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Substrate Activation at the Ni,Fe Cluster of CO Dehydrogenases: The Influence of the Protein Matrix
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Substrate Activation at the Ni,Fe Cluster of CO Dehydrogenases: The Influence of the Protein Matrix
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ACS Catalysis

Cite this: ACS Catal. 2022, 12, 20, 12711–12719
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https://doi.org/10.1021/acscatal.2c02922
Published October 6, 2022

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Abstract

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Carbon monoxide dehydrogenases catalyze the reversible conversion of CO2 with two electrons to CO and water at a unique Ni- and Fe-containing cluster (cluster C). Structural studies indicate that several highly conserved amino acids in the second coordination sphere of cluster C support the activation of the substrates, CO/CO2 and water, and may be mandatory for catalytic turnover. However, their contribution to substrate activation has been poorly explored. We replaced the three residues with potential direct interaction with the substrates (I567, H93, and K563) and one residue essential for proton/water transfer (H96) and analyzed the associated changes in the structure and reactivity of the enzyme. In addition to the expected exchange of side chains, we observed rearrangements of water molecules as well as the appearance of additional water molecules at the active site. These changes also affect the coordination of cluster C and the hydroxo ligand at Fe, with additional hydroxo/water ligands at Ni. Subsequently, we were able to convert cluster C from a [NiFe4(OH)(μ3-S)4] cluster to a [Fe4(μ3-S)4] cluster by exchanging K563 and a primary coordinating C295. Therefore, the second coordination sphere is important not only for the affinity of the substrates but also for the stability of cluster C. Thus, beyond substrate activation, the residues in the second coordination sphere of cluster C also determine its coordination and stability.

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Introduction

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Carbon monoxide dehydrogenases (CODHs) catalyze the reversible interconversion of CO with water to CO2, two electrons, and two protons. (1−3) We distinguish two non-homologous types of CODHs that use different cofactors. (1) Cu,Mo-CODHs catalyze only CO oxidation, whereas Ni,Fe-CODHs support the reaction in both directions. Ni,Fe-CODHs are widely distributed in anaerobic microorganisms, including acetogenic, phototrophic, and hydrogenic bacteria, sulfate-reducing bacteria and archaea, and methanogenic archaea. (4−6) Their widespread distribution and phylogeny suggest that Ni,Fe-CODHs existed in the last common ancestor LUCA, placing them among the oldest known enzymes. (4)
According to their structure, we distinguish two types of Ni,Fe-CODHs (in the following referred to as CODHs for short): cooS- and cdh-type CODHs. (7) CooS-type CODHs are homodimers traversed by a chain of five Fe/S centers, two of which belong to the active site. (8−11) In the active site, we find a [NiFe4(OH)(μ3-S)4] cluster, which is referred to as cluster C, consisting of a distorted NiFe3S4 heterocubane connected to a ferrous ion in exo. (8,9,12) Compared to the basic cooS-type structure, the cdh-type CODHs display an extended main subunit with two additional [Fe4S4] centers and a second smaller subunit. (13,14) Despite these differences, the structure of the [NiFe4(OH)(μ3-S)4] cluster and the immediate environment around the active site is highly conserved in both CODH types. (3,15)
Binding and activation of substrates and products of the reversible reaction─CO, water, CO2, electrons, and protons─take place within the conserved environment of cluster C. At least four redox states have been described for cluster C: Cox, Cred1, Cint, and Cred2. (16,17) Cox is a catalytically inactive, diamagnetic oxidized state, which is reduced to Cred1 at potentials of below −100 mV. (16) Cred1 is capable of oxidizing CO, whereby a two-electron more reduced state called Cred2 is generated with a midpoint potential for the Cred1/Cred2 couple below −500 mV, close to the midpoint potential of CO/CO2. (18−22) Cred2 undergoes two one-electron oxidation steps, regenerating Cred1 via the diamagnetic Cint state. (16) Midpoint potentials for the transition between oxidation states are modulated by the presence of substrates. (20)
The presently accepted mechanism for CO oxidation involves the binding of CO to Ni2+ in the Cred1 state, in which a water molecule is activated by binding to the ferrous ion (Fe1) in exo. (12,23) Deprotonation of the water ligand creates a nucleophilic hydroxo-ligand at Fe1, while binding of CO to the only weakly back-bonding Ni2+ ion further favors the nucleophilic attack to form a Ni,Fe1-bound carboxylate. (12,24) The subsequent deprotonation and shift of electrons from the metal-carboxylate via clusters C and B, and potentially cluster D, to external electron acceptors regenerates the Cred1 state and closes the catalytic cycle. (12,17) In addition to cluster C, it is mainly the conserved residues around the Ni ion and the asymmetrically coordinated Fe ion (Figure 1) that could contribute to substrate activation. (25) In particular, the structures with the bound substrates, water and CO2, (12,26) indicate the stabilization of intermediates via H-bridge interactions by the residues K563 and H93.

Figure 1

Figure 1. The active site structures of CODH show (A) water bound to cluster C (PDB ID: 3B53) and (B) CO2 bound to cluster C (PDB ID: 4UDX). Bold-labeled amino acids have been exchanged and studied in this work. Cys residues coordinating the [3Fe4S] site are not shown for clarity. The alternative Fe1 position is shown as a transparent gray sphere.

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CODHs are attractive models for catalytic CO2 reduction as they catalyze the reaction with virtually no overpotential. (20,27,28) A complete chemical synthetic reconstruction of the [NiFe4(OH)(μ3-S)4] cluster is still pending, (29,30) leaving the relative contributions of the metal cluster and the protein matrix to the catalysis unresolved. The likely essential role of the protein matrix for the activity of CODHs already motivated mutation studies. (31−34) For example, in 2004, Kim et al. studied a potential proton transfer network and exchanged two residues at the CO2- and H2O-binding sites K563 and H93. (31) Contrary to what the strict conservation and central position of the residues might suggest, the activity of the enzyme variants in which one of the two residues was exchanged for alanine dropped only to about 40% of the initial activity. (31) However, since the structures of the enzyme variants were not investigated, possible compensation by structural rearrangements or restructuring of the water network could not be excluded.
Here, we use the cooS-type CODH-II from Carboxydothermus hydrogenoformans (CODH-IICh) as a model system to investigate the role of conserved residues around the substrate binding sites on the structure and function of CODHs. By exchanging all residues bold-labeled in Figure 1, we show that their exchange restructures the water network at the active site, changes the coordination and stability of cluster C, and modulates the affinity for CO. Exchanges of the conserved lysine residue particularly reduced catalytic efficiency, underpinning the central role of this residue in the stabilization of reaction intermediates and/or the transfer of protons.

Results and Discussion

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WT-CODH-IICh as Reference

As a reference for the Ni-dependent reactivity of CODHs, we determined the crystal structure of WT-CODH-IICh at a resolution (dmin) of 1.23 Å (Table S1). As none of the produced CODH variants had the full Ni content (Table 1), this reference was necessary to correlate the turnover number with the Ni content in cluster C. WT-CODH-IICh displays the same [NiFe4(OH)(μ3-S)4] cluster composition and arrangement as previously published (12) (Figure S1). The Ni ion has an occupancy of 56%, and Fe1 is observed in two positions, Fe1A (45%) with a bound OH-ligand and Fe1B (40%). Conserved residues in the second coordination sphere of cluster C occupy the positions shown in Figure 1.
Table 1. Kinetic Parameters of Wild-Type and CODH-IICh Variants
variantNi-contentkcat (s–1)akcatn (s–1)bKm (μM)kcatn/Km (s–1 M–1)
WT0.5498918318.7 ± 1.42.1 × 108
K563A0.32185620.9 ± 2.52.7 × 106
H93A0.2624393518.0 ± 1.95.2 × 107
H96D0.4020502.7 ± 0.31.9 × 107
I567L0.51725142286.8 ± 10.91.6 × 107
I567T0.5755096520.7 ± 3.24.7 × 107
I567A0.47 (0.22 + 0.25)40185312.4 ± 1.76.9 × 107
K563Hcn.d.13.8 2.7 ± 0.5 
C295Dcn.d.n.d.n.d.n.d.n.d.
a

kcatn is a normalized turnover number based on the Ni content.

b

Uncertainties from data approximation for Km are shown.

c

n.d. refers to “not detected”. Either for crystallographically determined Ni content (K563H and C295D) or for CO oxidation activity (C295D) under our assay conditions.

We determined steady-state kinetic constants for wild-type and CODH-IICh variants in the CO-oxidation reaction at 25 °C (Table 1 and Figure S2). For WT-CODH-IICh, the Michaelis constant Km of 8.7 μM for CO is similar to the values previously reported for CODH-IICh in solution (18 μM) (35) and when immobilized on an electrode (8 μM). (36) WT-CODH-IICh exhibited a CO oxidation rate (kcatn) of 1831 s–1, where kcatn is kcat normalized to the crystallographic Ni occupancy. With an Arrhenius activation energy of 40 kJ mol–1, (36) this corresponds to ∼17,211 s–1 at 70 °C, similar to what has been reported before. (35) Thus, the WT-CODH-IICh has a catalytic efficiency (keff = kcatn/Km) of 2.09 × 108 s–1 M–1 for CO oxidation (Table 1).

