols. transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which det. the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochem., structural, spectroscopic, theor., and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extg. quant. information from the comparison of data and kinetic models and by exploring the combination of exptl. kinetics and theor. chem. In studies of these catalytic mechanisms, direct electrochem., the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an elec. current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chem. and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochem. can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramol. diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodn. of intramol. electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equil. potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examg. the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochem. investigations and MD, DFT, and TD-DFT calcns. is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O2, and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and detg. their structure using spectroscopic or structural methods.
- 40Léger, C.; Dementin, S.; Bertrand, P.; Rousset, M.; Guigliarelli, B. Inhibition and Aerobic Inactivation Kinetics of Desulfovibrio Fructosovorans NiFe Hydrogenase Studied by Protein Film Voltammetry. J. Am. Chem. Soc. 2004, 126, 12162– 12172, DOI: 10.1021/ja046548d[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntFGhs74%253D&md5=919d588f69d5af2f75a716b826cf0781Inhibition and Aerobic Inactivation Kinetics of Desulfovibrio fructosovorans NiFe Hydrogenase Studied by Protein Film VoltammetryLeger, Christophe; Dementin, Sebastien; Bertrand, Patrick; Rousset, Marc; Guigliarelli, BrunoJournal of the American Chemical Society (2004), 126 (38), 12162-12172CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addn. in the electrochem. cell of H2, CO, or O2 allow simple and virtually instantaneous detns. of the Michaelis const., inhibition const., or rate of inactivation, resp., thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we det. and discuss how its affinity for the active site changes as the driving force for the H+/H2 reaction is continuously varied. Inactivation by O2 is a slow, bimol. process (with pH-independent rate const. ≈ 3×104 s-1 M-1 at 40°, under one atm of H2) that leads to a mixt. of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H2. This exptl. approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.
- 41Fourmond, V.; Lautier, T.; Baffert, C.; Leroux, F.; Liebgott, P.; Dementin, S.; Rousset, M.; Arnoux, P.; Pignol, D.; Meynial-Salles, I.; Soucaille, P.; Bertrand, P.; Léger, C. Correcting for Electrocatalyst Desorption and Inactivation in Chronoamperometry Experiments. Anal. Chem. 2009, 81, 2962, DOI: 10.1021/ac8025702[ACS Full Text], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtl2qsr0%253D&md5=4c664f44beca6f3a84d7b55d49e8c0fdCorrecting for Electrocatalyst Desorption and Inactivation in Chronoamperometry ExperimentsFourmond, Vincent; Lautier, Thomas; Baffert, Carole; Leroux, Fanny; Liebgott, Pierre-Pol; Dementin, Sebastien; Rousset, Marc; Arnoux, Pascal; Pignol, David; Meynial-Salles, Isabelle; Soucaille, Phillippe; Bertrand, Patrick; Leger, ChristopheAnalytical Chemistry (Washington, DC, United States) (2009), 81 (8), 2962-2968CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Chronoamperometric expts. with adsorbed electrocatalysts are commonly performed either for anal. purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concn. is being increased to det. a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such expts., the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. The authors propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric expts. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control expt. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. The authors describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case the authors discuss the trustfulness and the benefit of the correction. This method also applies to expts. where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concn. of catalyst.
- 42del Barrio, M.; Guendon, C.; Kpebe, A.; Baffert, C.; Fourmond, V.; Brugna, M.; Léger, C. A Valine-to-Cysteine Mutation Further Increases the Oxygen Tolerance of Escherichia Coli NiFe Hydrogenase Hyd-1. ACS Catal. 2019, 9, 4084– 4088, DOI: 10.1021/acscatal.9b00543[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtFGnurc%253D&md5=37545f094f547cdcb0b1b91884744c33Valine-to-cysteine mutation further increases the oxygen tolerance of Escherichia coli NiFe hydrogenase Hyd-1del Barrio, Melisa; Guendon, Chloe; Kpebe, Arlette; Baffert, Carole; Fourmond, Vincent; Brugna, Myriam; Leger, ChristopheACS Catalysis (2019), 9 (5), 4084-4088CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Some NiFe hydrogenases are particularly resistant to O2 as a result of either the natural presence of a particular FeS cluster or the artificial replacement of a conserved valine residue near the Ni site. We show that the two protective effects can be combined in a single enzyme by constructing and characterizing the V78C variant of the naturally O2-tolerant Escherichia coli NiFe hydrogenase Hyd-1. We elucidated the effect of the mutation by comparing the kinetics of inhibition by CO and O2 of a no. of wild-type forms and valine-to-cysteine variants of NiFe hydrogenases.