CO Binding Affinity

As a first step, we focused on CO binding and the potential influence of the protein matrix on CO affinity. In addition to determining the Km value for CO, we also needed an estimate for the affinity (Kd) of CO and the on/off rate constants. For this, we applied the Briggs–Haldane steady-state kinetics model with Km = (k–1 + kcat)/k+1. Employing an enzyme variant whose kcat can change but whose binding affinity for CO is held constant would allow us to obtain an estimate for Kd and the individual rate constants of CO binding. In their analysis of potential proton transfer networks in CODHs, Kim et al. (31) found that the exchange of His96 to aspartate (H96D, CODH-IICh-numbering), despite being more than 7 Å away from the active site, lowered the specific activity of the CODH from Moorella thermoacetica to 0.4%.
We created and analyzed the corresponding exchange in CODH-IICh. As expected from the earlier study, the turnover number decreased substantially, here approximately 37-fold (kcatn = 50 s–1, 2.7% of WT-CODH-IICh) (Figure S2 and Table 1).
The crystal structure of H96D-CODH-IICh revealed a cluster C structure indistinguishable from that of WT-CODH-IICh (Figure 2 and Figure S1). Residues of the first and second coordination sphere of cluster C appear unperturbed by the exchange. However, in addition to the water molecules (w1–w4) present in the WT, a new water molecule (wa) occupies the open space created by the mutation within a short H-bonding distance (2.6 Å) to the exchanged side chain (H96D, Figure 2).

Figure 2

Figure 2. Crystal structures of H96D-CODH-IICh. A stereo view of cluster C with its outer sphere environment. 2Fobs – Fcalc electron density map (gray mesh) is contoured at 1.5 σ. H-bond interactions are shown in blue lines. Atoms are color-coded: green for C, blue for N, red for O, gold for S, brown for Fe, light blue for Ni. The alternative Fe1 position is shown as a transparent gray sphere. Waters (w1–w4) are numbered as in WT-CODH-IICh (Figure S1), whereas wa indicates the newly observed water.

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In contrast to all other variants studied here, the H96D exchange does not increase but decreases the Km for CO (Km = 2.7 μM). The lower Km may seem surprising but is expected for an enzyme following the Briggs–Haldane mechanism. By lowering kcat, the Km value would need to decrease if k+1 and k–1 are unchanged. Taking the data from WT-CODH-IICh and H96D-CODH-IICh together and assuming that the rate constants (k+1 and k–1) of CO-binding to cluster C are unperturbed, we get k+1 = 2.97 × 108 M–1 s–1 and k–1 = 751 s–1. This results in a Kd = k–1/ k+1 for CO of 2.5 μM, corresponding to a binding energy of −32 kJ/mol.
The lower Km value partially compensates for the loss in kcat, resulting in a keff of 1.9 × 107 s–1 M–1, only tenfold lower than WT-CODH-IICh (Table 1). The loss of activity would support a non-redundant role for H96 in proton transfer. (31) As the earlier study was not complemented by structural analysis, not all effects of the exchange could be taken into account. Our structure shows that the H96D exchange also altered the surrounding water structure. This additional effect may add a further twist to the proton transfer not only via the side-chains but also through the water network. In addition to proton transfer, it may also interfere with water transport to the active site, as the additional water (wa) is part of a water chain ending at the OHx ligand of Fe1 (Figure 2). The wa is in short H-bonding distance to the Asp side chain, and thus, its tighter binding may reduce water mobility along the chain of water molecules from the surface to cluster C.

CO Binding Site

CODHs are characterized by long hydrophobic tunnels, supporting the transport of CO and likely CO2 as well. (8,10,37−39) A branch of these hydrophobic channels ends directly above the apical coordination site of Ni in cluster C. The end of the channel and the space around the apical binding site of Ni is shaped by the conserved side chain of Ile567 with the shortest distance of 5.4 Å between Ni and Ile567. Its crucial position suggests that Ile567 may guide the binding of CO in the CO-oxidation reaction. Notably, Ile567 is highly conserved within the CODHs (Figure S3).
In the structure of the cdh-type CODH from Methanosarcina barkeri, a residual density above Ni was modeled with a CO ligand in a bent conformation, atypical for a metal-carbonyl, (13) and a similarly bent conformation was reported for the CN-bound structure of CODH/ACSMt. (40) The authors suggested that Ile567 may enforce this bending as its side chain would sterically hinder the expected linear arrangement. A bent Ni-carbonyl could decrease π-backbonding in the metal carbonyl, rendering CO more susceptible to a nucleophilic attack by OH, and would thus increase the overall reactivity of CODHs. (41)
To test this hypothesis, we produced three variants of CODH-IICh (I567L, I567T, and I567A) and analyzed their structures and kinetics (Figure 3 and Figure S2 ). Exchange of Ile567 to leucine (I567L) or threonine (I567T) neither perturbed the structure of the side chains around cluster C nor did it decrease the Ni content compared to WT (Table 1). Two new water molecules (wb and wc) occupy the void created by the exchanges of Ile (I567L and I567T) (Figure 3A,B). The I567L variant has a side-chain torsion angle (χ1-angle) for leucine directing the side chain opposite to that of the WT isoleucine, but no changes are observed at Ni and Fe1, which keeps its OH ligand (Figure 3A). In contrast, I567T-CODH-IICh has a μ-OH ligand (we) bound between Ni (Ni-we = 2.32 Å) and Fe1A (Fe1A-we = 2.39 Å), which completes the square-planar coordination of the Ni ion (Figure 3B). While the I567T exchange only had a mild effect on the kinetics (Km = 19.8 μM, kcatn = 965 s–1), the I567L exchange increased the Km almost 10-fold (Km = 86.8 μM) but did not affect the CO-oxidation rate (kcatn = 1422 s–1) (Table 1). The increase in Km in I567L-CODH-IICh resulted in an order of magnitude decrease in keff (1.6 × 107 s–1 M–1) (Table 1). This effect is most likely due to structural hindrance originating from the substitution, as the exchange of Ile to Leu altered the branching at Cγ, restricting CO access to the Ni (Figure 4B).

Figure 3

Figure 3. Crystal structures of the CODH-IICh I567 variants. Structures of cluster C with the 2Fobs – Fcalc electron density map (gray mesh) contoured at 1.5 σ for (A) I567L-CODH-IICh, (B) I567T-CODH-IICh, and (C) I567A-CODH-IICh are shown as stereo view. Atoms are color-coded as in Figure 2. Wild-type Leu is shown as transparent sticks with red outline, and the second minor conformation of H93 in C is shown in the atom-colored transparent stick model. H-bond interactions are indicated by blue lines. While conserved waters of WT are numbered in the same order, newly observed waters are shown in alphabetic order and given equivalent names when the shift of position is less than 1.1 Å between variants.

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Figure 4

Figure 4. Restricting access to the active site. (A) WT-CODH-IICh. (12) (B) I567L-CODH-IICh. (C) I567T-CODH-IICh. (D) I567A-CODH-IICh. Residues above the Ni ion delineating the gas channel are shown with their van der Waals spheres including hydrogen atoms (white spheres). Cluster C of the WT is shown to indicate the Ni position (blue sphere). Atoms are color-coded as in Figure S1.

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Like I567T, I567A caused a significant change in the structure of cluster C. The Ni ion, which otherwise is found solely in-plane with S1, S2, and Sg-Cys526 (Figure S1), exhibits two distinct geometries: (1) NiA (22%), found in the typically observed in-plane position, and (2) NiB (25%), with an as yet unobserved trigonal pyramidal coordination with wd as an additional weak ligand. In contrast to the structural perturbation, there is only a small change in the CO oxidation kinetics. If we consider that the CO-oxidation activity is accounted for by both Ni positions, I567A-CODH-IICh has a kcatn of 853 s–1, about 50% of WT, and a Km comparable to WT (12.4 μM).

Residues at the CO2 Binding Site

Two residues are in short H-bonding distance to the CO2-bound cluster C: H93 and K563 (Figure 1). (12,26) Both are strictly conserved and, based on the H-bonding interactions and charge(s) they contribute, ought to be indispensable for catalytic CO oxidation/CO2 reduction. When we substituted H93 for the short, nonpolar, aliphatic side chain of Ala (H93A, Table 1), kcatn decreased 50% and Km doubled in comparison to WT-CODH-IICh, decreasing keff four-fold (5.2 × 107 s–1 M–1). In contrast to these small kinetic changes, the structural analysis of the H93A variant (H93A-CODH-IICh) revealed a surprising difference. The gap left by the omission of the imidazole side chain is now occupied by Lys563, which adopts two new conformations (Figure 5A). The hydroxyl ligand at Fe1 is still present but with lower occupancy than in WT-CODH-IICh.