- 43Almeida, M. G.; Silveira, C. M.; Guigliarelli, B.; Bertrand, P.; Moura, J. J. G.; Moura, I.; Léger, C. A Needle in a Haystack: The Active Site of the Membrane-Bound Complex Cytochrome c Nitrite Reductase. FEBS Lett. 2007, 581, 284– 288, DOI: 10.1016/j.febslet.2006.12.023[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslyqtQ%253D%253D&md5=27acbb3f784d63ef94913955b213507fA needle in a haystack: The active site of the membrane-bound complex cytochrome c nitrite reductaseAlmeida, M. Gabriela; Silveira, Celia M.; Guigliarelli, Bruno; Bertrand, Patrick; Moura, Jose J. G.; Moura, Isabel; Leger, ChristopheFEBS Letters (2007), 581 (2), 284-288CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron redn. of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The no. of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously det. the redn. potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.
- 44Chovancova, E.; Pavelka, A.; Benes, P.; Strnad, O.; Brezovsky, J.; Kozlikova, B.; Gora, A.; Sustr, V.; Klvana, M.; Medek, P.; Biedermannova, L.; Sochor, J.; Damborsky, J. CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures. PLoS Comput. Biol. 2012, 8, e1002708 DOI: 10.1371/journal.pcbi.1002708[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1ansbfI&md5=aff24be751fef33d531b446cf6ab86c5CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structuresChovancova, Eva; Pavelka, Antonin; Benes, Petr; Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, JiriPLoS Computational Biology (2012), 8 (10), e1002708CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)Tunnels and channels facilitate the transport of small mols., ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chem. properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromol. structures. Herein we present a new version of CAVER enabling automatic anal. of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calcn. and clustering of pathways. A trajectory from a mol. dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the anal. of mol. dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estd. the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that anal. of mol. dynamics simulation is essential for the estn. of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochem. phenomena in the area of mol. transport, mol. recognition and enzymic catalysis. The software is freely available as a multiplatform command-line application online.
- 45Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A.; Moriarty, N.; Oeffner, R.; Read, R.; Richardson, D.; Richardson, J.; Terwilliger, T.; Zwart, P. PHENIX: A Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221, DOI: 10.1107/S0907444909052925[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisbc%253D&md5=67b439ff4bd61c659cae37ca4209b7bcPHENIX: a comprehensive Python-based system for macromolecular structure solutionAdams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
- 46Ogata, H.; Nishikawa, K.; Lubitz, W. Hydrogens Detected by Subatomic Resolution Protein Crystallography in a [NiFe] Hydrogenase. Nature 2015, 520, 571– 574, DOI: 10.1038/nature14110[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MvpvVanuw%253D%253D&md5=0bfe17f42242357775645917812a4f5bHydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenaseOgata Hideaki; Nishikawa Koji; Lubitz WolfgangNature (2015), 520 (7548), 571-4 ISSN:.The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-angstrom resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 ÅA resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 ÅA and 1.78ÅA, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
- 47Buhrke, T.; Lenz, O.; Krauss, N.; Friedrich, B. Oxygen Tolerance of the H2-Sensing [NiFe] Hydrogenase from Ralstonia Eutropha H16 Is Based on Limited Access of Oxygen to the Active Site. J. Biol. Chem. 2005, 280, 23791– 23796, DOI: 10.1074/jbc.M503260200[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXltlKhsrc%253D&md5=557d20410fde7e57e3820aa1240014b3Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active SiteBuhrke, Thorsten; Lenz, Oliver; Krauss, Norbert; Friedrich, BaerbelJournal of Biological Chemistry (2005), 280 (25), 23791-23796CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton redn. Hydrogen metab. predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. The authors have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the mol. background of its oxygen tolerance. Evidence is presented that the shape and size of the intramol. hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivs. that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of std. [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnol. application.
- 48Duché, O.; Elsen, S.; Cournac, L.; Colbeau, A. Enlarging the Gas Access Channel to the Active Site Renders the Regulatory Hydrogenase HupUV of Rhodobacter Capsulatus O2 Sensitive without Affecting Its Transductory Activity. FEBS J. 2005, 272, 3899– 3908, DOI: 10.1111/j.1742-4658.2005.04806.x[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MzoslOjsA%253D%253D&md5=27d9bdabfeb56608dc294b43f50cc19dEnlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activityDuche Ophelie; Elsen Sylvie; Cournac Laurent; Colbeau AnnetteThe FEBS journal (2005), 272 (15), 3899-908 ISSN:1742-464X.In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.
- 49Abou Hamdan, A.; Liebgott, P.; Fourmond, V.; Gutiérrez-Sanz, Ó.; De Lacey, A. L.; Infossi, P.; Rousset, M.; Dementin, S.; Léger, C. Relation between Anaerobic Inactivation and Oxygen Tolerance in a Large Series of NiFe Hydrogenase Mutants. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19916– 19921, DOI: 10.1073/pnas.1212258109[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslKhtA%253D%253D&md5=5a7fb740150648d2b1c4b67bef976f14Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutantsAbou Hamdan, Abbas; Liebgott, Pierre-Pol; Fourmond, Vincent; Gutierrez-Sanz, Oscar; De Lacey, Antonio L.; Infossi, Pascale; Rousset, Marc; Dementin, Sebastien; Leger, ChristopheProceedings of the National Academy of Sciences of the United States of America (2012), 109 (49), 19916-19921, S19916/1-S19916/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Ni-contg. hydrogenases, the biol. catalysts of H2 oxidn. and prodn., reversibly inactivate under anaerobic, oxidizing conditions. Here, the authors aimed at understanding the mechanism of (in)activation and what dets. its kinetics, because there is a correlation between fast reductive reactivation and O2 tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnol. Direct electrochem. is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here, the authors describe simple anal. models that were used to characterize and compare 16 mutants, obtained by substituting the position-74 Val residue of O2-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans. The authors obsd. that this substitution could accelerate reactivation up to 1000-fold, depending on the polarity of the position 74 residue side-chain. In terms of kinetics of anaerobic (in)activation and O2 tolerance, the Val-to-His (V74H) mutation had the most spectacular effect: The V74H mutant compared favorably with the O2-tolerant hydrogenase from Aquifex aeolicus, which was used here as a benchmark.
- 50Durrant, M. C. Controlled Protonation of Iron–Molybdenum Cofactor by Nitrogenase: A Structural and Theoretical Analysis. Biochem. J. 2001, 355, 569– 576, DOI: 10.1042/bj3550569[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjslCntb0%253D&md5=4138f63f02aa70c2d582c2f80ce9dda4Controlled protonation of iron-molybdenum cofactor by nitrogenase: a structural and theoretical analysisDurrant, Marcus C.Biochemical Journal (2001), 355 (3), 569-576CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Qual. mol. modeling has been used to identify possible routes for transfer of protons from the surface of the nitrogenase protein to the iron-molybdenum cofactor (FeMoco) and to substrates during catalysis. Three proton-transfer routes have been identified; a water-filled channel running from the protein exterior to the homocitrate ligand of FeMoco, and two hydrogen-bonded chains to specific FeMoco sulfur atoms. It is suggested that the water channel is used for multiple proton deliveries to the substrate, as well as in diffusion of products and substrates between FeMoco and the bulk solvent, whereas the two hydrogen-bonded chains each allow a single proton to be added to, and subsequently depart from, FeMoco during the catalytic cycle. Possible functional differences in the proton-transfer channels are discussed in terms of assessment of the protein environment and specific hydrogen-bonding effects. The implications of these observations are discussed in terms of the suppression of wasteful prodn. of dihydrogen by nitrogenase and the Lowe-Thorneley scheme for dinitrogen redn.
- 51Barney, B. M.; Yurth, M. G.; Santos, P. C.; Dean, D. R.; Seefeldt, C.; Carolina, N. A Substrate Channel in the Nitrogenase MoFe Protein. JBIC, J. Biol. Inorg. Chem. 2009, 14, 1015– 1022, DOI: 10.1007/s00775-009-0544-2[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmt1SktLo%253D&md5=99d668f2f9e91f56817ba27891399497A substrate channel in the nitrogenase MoFe proteinBarney, Brett M.; Yurth, Michael G.; Dos Santos, Patricia C.; Dean, Dennis R.; Seefeldt, Lance C.JBIC, Journal of Biological Inorganic Chemistry (2009), 14 (7), 1015-1022CODEN: JJBCFA; ISSN:0949-8257. (Springer)Nitrogenase catalyzes the six electron/six proton redn. of N2 to two ammonia mols. at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the α-subunit of the MoFe protein, with no obvious access for substrates. Examn. of high-resoln. X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate redn. kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the redn. of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, Vmax values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the Km values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.