Figure 5

Figure 5. Crystal structures of CODH-IICh variants at the CO2 binding site. Structures of (A) H93A-CODH-IICh and (B) K563A-CODH-IICh are shown in stereo view with the 2Fobs – Fcalc electron density map (gray mesh) contoured at 1.5 σ. Transparent, red-outlined sticks indicate superimposed wild-type residues. Waters conserved in WT-CODH-IICh are numbered, additional waters (wf and we) are also observed in I567T-CODH-IICh (Figure 3B). The second minor conformations of K563 in (A) and H93 in (B) are shown as transparent sticks. Atoms are color-coded as in Figure 2. H-bond interactions are indicated by blue lines.

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The exchange of K563 to alanine (K563A) decreased catalytic CO oxidation more than any of the other exchanges. The K563A variant (K563A-CODH-IICh) retained only 3% WT activity, reducing the catalytic efficiency by two orders of magnitude (keff = 2.7 × 106 s–1 M–1). Interestingly, the K563A exchange does not create any larger structural reorganization of cluster C or in its vicinity. The side chain of K563 also seems important to stabilize the OH ligand at Fe1, which in K563A is replaced by a water ligand, binding equidistant between Fe1 and Ni (H2O-Ni,Fe1 distance of 2.75 Å), thereby completing the square-planar coordination of Ni (Figure 5B).
The exchange K563A increases the transition state energy by 10.8 kJ/mol (ΔΔGES⧧ and eq S2 in the SI) (42) compared to WT-CODH-IICh (Figure 6), which would be consistent with a strong hydrogen-bonding interaction of the K563 side chain stabilizing the transition state (Figure S1).

Figure 6

Figure 6. Kinetic parameters of CODH-IICh variants. Only exchanged side chains are shown with the corresponding kinetic parameters, Km and kcat, and transition state energy difference ΔΔGES⧧ (42) for K563A.

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The exchanges of K563A and H93A are also remarkable in that they have the lowest Ni content, even though all CODH-IICh variants were produced under identical conditions. Larger differences in Ni content therefore reflect changed properties of the protein matrix. Thus, K563 and H93 may have an influence on maturation or may be important for cluster C stability. As both residues are not coordinating Ni but form H-bonds to bridging ligands such as CO2 and the weakly Ni-coordinating OH-ligand at Fe1, it is possible that these bridging ligands, by completing the square-planar coordination, are central to stabilizing the Ni ion in cluster C.
As the H93A exchange was partly compensated by a movement of the lysine side chain, we were interested to know if an exchange of the lysine for histidine (K563H) could cause a higher activity than K563A. However, K563H-CODH-IICh exhibited a CO oxidation kcat of 13.8 s–1 (∼133-fold lower than kcatn of WT-CODH-IICh). At first, it was thought to be as active as K563A-CODH-IICh and that both exchanges are similarly destructive for activity. However, when we analyzed the structure of K563H-CODH-IICh, we found that cluster C had been converted into a [Fe4S4]-like cluster (see the following section for details).

A Catalytically Active [Fe4S4] Cluster?

Substitutions of residues coordinating Fe1 at cluster C (H261 and C295) are known to cause a complete loss of CO-oxidation activity, Ni-content, and the typical EPR spectroscopic signature of cluster C, (43,44) but structures of these variants have not yet been resolved.
However, exchanges of second coordination sphere residues were, so far, not observed to convert cluster C into a [Fe4S4] cluster. Thus, the observation of a [Fe4S4]-like cluster ([Fe4(μ3-S)4] cluster) in K563H-CODH-IICh was unexpected. The presence of the [Fe4(μ3-S)4] cluster was clearly confirmed by anomalous difference Fourier maps for S and Fe (Figure 7A). The substituted H563 residue exhibited two conformations (51% in a and 49% in b) with no direct coordination to iron atoms of cluster C. Fe1a (45%) was located below the usual Ni position to form the cubane cluster, while Fe1b (55% occupancy) coordinated by C295 was positioned a bit too far (2.98 and 2.56 Å) from the S atoms (S3 and S4, respectively) to be a component of the cluster.

Figure 7

Figure 7. Transformed cluster of (A) K563H-CODH-IICh and (B) C295D-CODH-IICh shown in stereo view. 2Fobs – Fcalc electron density maps (gray mesh) were contoured at 1.5 σ. Anomalous difference Fourier maps are shown in transparent surface for Fe (cyan) with a contour of 6.0 σ and for S (purple) with a contour of 2.5 σ. Atoms are color-coded as in Figure 2. Fe atoms are numbered. Alternative positions are labeled (a) and (b). H-bonds are shown in blue lines.

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Even though no anomalous scattering contribution from Ni could be detected, K563H-CODH-IICh exhibited a kcat of 13.8 s–1. This may be taken as an indication that even a [Fe4S4] cluster in a CODH protein matrix might be able to oxidize CO. However, while K563H-CODH-IICh shows a more than 100-fold lower turnover than WT-CODH-IICh, it has a similar Km value, which would argue for a similar binding site for CO, including a Ni2+ ion, thus suggesting an intrinsic heterogeneity of our preparation with low Ni-content. This explanation is also supported by our study of the C295D-CODH-IICh variant (see below), which also shows a conversion of the [NiFe4(OH)(μ3-S)4] cluster to a [Fe4S4]-like cluster.
Cys295 coordinates Fe1 but may also act as a weak ligand of Ni. The mutations C295A and C295E in CODH-IICh have already been shown to abolish Ni incorporation into cluster C. (32) Interestingly, the exchange of Cys295 to glutamate is the hallmark of a recently discovered enzyme class closely related to CODHs but with a morphing Fe/S/O cluster in place of cluster C and is unable to catalyze either CO oxidation or CO2 reduction. (45) We created and investigated a new variant of CODH-IICh, C295D-CODH-IICh. As expected, the C295D exchange transforms the [NiFe4(OH)(μ3-S)4] cluster into a [Fe4(μ3-S)4] cluster (Figure 7B). Anomalous difference Fourier maps confirmed the positions of iron and sulfur atoms (Figure 7B) with Fe–S distances of 2.2–2.3 Å (Table S2), similar to that of a typical [Fe4S4] cluster. The [Fe4(μ3-S)4] cluster is formed by displacement of the Ni by an Fe ion (Fe1 in Figure 7B) and shows no observable anomalous scattering contribution of Ni. Additional electron density between H261 and C295D did not show any anomalous signal for S, Fe, or Ni. Thus, we modeled a water molecule, coordinating Fe1 (2.2 Å) and being in H-bonding distance to C295D (2.53 Å) and H261 (2.60 Å). Consequently, Fe1 exhibited a distorted square pyramidal rather than tetrahedral coordination. Notably, in terms of coordination and structure, the [Fe4(μ3-S)4] cluster combines features of a classical cubane-type [Fe4S4] cluster with 4× Cys-coordination and an aconitase-type [Fe4S4] cluster with 3× Cys coordination plus an open coordination site occupied by a water molecule. The distorted coordination at Fe1 together with the H-bond between the water ligand and C295D seems to allow for this compromise with 4× Cys-coordination plus an open coordination site with a water ligand. We also analyzed C295D-CODH-IICh for its ability to oxidize CO, which could not be detected. Thus, as expected, the [Fe4(μ3-S)4] cluster variant created here has either very low (below detectable levels) or no CO oxidizing activity. The C295D variant only has one major [Fe4(μ3-S)4] cluster species and is more homogeneous than its K563H counterpart. Finally, although the [Fe4S4]-like clusters of C295D-CODH-IICh and K563H-CODH-IICh are not identical, the lack of CO oxidation under our assay conditions found in C295D-CODH-IICh supports the essential role of Ni for efficient CO oxidation.