- 52Amara, P.; Andreoletti, P.; Jouve, H. M.; Field, M. J. Ligand Diffusion in the Catalase from Proteus Mirabilis: A Molecular Dynamics Study. Protein Sci. 2001, 10, 1927– 1935, DOI: 10.1110/ps.14201[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXntFeqsLo%253D&md5=c5b9a7949c64211261c4e5dcf985e936Ligand diffusion in the catalase from Proteus mirabilis: a molecular dynamics studyAmara, Patricia; Andreoletti, Pierre; Jouve, Helene Marie; Field, Martin J.Protein Science (2001), 10 (10), 1927-1935CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)The role of the channels and cavities present in the catalase from Proteus mirabilis (PMC) was investigated using mol. dynamics (MD) simulations. The reactant and products of the reaction, H2O2 → 1/2 O2 + H2O, catalyzed by the enzyme were allowed to diffuse to and from the active site. Dynamic fluctuations in the structure are found necessary for the opening of the major channel, identified in the X-ray model, which allows access to the active site. This channel is the only pathway to the active site obsd. during the dynamics, and both the products and reactant use it. H2O and O2 are also detected in a cavity defined by the heme and Ser196, which could play an important role during the reaction. Free energy profiles of the ligands diffusing through the major channel indicate that the barriers to ligand diffusion are less than 20 kJ mol-1 for each of the species. It is not clear from our study that minor channels play a role for access to the protein active site or to the protein surface.
- 53Fritsch, J.; Scheerer, P.; Frielingsdorf, S.; Kroschinsky, S.; Friedrich, B.; Lenz, O.; Spahn, C. M. T. The Crystal Structure of an Oxygen-Tolerant Hydrogenase Uncovers a Novel Iron-Sulphur Centre. Nature 2011, 479, 249– 252, DOI: 10.1038/nature10505[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlWksr7L&md5=0baaeed344d6182959296d5dec1627c6The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulfur centerFritsch, Johannes; Scheerer, Patrick; Frielingsdorf, Stefan; Kroschinsky, Sebastian; Friedrich, Baerbel; Lenz, Oliver; Spahn, Christian M. T.Nature (London, United Kingdom) (2011), 479 (7372), 249-252CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Hydrogenases are abundant enzymes that catalyze the reversible interconversion of H2 into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O2 are considered to be central to H2-based technologies, such as enzymic fuel cells and for light-driven H2 prodn. Despite comprehensive genetic, biochem., electrochem., and spectroscopic investigations, the mol. background allowing a structural interpretation of how the catalytic center is protected from irreversible inactivation by O2 has remained unclear. Here, the authors present the crystal structure of an O2-tolerant [NiFe]-hydrogenase from the aerobic H2 oxidizer, Ralstonia eutropha H16 at 1.5 Å resoln. The heterodimeric enzyme consisted of a large subunit harboring the catalytic center in the H2-reduced state and a small subunit contg. an electron relay consisting of 3 different Fe-S clusters. The cluster proximal to the active site displayed an unprecedented [4Fe-3S] structure and was coordinated by 6 Cys residues. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas mol. approaching the active site. It serves as an electron acceptor in the course of H2 oxidn. and as an electron-delivering device upon O2 attack at the active site. This dual function was supported by the capability of the novel Fe-S cluster to adopt 3 redox states at physiol. redox potentials. The 2nd structural feature was a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biol. and chem. H2-converting catalysts that are capable of cycling H2 in air.
- 54Dementin, S.; Burlat, B.; De Lacey, A. L.; Pardo, A.; Adryanczyk-Perrier, G.; Guigliarelli, B.; Fernández, V. M.; Rousset, M. A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] Hydrogenase. J. Biol. Chem. 2004, 279, 10508– 10513, DOI: 10.1074/jbc.M312716200[Crossref], [PubMed], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvFChs7c%253D&md5=38c266f2161f170bf8acbf3ccad891b3A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] HydrogenaseDementin, Sebastien; Burlat, Benedicte; De Lacey, Antonio L.; Pardo, Alejandro; Adryanczyk-Perrier, Geraldine; Guigliarelli, Bruno; Fernandez, Victor M.; Rousset, MarcJournal of Biological Chemistry (2004), 279 (11), 10508-10513CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Kinetic, EPR, and Fourier transform IR spectroscopic anal. of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H2/ortho-H2 conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover nos. for the oxidn. of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H2 oxidn. and hydrogen isotope exchange activities, but did not change the para-H2/ortho-H2 conversion activity. The spectroscopic properties of this mutant were totally different, esp. in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.