Conclusions

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We exchanged several strictly conserved residues around cluster C and observed that the activity changed to different degrees, with some of the exchanges showing only little effect on the activity. In all cases though, we observed non-local structural changes including rearrangements in the water structure around cluster C. The most dramatic changes are triggered by the K563H and C295D exchanges. Both destabilize cluster C, transforming it into a [Fe4(μ3-S)4] cluster. Interestingly, in both cases, not only is Ni lost, but so is Fe1 in exo. C295 stabilizes Fe1 in the exo-position, and its absence seems to allow the Fe ion to occupy the Ni position, forming the more stable [Fe4(μ3-S)4] cluster. Notably, while the loss of Ni results in a dramatic loss of CO oxidizing activity, we cannot exclude that the [Fe4(μ3-S)4] cluster may have some CO oxidizing activity, as recently shown for the [Fe4(μ3-S)4] cluster of NifH and VnfH. (46)
The number and variety of structural changes that we observed also underline that every exchange alters more than one functional aspect. For example, the H96D exchange does not just alter a sidechain but also the arrangement and interaction between water molecules. Although the effects of exchanging I567 on the reactivity were relatively minor and argue against a larger contribution to CO activation, we still see movements at the Ni site, where we alter the water structure and either introduce heterogeneity (I567T) or change the coordination of Ni. These changes again exclude monocausal explanations.
The role of K563 is another example of a residue likely serving more than one catalytic function. K563 was already suggested to be part of the proton transport pathway, (31) but it also is in H-bonding distance to the Ni,Fe-bound CO2, stabilizing with its positive charge the negatively charged CO22– ligand. (12,26) Another contribution of K563 to catalysis becomes apparent in the K563A structure: stabilizing the OH ligand at Fe1. The OH ligand at Fe1 is the nucleophile attacking the Ni2+-bound CO to form, after deprotonation, CO22–. (12) K563A-CODH-IICh has a water ligand instead of a hydroxy ligand, which is a substantially weaker nucleophile. It appears, therefore, that the positive charge of K563 is required to stabilize the deprotonated OH ligand at Fe1, and once K563 is removed, a neutral water ligand is favored in its position. We cannot entangle how much each of these three potential catalytic contributions of K563 contributes to the observed loss of activity.
Overall, modifications in the water structure around cluster C or leading to cluster C seem predominant among the effects. The role of water is especially noteworthy as it is a substrate, may form part of the proton wire, or may act directly as a proton donor or acceptor in the catalytic reaction. Changes in the water structure are likely linked to changes in water mobility, especially when short H-bonds are formed, as seen with the H96D exchange.
Particularly surprising are the switches in Ni coordination, especially the water/OH ligand completing the square-planar coordination, which is in contrast to the T-shaped coordination and longer Ni–OH distance observed in WT-CODH-IICh. As 3d8 ions like Ni2+ have a strong tendency to complete their square-planar coordination, a ligand at this position was expected, and a computational study suggested that its absence in the crystal structures may be due to photoreduction by exposure to intense X-ray radiation. (47) Now, we observe this ligand in two CODH variants, I567T-CODH-IICh and K563A-CODH-IICh. The crystallographic measuring protocol at the synchrotron was the same for all variants, and even WT-CODH-IICh crystals measured using the low X-ray dose of a rotating anode X-ray source have the T-shaped coordination. Thus, the presence or absence of a Ni-OH or Ni-OH2 ligand seems to depend primarily on the local environment and its control over the water arrangement. The K563 exchange likely destabilizes the Fe-OH ligand so that an upward shift of the water to become a Ni ligand is favorable. The I567T exchange leaves the Fe1-OH stabilization intact but introduces a new water molecule (we in Figure 3B) above the Ni site, which may again stabilize the Ni-OH ligand.
Our study shows how precariously the water structure at cluster C depends on the surrounding amino acids and how even seemingly small changes destabilize the cluster to such an extent that asymmetrically coordinated [Fe4(μ3-S)4] clusters can form, underscoring the plasticity of cluster C.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.2c02922.

  • Materials and methods, diffraction data collection, structure determination, structure refinement, statistics of data collection and structure refinement, Fe–S distances and angles of the [Fe4(μ3-S)4] cluster from C295D-CODH-IICh and K563H-CODH-IICh, stereo view of cluster C and its surroundings, steady state kinetics of CO oxidation, and sequence alignment (PDF)

Accession Codes

The coordinates and structure factor amplitudes of CODH-IICh variants were deposited in the Protein Data Bank under the accession names of 7ZX3 for C295D, 7ZX5 for I567T, 7ZX6 for I567L, 7ZXC for H96D, 7ZXJ for K563A, 7ZXL for H93A, 7ZXX for K563H, and 7ZY1 for I567A.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Yudhajeet Basak - Institute of Biology, Humboldt-Universität zu Berlin, Unter den Linden 6, Berlin 10099, Germany;  Orcidhttps://orcid.org/0000-0002-9547-4414
    • Jae-Hun Jeoung - Institute of Biology, Humboldt-Universität zu Berlin, Unter den Linden 6, Berlin 10099, Germany;  Orcidhttps://orcid.org/0000-0003-0546-8342
    • Lilith Domnik - Institute of Biology, Humboldt-Universität zu Berlin, Unter den Linden 6, Berlin 10099, Germany
    • Jakob Ruickoldt - Institute of Biology, Humboldt-Universität zu Berlin, Unter den Linden 6, Berlin 10099, Germany
  • Author Contributions

    Y.B. and J.-H.J. contributed equally to this paper.

    Author Contributions

    Y.B. and J.-H.J. refined crystal structures and analyzed data. L.D. carried out initial data collection. J.R. measured and analyzed kinetic data. Y.B., H.D. and J.-H.J. wrote and revised the manuscript. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We acknowledge access to beamlines of the BESSY II storage ring (Berlin) through the Joint Berlin MX-Laboratory sponsored by Helmholtz-Zentrum Berlin für Materialien und Energie, Freie Universität Berlin, Humboldt-Universität zu Berlin, Max-Delbrück-Centrum, and the Leibniz-Institut für Molekulare Pharmakologie. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2008 – 390540038 (UniSysCat) and DFG project DO 785/6-2.

Abbreviations

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CODH

Ni,Fe–carbon monoxide dehydrogenase

LUCA

last universal common ancestor

Ch

Carboxydothermus hydrogenoformans

Rr

Rhodospirillum rubrum

Mt

Moorella thermoacetica

WT

wild-type

References

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This article references 47 other publications.

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    Gong, Weimin; Hao, Bing; Wei, Zhiyi; Ferguson, Donald J., Jr.; Tallant, Thomas; Krzycki, Joseph A.; Chan, Michael K.
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (28), 9558-9563CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallog. of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidn. has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α2ε2 component from the α8β8γ8δ8ε8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane prodn. on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, resp., and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
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  14. 14
    Gencic, S.; Duin, E. C.; Grahame, D. A. Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila. J. Biol. Chem. 2010, 285, 15450– 15463,  DOI: 10.1074/jbc.M109.080994
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    14
    Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila: Acetyl c-c bond fragmentation at the a cluster promoted by protein conformational changes
    Gencic, Simonida; Duin, Evert C.; Grahame, David A.
    Journal of Biological Chemistry (2010), 285 (20), 15450-15463CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria and by the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex in Archaea. In both systems, a nickel- and Fe/S-contg. active site metal center, the A cluster, catalyzes acetyl C-C bond formation/breakdown. Carbonyl group exchange of [1-14C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO2 was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidn. to CO2 at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by anal. of three recombinant A cluster proteins, ACDS β subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACSCh), and truncated ACSCh lacking its 317-amino acid N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni+-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C-C bond fragmentation. Protein conformational changes, related to "open/closed" states previously identified crystallog., were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni2+-acetyl intermediate, controlling Ni2+-acetyl fragmentation and Ni2+(CO)(CH3) condensation. EPR spectral changes likely reflect variations in the Ni+-CO equatorial coordination environment in closed buried hydrophobic and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, vs. interdomain contacts in ACS, ensures that CO is not released from the ACDS β subunit in the absence of appropriate interactions with the α2ε2 CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO2.
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  15. 15
    Staples, C. R.; Heo, J.; Spangler, N. J.; Kerby, R. L.; Roberts, G. P.; Ludden, P. W. Rhodospirillum rubrum CO-dehydrogenase. Part 1. Spectroscopic studies of CODH variant C531A indicate the presence of a binuclear [FeNi] cluster. J. Am. Chem. Soc. 1999, 121, 11034– 11044,  DOI: 10.1021/ja990396i
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    15
    Rhodospirillum rubrum CO-Dehydrogenase. Part 1. Spectroscopic Studies of CODH Variant C531A Indicate the Presence of a Binuclear [FeNi] Cluster
    Staples, Christopher R.; Heo, Jongyun; Spangler, Nathan J.; Kerby, Robert L.; Roberts, Gary P.; Ludden, Paul W.
    Journal of the American Chemical Society (1999), 121 (48), 11034-11044CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A variant of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum was constructed by site-directed mutagenesis of the cooS gene to yield a CODH with ala in place of cys-531. This variant form of CODH (C531A) has a metal content identical to that of wild-type CODH but has an extremely slow turnover rate. Cys-531 is not essential for construction of the [Fe4S4] clusters or for incorporation of nickel. The Km for Me viologen is identical to that of wild-type CODH, but the Km for CO is approx. 30% that of wild-type CODH. The data suggest that in C531A CODH a rate-limiting step has been introduced at the point of electron transfer from the Ni site to an assocd. [Fe4S4]C cluster. Examn. of indigo carmine-poised, CO-pretreated C531A CODH revealed the presence of a paramagnetic species (g = 2.33, 2.10, 2.03; gave = 2.16), which was also obsd. in dithionite-treated samples. This species was shown to represent as much as 0.90±0.10 spins/mol., yet prodn. of the species from fully oxidized C531A CODH did not involve a concurrent decrease in the molar extinction coeff. at 420 nm, indicating that the [Fe4S4] clusters remained in the 2+ oxidn. state. 61Ni-substituted CO-pretreated C531A CODH, when poised with indigo carmine, showed no broadening of the resonances, indicating that no detectable spin d. resides upon Ni. Comparisons of the EPR spectrum of the gave = 2.16 species to Ni-C(CO) and Ni-C of Alcaligenes eutrophus [NiFe] hydrogenase are presented. On the basis of these comparisons and on the lack of 61Ni broadening, the gave = 2.16 resonance is interpreted as arising from a [(COL)Fe3+-Ni2+-H-]4+ (S = 1/2) system, where COL is an activating nonsubstrate CO ligand. On the basis of the absence of spectroscopic features present in wild-type CODH, and representing coupled forms of the putative [FeNi] cluster with a [Fe4S4], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the assocd. [Fe4S4] cluster.
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  16. 16
    Fraser, D. M.; Lindahl, P. A. Evidence for a proposed intermediate redox state in the CO/CO2 active site of acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermoaceticum. Biochemistry 1999, 38, 15706– 15711,  DOI: 10.1021/bi990398f
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    16
    Evidence for a Proposed Intermediate Redox State in the CO/CO2 Active Site of Acetyl-CoA Synthase (Carbon Monoxide Dehydrogenase) from Clostridium thermoaceticum
    Fraser, Daniel M.; Lindahl, Paul A.
    Biochemistry (1999), 38 (48), 15706-15711CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    When samples of the enzyme in the Cred1 state were reduced with Ti3+ citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded Cred2. The EPR-silent state formed within 1 min of adding Ti3+ citrate, while Cred2 formed after 60 min. Ti3+ citrate appeared to slow the rate by which Cred2 formed from Cred1 and stabilize the C-cluster in the previously proposed Cint state. This is the first strong evidence for Cint, and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti3+ citrate had a different effect on the enzyme in a CO2 atmosphere; it shifted redn. potentials of metal centers (relative to those obtained using CO) and did not stabilize Cint. Different redox behavior was also obsd. when Me viologen and benzyl viologen were used as reductants. This variability was exploited to prep. enzyme samples in which EPR from Cred2 was present without interfering signals from Bred. The satn. properties of Bred depended upon the redox state of the enzyme. Three satn. "modes", called Sat1-Sat3, were obsd. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2.04 and 1.90 resonances, and was obsd. in samples poised at slightly neg. potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly assocd. with intermediate redox states and the presence of CO2. Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was obsd. at the most neg. potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.
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  17. 17
    Lindahl, P. A. Implications of a carboxylate-bound C-cluster structure of carbon monoxide dehydrogenase. Angew. Chem., Int. Ed. 2008, 47, 4054– 4056,  DOI: 10.1002/anie.200800223
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    Implications of a carboxylate-bound C-cluster structure of carbon monoxide dehydrogenase
    Lindahl, Paul A.
    Angewandte Chemie, International Edition (2008), 47 (22), 4054-4056CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Seeing the C: Nickel-contg. carbon monoxide dehydrogenases reversibly oxidize CO to CO2 at a {[Fe3S4]:[Ni···Fea]} active site known as the C-cluster. Recently reported structures of the enzyme by Jeoung and Dobbek, including those of CO2-bound and OH-bound intermediates, shed new light on the enzyme's catalytic mechanism. This highlight describes these developments and their implications.
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  18. 18
    Grahame, D. A.; DeMoll, E. Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri. Biochemistry 1995, 34, 4617– 4624,  DOI: 10.1021/bi00014a015
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    18
    Substrate and Accessory Protein Requirements and Thermodynamics of Acetyl-CoA Synthesis and Cleavage in Methanosarcina barkeri
    Grahame, David A.; DeMoll, Edward
    Biochemistry (1995), 34 (14), 4617-24CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Enzymol. studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted to identify and characterize physiol. relevant substrates and reactions in acetyl-CoA synthesis and decompn. in methanogens. Whereas previous investigations employed carbon monoxide as substrate and reducing agent for acetyl-CoA synthesis, the authors discovered that bicarbonate (or CO2) acts as a highly efficient carbonyl group precursor substrate in the presence of either hydrogen or Ti3+·EDTA as reducing agent. In reactions with Ti3+·EDTA, synthesis of acetyl-CoA was strongly dependent on ferredoxin, and in reactions with H2, dependence on ferredoxin was abs. Two major hydrogenases were resolved from the enzyme complex prepn. by HPLC gel filtration. One of these hydrogenases was shown to be active in reconstitution of acetyl-CoA synthesis in CO2-contg. reactions with H2 as reducing agent. The hydrogenase active in reconstitution was capable of reducing ferredoxin, but was unreactive toward the 8-hydroxy-5-deazaflavin deriv. coenzyme F420. In contrast, the hydrogenase that did not reconstitute acetyl-CoA synthesis was reactive with F420 but was unable to reduce ferredoxin. Further expts. were performed in which the value of the equil. const. (Keq) was detd. for the reaction: H2 + CO2 + CH3-H4SPt + CoASH .dblharw. acetyl-CoA + H4SPt + H2O (where CH-HSPt and HSPt stand for N-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, resp.). Keq for this reaction was 2.09×106 M-1ATMH2-1 at 37°. Calcns. of thermodn. values for addnl., related reactions were made and are discussed. The findings indicate that the hydrogen partial pressure is crit. in detg. whether net synthesis or cleavage of acetyl-CoA is favored. As partial pressures of H2 drop below approx. 10-3 atm, acetyl-CoA synthesis becomes more and more unfavorable. The results support the theory that redox potential inside the cell or hydrogen availability may regulate carbon flow through the ACDS complex in methanogens.