- 55Bertrand, P.; Dole, F.; Asso, M.; Guigliarelli, B. Is There a Rate-Limiting Step in the Catalytic Cycle of [Ni-Fe] Hydrogenases?. JBIC, J. Biol. Inorg. Chem. 2000, 5, 682– 691, DOI: 10.1007/s007750000152[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXovV2hsL4%253D&md5=dd2371710d29fd6647321fbb8a9237d6Is there a rate-limiting step in the catalytic cycle of [Ni-Fe] hydrogenases?Bertrand, Patrick; Dole, Francois; Asso, Marcel; Guigliarelli, BrunoJBIC, Journal of Biological Inorganic Chemistry (2000), 5 (6), 682-691CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopersicina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, resp. This proposal is consistent with the data available from other Ni-Fe enzymes.
- 56Marques, M. F. C. Ph.D. Thesis, Structural and Functional Studies of a High Activity NiFeSe Hydrogenase; Universidade Nova de Lisboa: Oeiras, 2014.Google ScholarThere is no corresponding record for this reference.
- 57Keller, K. L.; Wall, J. D.; Chhabra, S.; Voigt, C. Methods Enzymol. 2011, 497, 503, DOI: 10.1016/B978-0-12-385075-1.00022-6[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpsVGksr4%253D&md5=352517f8a4ab96546bb12c6fcccc3794Methods for engineering sulfate reducing bacteria of the genus DesulfovibrioKeller, Kimberly L.; Wall, Judy D.; Chhabra, SwapnilMethods in Enzymology (2011), 497 (Synthetic Biology, Part A), 503-517CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. Sulfate reducing bacteria (SRB) are physiol. important given their nearly ubiquitous presence and have important applications in the areas of bioremediation and bioenergy. This chapter provides details on the steps used for homologous-recombination mediated chromosomal manipulation of Desulfovibrio vulgaris Hildenborough, a well-studied sulfate reducer. More specifically, we focus on the implementation of a "parts" based approach for suicide vector assembly, important aspects of anaerobic culturing, choices for antibiotic selection, electroporation-based DNA transformation, as well as tools for screening and verifying genetically modified constructs. These methods, which in principle may be extended to other SRB, are applicable for functional genomics investigations, as well as metabolic engineering manipulations.
- 58Bertrand, P.; Frangioni, B.; Dementin, S.; Sabaty, M.; Arnoux, P.; Guigliarelli, B.; Pignol, D.; Léger, C. Effects of Slow Substrate Binding and Release in Redox Enzymes: Theory and Application to Periplasmic Nitrate Reductase. J. Phys. Chem. B 2007, 111, 10300– 10311, DOI: 10.1021/jp074340j[ACS Full Text], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos1Sks7c%253D&md5=4741b536281eb2ebaede3caa699fd39fEffects of Slow Substrate Binding and Release in Redox Enzymes: Theory and Application to Periplasmic Nitrate ReductaseBertrand, Patrick; Frangioni, Bettina; Dementin, Sebastien; Sabaty, Monique; Arnoux, Pascal; Guigliarelli, Bruno; Pignol, David; Leger, ChristopheJournal of Physical Chemistry B (2007), 111 (34), 10300-10311CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)For redox enzymes, the technique of protein film voltammetry (PFV) makes it possible to det. the entire profile of activity against driving force by allowing the enzyme to directly exchange electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chem. steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully anal. treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quant. conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concn. dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the redn. potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional expts.; the position of the wave (i.e., the driving force that is required to elicit catalysis) departs from the redn. potential of the active site even at the lowest substrate concn., and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramol. electron transfer from the proximal [4Fe4S]+ cluster to the active site MoV.
- 59Fourmond, V. QSoas: A Versatile Software for Data Analysis. Anal. Chem. 2016, 88, 5050– 5052, DOI: 10.1021/acs.analchem.6b00224[ACS Full Text], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsVKksrk%253D&md5=d22e37a8f55f5b5e579f5711f0f713ebQSoas: A Versatile Software for Data AnalysisFourmond, VincentAnalytical Chemistry (Washington, DC, United States) (2016), 88 (10), 5050-5052CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Undoubtedly, the most natural way to confirm a model is to quant. verify its predictions. However, this is not done systematically, and one of the reasons for that is the lack of appropriate tools for analyzing data, because the existing tools do not implement the required models or they lack the flexibility required to perform data anal. in a reasonable time. We present QSoas, an open-source, cross-platform data anal. program written to overcome these problems. In addn. to std. data anal. procedures and full automation using scripts, QSoas features a very powerful data fitting interface with support for arbitrary functions, differential equation and kinetic system integration, and flexible global fits. QSoas is available from http://www.qoas.org.
- 60Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132, DOI: 10.1107/S0907444909047337[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1SisLc%253D&md5=1aa9a38aeb3ce95af4ffb7d8b8a142bdSoftware XDS for image rotation, recognition and crystal symmetry assignmentKabsch, WolfgangActa Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
- 61Vonrhein, C.; Flensburg, C.; Keller, P.; Sharff, A.; Smart, O.; Paciorek, W.; Womack, T.; Bricogne, G. Data Processing and Analysis with the AutoPROC Toolbox. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 293– 302, DOI: 10.1107/S0907444911007773[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtLs%253D&md5=a0a3579f6b371b6312819883864aa80dData processing and analysis with the autoPROC toolboxVonrhein, Clemens; Flensburg, Claus; Keller, Peter; Sharff, Andrew; Smart, Oliver; Paciorek, Wlodek; Womack, Thomas; Bricogne, GerardActa Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 293-302CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A typical diffraction expt. will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and nos. that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concg. on difficulties that may often show up during the first steps along the path of turning the expt. (i.e. data collection) into a model (i.e. interpreted electron d.). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analyzing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the expt. is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
- 62Evans, P. Scaling and Assessment of Data Quality. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72– 82, DOI: 10.1107/S0907444905036693[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7jM&md5=293d3876e534c0c57813990515bb3c76Scaling and assessment of data qualityEvans, PhilipActa Crystallographica, Section D: Biological Crystallography (2006), D62 (1), 72-82CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)The various phys. factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analyzed to set the real resoln. of the data set, to detect bad regions (e.g. bad images), to analyze radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation anal. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be detd. from scores such as correlation coeffs. between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.
- 63Tickle, I. J.; Flensburg, C.; Keller, P.; Paciorek, W.; Sharff, A.; Vonrhein, C.; Bricogne, G. STARANISO; Global Phasing: Cambridge, United Kingdom, 2018.Google ScholarThere is no corresponding record for this reference.
- 64Evans, P. R.; Murshudov, G. N. How Good Are My Data and What Is the Resolution?. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1204– 1214, DOI: 10.1107/S0907444913000061[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVejsL8%253D&md5=3f649426c21e9d423f94ef8d7c568233How good are my data and what is the resolution?Evans, Philip R.; Murshudov, Garib N.Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1204-1214CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Following integration of the obsd. diffraction spots, the process of data redn.' initially aims to det. the point-group symmetry of the data and the likely space group. This can be performed with the program POINTLESS. The scaling program then puts all the measurements on a common scale, avs. measurements of symmetry-related reflections (using the symmetry detd. previously) and produces many statistics that provide the first important measures of data quality. A new scaling program, AIMLESS, implements scaling models similar to those in SCALA but adds some addnl. analyses. From the analyses, a no. of decisions can be made about the quality of the data and whether some measurements should be discarded. The effective resoln.' of a data set is a difficult and possibly contentious question (particularly with referees of papers) and this is discussed in the light of tests comparing the data-processing statistics with trials of refinement against obsd. and simulated data, and automated model-building and comparison of maps calcd. with different resoln. limits. These trials show that adding weak high-resoln. data beyond the commonly used limits may make some improvement and does no harm.
- 65McCoy, A. J. Solving Structures of Protein Complexes by Molecular Replacement with Phaser. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 32– 41, DOI: 10.1107/S0907444906045975[Crossref], [PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlSqu77F&md5=323a5297bdf622ebaf398a87c39dc794Solving structures of protein complexes by molecular replacement with PhaserMcCoy, Airlie J.Acta Crystallographica, Section D: Biological Crystallography (2007), 63 (1), 32-41CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Mol. replacement (MR) generally becomes more difficult as the no. of components in the asym. unit requiring sep. MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asym. units to be built up from individual components with a 'tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a no. of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large no. of copies of the same component in the asym. unit or where the components of the asym. unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the std. 'automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.