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  19. 19
    Thauer, R. K. Energy metabolism of methanogenic bacteria. Biochim. Biophys. Acta, Bioenerg. 1990, 1018, 256– 259,  DOI: 10.1016/0005-2728(90)90261-2
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    Energy metabolism of methanogenic bacteria
    Thauer, Rudolf K.
    Biochimica et Biophysica Acta, Bioenergetics (1990), 1018 (2-3), 256-9CODEN: BBBEB4; ISSN:0005-2728.
    A review with 41 refs.
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    Lindahl, P. A.; Münck, E.; Ragsdale, S. W. CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres. J. Biol. Chem. 1990, 265, 3873– 3879,  DOI: 10.1016/S0021-9258(19)39675-9
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    20
    Carbon monoxide dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in carbon dioxide and argon atmospheres
    Lindahl, Paul A.; Munck, Eckard; Ragsdale, Stephen W.
    Journal of Biological Chemistry (1990), 265 (7), 3873-9CODEN: JBCHA3; ISSN:0021-9258.
    The EPR and redox properties of the metal clusters in carbon monoxide dehydrogenase (I) from C. thermoaceticum were studied. Controlled potential coulometric reductive titrns. of I were performed under Ar and CO2 atmospheres. In the titrns. performed under Ar, 5-8 electrons/dimer were required for redn., and 4 distinct EPR signals appeared. These included a signal with gave = 1.82 (Em ∼ -220 mV), 2 signals with the same g values but different linewidths at gave = 1.94 (Em ∼ -440 mV), and a signal at gave = 1.86 (Em ∼ -530 mV). All of the S = 1/2 EPR signals had low spin concns.; values of 0.2-0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also obsd., and these may account, at least to some degree, for the low spin concn. values. Under CO2, and at neg. potentials, I served as an electrocatalyst in the redn. of CO2 to CO. The apparent half-maximal activity for this redn. at pH 6.3 occurred at -430 mV, a potential near the thermodn. value. An EPR signal, arising from a complex contg. Ni, Fe, and the C from CO/CO2 developed along with this activity. The redn. of this complex is probably the last step to occur prior to the catalysis of CO2 redn.
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  21. 21
    Lindahl, P. A.; Ragsdale, S. W.; Münck, E. Mössbauer study of CO dehydrogenase from Clostridium thermoaceticum. J. Biol. Chem. 1990, 265, 3880– 3888,  DOI: 10.1016/S0021-9258(19)39676-0
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    21
    Moessbauer study of carbon monoxide dehydrogenase from Clostridium thermoaceticum
    Lindahl, Paul A.; Ragsdale, Stephen W.; Munck, Eckard
    Journal of Biological Chemistry (1990), 265 (7), 3880-8CODEN: JBCHA3; ISSN:0021-9258.
    Moessbauer spectroscopy was used to study the metal clusters of carbon monoxide dehydrogenase from C. thermoaceticum. At potentials of >-200 mV, all of the ∼12 Fe atoms were found to reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials, a variety of components were obsd. About 40% of the Fe appeared to belong to 1 [Fe4S4]1+ cluster. The Moessbauer spectrum (∼1.8% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65 was also obsd. Also present was a doublet (9% of Fe) with ΔEQ = 2.90 mm/s and δ = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component appeared to interact with a Ni site to form an EPR-silent complex with half-integral electronic spin. The Fe environments of the S = 1/2 NiFeC complex were also characterized. This complex contributed ∼20% of the total Moessbauer absorption when the EPR signal had ∼0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, it is speculated that the NiFeC complex may consist of a Ni site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the Moessbauer and EPR data, taken together, led to the conclusion that current prepns., whereas homogeneous according to purifications stds., are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compns. in this enzyme premature.
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  22. 22
    Spangler, N. J.; Lindahl, P. A.; Bandarian, V.; Ludden, P. W. Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum. J. Biol. Chem. 1996, 271, 7973– 7977,  DOI: 10.1074/jbc.271.14.7973
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    22
    Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum
    Spangler, Nathan J.; Lindahl, Paul A.; Bandarian, Vahe; Ludden, Paul W.
    Journal of Biological Chemistry (1996), 271 (14), 7973-7CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe4S4]2+/1+ cluster (C-center) that serves as the CO-oxidn. site and a std. [Fe4S4]2+/1+ cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in ESR (EPR) and Moessbauer studies. In this report, EPR-redox titrns. demonstrate that the fully oxidized, diamagnetic form of the C-center (Cox) undergoes a one-electron redn. to the Cred1 state (gav = 1.87) with a midpoint potential of -110 mV. The redn. of Cox to Cred1 is shown to coincide with the redn. of an [Fe4S4]2+/1+ cluster in redox-titrn. expts. monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe4S4]2+/1+ clusters, does not exhibit EPR-active states or reduced Fe4S4 clusters at potentials more pos. than -350 mV.
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  23. 23
    DeRose, V. J.; Telser, J.; Anderson, M. E.; Lindahl, P. A.; Hoffman, B. M. A multinuclear ENDOR study of the C-cluster in CO dehydrogenase from Clostridium thermoaceticum: Evidence for HxO and histidine coordination to the [Fe4S4] center. J. Am. Chem. Soc. 1998, 120, 8767– 8776,  DOI: 10.1021/ja9731480
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    23
    A Multinuclear ENDOR Study of the C-Cluster in CO Dehydrogenase from Clostridium thermoaceticum: Evidence for HxO and Histidine Coordination to the [Fe4S4] Center
    DeRose, Victoria J.; Telser, Joshua; Anderson, Mark E.; Lindahl, Paul A.; Hoffman, Brian M.
    Journal of the American Chemical Society (1998), 120 (34), 8767-8776CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The C-cluster of carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidn. of CO to form CO2. This study reports electron nuclear double resonance (ENDOR) spectroscopy of the one-electron reduced (Cred1), the CN--inhibited, and the CO (or dithionite)-reduced (Cred2) forms of the C-cluster from Clostridium thermoaceticum CODH (CODHCt). The obsd. hyperfine interactions of 1,2H, 14N, 13C, and 57Fe support and extend the current Ni-X-[Fe4S4] C-cluster model in which a [Fe4S4] center is linked to a Ni ion through a unique iron, FCII. The unpaired electron spin apparently is localized on the [Fe4S4] component of the cluster, and thus the hyperfine interactions obsd. by ENDOR most probably reflect species assocd. with that component. A solvent-exchangeable proton with a max. hyperfine coupling of A(1H) = 16 MHz is detected in the Cred1 form, but not in the CN--inhibited or Cred2 forms. The exchangeable proton is assigned to a probable solvent-derived (HxO, x = 1, 2) ligand to FCII of the Cred1 [Fe4S4]1+ center and is predicted to be a substrate in CO/CO2 catalysis. For both Cred1 and Cred2, we find ENDOR features in the region expected for a nitrogen-donor ligand which likely arise from a histidine ligand to the [Fe4S4] center. 57Fe ENDOR detects at least two classes of Fe in Cred1 that most likely arise from the (Fe2.5+)2 mixed-valence pair. Their large max. couplings of A(57Fe) > 40 MHz support the unusual nature of the cluster; these do not change dramatically between the Cred1 and Cred2 forms of the enzyme. Cred2 formed by redn. with 13CO reveals no new 13C features, strongly suggesting that neither CO nor its oxidized products are bound to the [Fe4S4] center in Cred2. Taken together, these ENDOR assignments suggest that in the Cred1 state, the unique Fe ion of the CODH C-cluster has an available coordination site that stably binds HxO or CN- and that redn. of the C-cluster results in rearrangement at that site, causing loss of the bound aq. ligand.
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    Macgregor, S. A.; Lu, Z.; Eisenstein, O.; Crabtree, R. H. Why nickel (II) binds CO best in trigonal bipyramidal and square pyramidal geometries and possible consequences for CO dehydrogenase. Inorg. Chem. 1994, 33, 3616– 3618,  DOI: 10.1021/ic00094a030
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    Why Nickel(II) Binds CO Best in Trigonal Bipyramidal and Square Pyramidal Geometries and Possible Consequences for CO Dehydrogenase
    Macgregor, Stuart A.; Lu, Zheng; Eisenstein, Odile; Crabtree, Robert H.
    Inorganic Chemistry (1994), 33 (16), 3616-18CODEN: INOCAJ; ISSN:0020-1669.
    Ni(II) is normally too weak a π-donor to bind CO, but rare examples of such species are known such as TBP [NiCl2(PMe3)2(CO)] (I). By means of Extended Huckel calcns., the authors show why (I) is able to bind CO. In contrast to other geometries typical of Ni(II), TBP has a filled high-lying M(dπ) b2 orbital and so encourages π-back donation. The distortion from ideal TBP by closing an equatorial X-Ni-X angle, due to the presence of π-donor chlorides, causes an addnl. destabilization of the filled metal dπ orbital (b2) and makes this Ni(II) an even better π-donor and capable of binding CO. These ideas suggest likely geometries and ligand types for the active site structure of the Ni enzyme, CO Dehydrogenase.
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    Terranova, U. Residues surrounding the active centre of carbon monoxide dehydrogenase are key in converting CO2 to CO. J. Biol. Inorg. Chem. 2021, 26, 617– 624,  DOI: 10.1007/s00775-021-01878-4
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    Residues surrounding the active center of carbon monoxide dehydrogenase are key in converting CO2 to CO
    Terranova, Umberto
    JBIC, Journal of Biological Inorganic Chemistry (2021), 26 (5), 617-624CODEN: JJBCFA; ISSN:0949-8257. (Springer)
    The enzyme carbon monoxide dehydrogenase is capable of efficiently converting CO2 to CO and, therefore, can enable an affordable CO2 recycling strategy. The redn. of CO2 occurs at a peculiar nickel-iron-sulfur cluster, following a mechanism that remains little understood. In this study, we have used ab initio mol. dynamics simulations to explore the free energy landscape of the reaction. We predict the existence of a COOH ligand that strongly interacts with the surrounding protein residues and favors a mechanism where a H2O mol. is eliminated before CO. We have taken advantages of the insights offered by our simulations to revisit the catalytic mechanism and the role of the residues surrounding the active center in particular, thus assisting in the design of inorg. catalysts that mimic the enzyme.
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    Fesseler, J.; Jeoung, J.-H.; Dobbek, H. How the [NiFe4S4] cluster of CO dehydrogenase activates CO2 and NCO–. Angew. Chem., Int. Ed. 2015, 54, 8560– 8564,  DOI: 10.1002/anie.201501778
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    26
    How the [NiFe4S4] Cluster of CO Dehydrogenase Activates CO2 and NCO-
    Fesseler, Jochen; Jeoung, Jae-Hun; Dobbek, Holger
    Angewandte Chemie, International Edition (2015), 54 (29), 8560-8564CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Ni,Fe-contg. CO dehydrogenases (CODHs) use a [NiFe4S4] cluster, termed cluster C, to reversibly reduce CO2 to CO with high turnover no. Binding to Ni and Fe activates CO2, but current crystal structures have insufficient resoln. to analyze the geometry of bound CO2 and reveal the extent and nature of its activation. The crystal structures of CODH in complex with CO2 and the isoelectronic inhibitor NCO- are reported at true at. resoln. (dmin≤1.1 Å). Like CO2, NCO- is a μ2,η2 ligand of the cluster and acts as a mechanism-based inhibitor. While bound CO2 has the geometry of a carboxylate group, NCO- is transformed into a carbamoyl group, thus indicating that both mols. undergo a formal two-electron redn. after binding and are stabilized by substantial π backbonding. The structures reveal the combination of stable μ2,η2 coordination by Ni and Fe2 with reductive activation as the basis for both the turnover of CO2 and inhibition by NCO-.
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  27. 27
    Parkin, A.; Seravalli, J.; Vincent, K. A.; Ragsdale, S. W.; Armstrong, F. A. Rapid and efficient electrocatalytic CO2/CO interconversions by Carboxydothermus hydrogenoformans CO dehydrogenase I on an electrode. J. Am. Chem. Soc. 2007, 129, 10328– 10329,  DOI: 10.1021/ja073643o
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    Rapid and Efficient Electrocatalytic CO2/CO Interconversions by Carboxydothermus hydrogenoformans CO Dehydrogenase I on an Electrode
    Parkin, Alison; Seravalli, Javier; Vincent, Kylie A.; Ragsdale, Stephen W.; Armstrong, Fraser A.
    Journal of the American Chemical Society (2007), 129 (34), 10328-10329CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The Ni-contg. carbon monoxide dehydrogenase I from Carboxydothermus hydrogenoformans adsorbed on a pyrolytic graphite "edge" electrode catalyzes rapid CO2/CO interconversions at the thermodn. potential.
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  28. 28
    Lazarus, O.; Woolerton, T. W.; Parkin, A.; Lukey, M. J.; Reisner, E.; Seravalli, J.; Pierce, E.; Ragsdale, S. W.; Sargent, F.; Armstrong, F. A. Water–gas shift reaction catalyzed by redox enzymes on conducting graphite platelets. J. Am. Chem. Soc. 2009, 131, 14154– 14155,  DOI: 10.1021/ja905797w
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    28
    Water-Gas Shift Reaction Catalyzed by Redox Enzymes on Conducting Graphite Platelets
    Lazarus, Oliver; Woolerton, Thomas W.; Parkin, Alison; Lukey, Michael J.; Reisner, Erwin; Seravalli, Javier; Pierce, Elizabeth; Ragsdale, Stephen W.; Sargent, Frank; Armstrong, Fraser A.
    Journal of the American Chemical Society (2009), 131 (40), 14154-14155CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The water-gas shift (WGS) reaction (CO + H2O ← CO2 + H2) is of major industrial significance in the prodn. of H2 from hydrocarbon sources. High temps. are required, typically in excess of 200 °C, using d-metal catalysts on oxide supports. In our study the WGS process is sepd. into two half-cell electrochem. reactions (H+ redn. and CO oxidn.), catalyzed by enzymes attached to a conducting particle. The H+ redn. reaction is catalyzed by a hydrogenase, Hyd-2, from Escherichia coli, and CO oxidn. is catalyzed by a carbon monoxide dehydrogenase (CODH I) from Carboxydothermus hydrogenoformans. This results in a highly efficient heterogeneous catalyst with a turnover frequency, at 30 °C, of at least 2.5 s-1 per min. functional unit (a CODH/Hyd-2 pair) which is comparable to conventional high-temp. catalysts.
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  29. 29
    Panda, R.; Zhang, Y.; McLauchlan, C. C.; Venkateswara Rao, P.; Tiago de Oliveira, F. A.; Münck, E.; Holm, R. H. Initial structure modification of tetrahedral to planar nickel(II) in a nickel–iron–sulfur cluster related to the C-cluster of carbon monoxide dehydrogenase. J. Am. Chem. Soc. 2004, 126, 6448– 6459,  DOI: 10.1021/ja030627s
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    29
    Initial Structure Modification of Tetrahedral to Planar Nickel(II) in a Nickel-Iron-Sulfur Cluster Related to the C-Cluster of Carbon Monoxide Dehydrogenase
    Panda, Rashmishree; Zhang, Yugen; McLauchlan, Craig C.; Rao, P. Venkateswara; Tiago de Oliveira, F. A.; Muenck, E.; Holm, R. H.
    Journal of the American Chemical Society (2004), 126 (20), 6448-6459CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A method was devised that creates a planar NiII site from a tetrahedral site in a NiFe3S4 cubane-type cluster. Reaction of [(Ph3P)NiFe3S4(LS3)]2- (2) with 1,2-bis(dimethylphosphino)ethane affords [(dmpe)NiFe3S4(LS3)]2- (3) (dmpe = 1,2-bis(dimethylphosphino)ethane, LS3 = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-)), isolated in ∼45% yield as (Et4N)2[3a]·2.5MeCN and (Et4N)2[3b]·0.25MeCN, both of which occur in triclinic space group P‾1. Each cryst. form contains two crystallog. inequivalent clusters with the same overall structure but slightly different dimensions. The cluster is bound by three thiolate terminal ligands to semirigid cavitand ligand LS3. The NiFe3S4 core contains three tetrahedral sites, one Fe(μ3-S)3(SR) and two Fe(μ3-S)2(μ2-S)(SR) with normal metric features, and one distorted square planar Ni(μ3-S)2P2 site in a Ni(μ3-S)2Fe face with mean bond lengths Ni-P = 2.147(9) Å and Ni-S = 2.29(2) Å. The opposite Fe2(μ3-S)(μ2-S) face places the μ2-S atom at nonbonding and variable distances (2.60-2.90 Å) above the Ni atom. Binding of the strong-field ligand dmpe results in a planar NiII site and deconstruction of the full cubane geometry. The structure approximates that established crystallog. in the C-cluster of C. Hydrogenoformans CO dehydrogenase whose NiFe4S4 core contains a planar NiS4 site and three tetrahedral FeS4 sites in a fragment that is bridged by sulfide atoms to an exo Fe atom. Mossbauer studies of polycryst. samples contg. both clusters 3a and 3b reveal at least two cluster types. The spectroscopically best defined cluster accounts for ∼54% of total Fe and exhibits hyperfine interactions quite similar to those reported for the S = 5/2 state of the protein-bound cubane-type cluster [ZnFe3S4]1+, whose Mossbauer spectrum revealed a high-spin Fe2+ site and a delocalized Fe2.5+Fe2.5+ pair. Development of reactions leading to a planar Ni and a sulfide-bridged Fe atom is requisite to attainment of a synthetic analog of this complex protein-bound cluster. This work demonstrates a tetrahedral (2) → planar (3) NiII stereochem. conversion can be effected by binding of ligands that generate a sufficiently strong in-plane ligand field.
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  30. 30
    Sun, J.; Tessier, C.; Holm, R. H. Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase. Inorg. Chem. 2007, 46, 2691– 2699,  DOI: 10.1021/ic062362z
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    30
    Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase
    Sun, Jibin; Tessier, Christian; Holm, R. H.
    Inorganic Chemistry (2007), 46 (7), 2691-2699CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    Substitution reactions at the Ni site of the cubane-type cluster [(Ph3P)NiFe3S4(LS3)]2- (2; LS3 = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-)) were studied in a synthetic approach to the C-clusters of CODH (CO dehydrogenase). Reaction of 2 with RS- or toluene-3,4-dithiolate (tdt) affords [(RS)NiFe3S4(LS3)]3- (R = Et (5), H (6)) or [(tdt)NiFe3S4(LS3)]3- (7), demonstrating that anionic S ligands can be bound at the NiII site. Clusters 5 and 6 contain tetrahedral Ni(μ3-S)3(SR) sites. Cluster 7 is of particular interest because it includes a cubanoid NiFe3(μ2-S)(μ3-S)3 core and an approx. planar Ni(tdt)(μ3-S)2 unit. The cubanoid structure is found in all C-clusters, and an NiS4-type unit is reported in C-hydrogenoformans CODH. Clusters 5/6 are formulated to contain the core [NiFe3S4]1+ ≡ Ni2+ (S = 1) + [Fe3S4]1- (S = 5/2) and 7 the core [NiFe3S4]2+ ≡ Ni2+ (S = 0) + [Fe3S4]0 (S = 2) from structure, 57Fe isomer shifts, and 1H NMR isotropic shifts. Also reported are [(EtS)CuFe3S4(LS3)]3- (9) and [Fe4S4(LS3)(tdt)]3- (11). The structures of 5-7, 9, and 11 are presented. Cluster 11, with a five-coordinate Fe(tdt)(μ3-S)3 site, provides a clear structural contrast with 7, which is currently the closest approach to a C-cluster but lacks the exo Fe atom found in the NiFe4S4,5 cores of the native clusters.
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  31. 31
    Kim, E. J.; Feng, J.; Bramlett, M. R.; Lindahl, P. A. Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases. Biochemistry 2004, 43, 5728– 5734,  DOI: 10.1021/bi036062u
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    Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases
    Kim, Eun Jin; Feng, Jian; Bramlett, Matthew R.; Lindahl, Paul A.
    Biochemistry (2004), 43 (19), 5728-5734CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidn. of CO to CO2 at a nickel-iron-sulfur active site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semiconserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. The activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or position 123. Activity was also rescued by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no ESR signals originating from the C-cluster. Electronic absorption and metal anal. suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or the stability of the C-cluster, and possibly for eliciting the redox chem. of the C-cluster required for catalytic activity.
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  32. 32
    Inoue, T.; Takao, K.; Yoshida, T.; Wada, K.; Daifuku, T.; Yoneda, Y.; Fukuyama, K.; Sako, Y. Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster. Biochem. Biophys. Res. Commun. 2013, 441, 13– 17,  DOI: 10.1016/j.bbrc.2013.09.143