- 66Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A Graphical User Interface to the CCP4 Program Suite Research Papers A Graphical User Interface to the CCP 4 Program Suite. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1131– 1137, DOI: 10.1107/S0907444903008126[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvFSksLs%253D&md5=25a0168dd7ea9f930036453a829e1e3dA graphical user interface to the CCP4 program suitePotterton, Elizabeth; Briggs, Peter; Turkenburg, Maria; Dodson, EleanorActa Crystallographica, Section D: Biological Crystallography (2003), D59 (7), 1131-1137CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)CCP4i is a graphical user interface that makes running programs from the CCP4 suite simpler and quicker. It is particularly directed at inexperienced users and tightly linked to introductory and scientific documentation. It also provides a simple project-management system and visualization tools. The system is readily extensible and not specific to CCP4 software.
- 67Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the Refinement of Macromolecular Crystal Structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355– 367, DOI: 10.1107/S0907444911001314[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtbk%253D&md5=f8f3202d246908500057ad7c71015b7bREFMAC5 for the refinement of macromolecular crystal structuresMurshudov, Garib N.; Skubak, Pavol; Lebedev, Andrey A.; Pannu, Navraj S.; Steiner, Roberto A.; Nicholls, Robert A.; Winn, Martyn D.; Long, Fei; Vagin, Alexei A.Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 355-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)This paper describes various components of the macromol. crystallog. refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS exptl. diffraction data. To ensure chem. and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolns. at least as low as 4 Å can be achieved thanks to low-resoln. refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, 'jelly-body' restraints and the use of novel long-range restraints on at. displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 addnl. offers TLS parameterization and, when high-resoln. data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resoln. spectrum encountered in macromol. crystallog.
- 68Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501, DOI: 10.1107/S0907444910007493[Crossref], [PubMed], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
- 69Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C.; Richardson, D. C. MolProbity: All-Atom Structure Validation for Macromolecular Crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 12– 21, DOI: 10.1107/S0907444909042073[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktg%253D%253D&md5=b5fc7574f43f01dd6e43c3663ca4f779MolProbity: all-atom structure validation for macromolecular crystallographyChen, Vincent B.; Arendall, W. Bryan, III; Headd, Jeffrey J.; Keedy, Daniel A.; Immormino, Robert M.; Kapral, Gary J.; Murray, Laura W.; Richardson, Jane S.; Richardson, David C.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 12-21CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact anal., complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallog. provides a wealth of biol. important mol. data in the form of at. three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystn. to data collection to phasing to model building to refinement, have made solving a structure using crystallog. easier than ever. However, despite these improvements, local errors that can affect biol. interpretation are widespread at low resoln. and even high-resoln. structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is crit. both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
- 70Berman, H.; Henrick, K.; Nakamura, H. Announcing the Worldwide Protein Data Bank. Nat. Struct. Mol. Biol. 2003, 10, 980– 980, DOI: 10.1038/nsb1203-980[Crossref], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXptFOmsbY%253D&md5=8146ff5fc67f64e31b2f8c475473879cAnnouncing the worldwide Protein Data BankBerman, Helen; Henrick, Kim; Nakamura, HarukiNature Structural Biology (2003), 10 (12), 980CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Group)There is no expanded citation for this reference.
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.9b02347.
View of the active site conformations in the as-isolated D. vulgaris Hildenborough [NiFeSe] hydrogenase, structure-based sequence alignments used to select the D. vulgaris Hildenborough [NiFeSe] hydrogenase mutants, X-ray data collection, processing, and refinement statistics, structural details at the active site and proximal [Fe4S4] cluster, views of the active site of the aerobically purified and crystallized [NiFeSe] hydrogenase G50T and G491S variants, H2 uptake activity of WT and variants, channels predicted by CAVER in [NiFe] and [NiFeSe] hydrogenase structures, detailed views of the hydrophilic channel and hydrophobic side channel, and graph showing the effect of CO on the H2 oxidation current of WT [NiFeSe] hydrogenase and variants adsorbed onto a graphite rotating electrode (PDF)
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