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    Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster
    Inoue, Takahiro; Takao, Kyosuke; Yoshida, Takashi; Wada, Kei; Daifuku, Takashi; Yoneda, Yasuko; Fukuyama, Keiichi; Sako, Yoshihiko
    Biochemical and Biophysical Research Communications (2013), 441 (1), 13-17CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
    A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-contg. carbon monoxide dehydrogenases (CODHs). His261, which coordinates one of the Fe atoms with Cys295, is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys295, we constructed CODH-II variants. Ala substitution for the Cys295 substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addn., the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys295 indirectly and His261 together affect Ni-coordination in the C-cluster.
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  33. 33
    Wittenborn, E. C.; Cohen, S. E.; Merrouch, M.; Léger, C.; Fourmond, V.; Dementin, S.; Drennan, C. L. Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase. J. Biol. Chem. 2019, 294, 13017– 13026,  DOI: 10.1074/jbc.RA119.009610
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    Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase
    Wittenborn, Elizabeth C.; Cohen, Steven E.; Merrouch, Meriem; Leger, Christophe; Fourmond, Vincent; Dementin, Sebastien; Drennan, Catherine L.
    Journal of Biological Chemistry (2019), 294 (35), 13017-13026CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel-iron-sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2. Like other complex metalloenzymes, CODH requires dedicated assembly machinery to form the fully intact and functional C-cluster. In particular, nickel incorporation into the C-cluster depends on the maturation factor CooC; however, the mechanism of nickel insertion remains poorly understood. Here, we compare X-ray structures (1.50-2.48 Å resoln.) of CODH from Desulfovibrio vulgaris (DvCODH) heterologously expressed in either the absence (DvCODH-CooC) or presence (DvCODH+CooC) of co-expressed CooC. We find that the C-cluster of DvCODH-CooC is fully loaded with iron but does not contain any nickel. Interestingly, the so-called unique iron ion (Feu) occupies both its canonical site (80% occupancy) and the nickel site (20% occupancy), with addn. of reductant causing further mismetallation of the nickel site (60% iron occupancy). We also demonstrate that a DvCODH variant that lacks a surface-accessible iron-sulfur cluster (the D-cluster) has a C-cluster that is also replete in iron but lacks nickel, despite co-expression with CooC. In this variant, all Feu is in its canonical location, and the nickel site is empty. This D-cluster-deficient CODH is inactive despite attempts to reconstitute it with nickel. Taken together, these results suggest that an empty nickel site is not sufficient for nickel incorporation. Based on our findings, we propose a model for C-cluster assembly that requires both CooC and a functioning D-cluster, involves precise redox-state control, and includes a two-step nickel-binding process.
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    Ciaccafava, A.; Tombolelli, D.; Domnik, L.; Fesseler, J.; Jeoung, J.-H.; Dobbek, H.; Mroginski, M. A.; Zebger, I.; Hildebrandt, P. When the inhibitor tells more than the substrate: The cyanide-bound state of a carbon monoxide dehydrogenase. Chem. Sci. 2016, 7, 3162– 3171,  DOI: 10.1039/C5SC04554A
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    When the inhibitor tells more than the substrate: the cyanide-bound state of a carbon monoxide dehydrogenase
    Ciaccafava, Alexandre; Tombolelli, Daria; Domnik, Lilith; Fesseler, Jochen; Jeoung, Jae-Hun; Dobbek, Holger; Mroginski, Maria Andrea; Zebger, Ingo; Hildebrandt, Peter
    Chemical Science (2016), 7 (5), 3162-3171CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Carbon monoxide dehydrogenase (CODH) is a key enzyme for reversible CO interconversion. To elucidate structural and mechanistic details of CO binding at the CODH active site (C-cluster), cyanide is frequently used as an iso-electronic substitute and inhibitor. However, previous studies revealed conflicting results on the structure of the cyanide-bound complex and the mechanism of cyanide-inhibition. To address this issue in this work, we have employed IR spectroscopy, crystallog., site directed mutagenesis, and theor. methods to analyze the cyanide complex of the CODH from Carboxydothermus hydrogenoformans (CODHIICh). IR spectroscopy demonstrates that a single cyanide binds to the Ni ion. Whereas the inhibitor could be partially removed at elevated temp., irreversible degrdn. of the C-cluster occurred in the presence of an excess of cyanide on the long-minute time scale, eventually leading to the formation of [Fe(CN)6]4- and [Ni(CN)4]2- complexes. Theor. calcns. based on a new high-resoln. structure of the cyanide-bound CODHIICh indicated that cyanide binding to the Ni ion occurs upon dissocn. of the hydroxyl ligand from the Fe1 subsite of the C-cluster. The hydroxyl group is presumably protonated by Lys563 which, unlike to His93, does not form a hydrogen bond with the cyanide ligand. A stable deprotonated ε-amino group of Lys563 in the cyanide complex is consistent with the nearly unchanged C-N stretching in the Lys563Ala variant of CODHIICh. These findings support the view that the proton channel connecting the soln. phase with the active site displays a strict directionality, controlled by the oxidn. state of the C-cluster.
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    Svetlitchnyi, V.; Peschel, C.; Acker, G.; Meyer, O. Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans. J. Bacteriol. 2001, 183, 5134– 5144,  DOI: 10.1128/JB.183.17.5134-5144.2001
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    Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans
    Svetlitchnyi, Vitali; Peschel, Christine; Acker, Georg; Meyer, Ortwin
    Journal of Bacteriology (2001), 183 (17), 5134-5144CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit mol. masses, N-terminal sequences, peptide maps, and immunol. reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in assocn. with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H2O → CO2 + 2 e- + 2 H+. Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) μmol of CO oxidized min-1 mg-1 of protein (Me viologen, pH 8.0, 70°). The two enzymes oxidize CO very efficiently, as indicated by kcat/Km values at 70° of 1.3·109 M-1 CO s-1 (CODH I) and 1.7·109 M-1 CO s-1 (CODH II). The apparent Km values at pH 8.0 and 70° are 30 and 18 μM CO for CODH I and CODH II, resp. Acetyl CoA synthase activity is not assocd. with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers contg. 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, resp. ESR (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.
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    Domnik, L.; Merrouch, M.; Goetzl, S.; Jeoung, J.-H.; Léger, C.; Dementin, S.; Fourmond, V.; Dobbek, H. CODH-IV: A high-efficiency CO-scavenging CO dehydrogenase with resistance to O2. Angew. Chem., Int. Ed. 2017, 56, 15466– 15469,  DOI: 10.1002/anie.201709261
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    CODH-IV: A high efficiency CO-scavenging CO dehydrogenase with resistance to O2
    Domnik, Lilith; Merrouch, Meriem; Goetzl, Sebastian; Jeoung, Jae-Hun; Leger, Christophe; Dementin, Sebastien; Fourmond, Vincent; Dobbek, Holger
    Angewandte Chemie, International Edition (2017), 56 (48), 15466-15469CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Carbon monoxide dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO2. Genomic anal. previously indicated that the metabolic functions of CODHs vary. The genome of Carboxydothermus hydrogenoformans encodes 5 CODHs (CODH-I-V), of which CODH-IV is found in a gene cluster near a peroxide-reducing enzyme. Our kinetic and crystallog. expts. revealed that CODH-IV differs from other CODHs in several characteristic properties: it has a very high affinity for CO, oxidizes CO at a diffusion-limited rate over a wide range of temps., and is more tolerant to O2 than CODH-II. Thus, our observations support the idea that CODH-IV is a CO scavenger in defense against oxidative stress and highlight that CODHs are more diverse in terms of reactivity than expected.
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  37. 37
    Doukov, T. I.; Blasiak, L. C.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. Xenon in and at the end of the tunnel of bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Biochemistry 2008, 47, 3474– 3483,  DOI: 10.1021/bi702386t
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    Xenon in and at the End of the Tunnel of Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase
    Doukov, Tzanko I.; Blasiak, Leah C.; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L.
    Biochemistry (2008), 47 (11), 3474-3483CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a Me group, CoA, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermol. CO transport. Here, we report a 2.5 A resoln. structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calcn. program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid compn. surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.
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  38. 38
    Jeoung, J.-H.; Dobbek, H. n-Butyl isocyanide oxidation at the [NiFe4S4OHx] cluster of CO dehydrogenase. J. Biol. Inorg. Chem. 2012, 17, 167– 173,  DOI: 10.1007/s00775-011-0839-y
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    n-Butyl isocyanide oxidation at the [NiFe4S4OHx] cluster of CO dehydrogenase
    Jeoung, Jae-Hun; Dobbek, Holger
    JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (2), 167-173CODEN: JJBCFA; ISSN:0949-8257. (Springer)
    Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidn. of carbon monoxide by reaction with water to yield carbon dioxide, two protons, and two electrons. Two principal types of CODHs can be distinguished. Ni,Fe-contg. CODHs contain a [NiFe4S4OHx] cluster within their active site, to which the direct binding of the substrates water and carbon dioxide has been revealed by protein X-ray crystallog. N-Bu isocyanide is a slow-turnover substrate of CODHs, whose oxidn. at the active site shows several parallels to the oxidn. of carbon monoxide. Here, we report the crystal structure of CODH-II from Carboxydothermus hydrogenoformans resulting from the enzymic oxidn. of Bu isocyanide to Bu isocyanate at its active site cluster. The high resoln. of the structure (dmin = 1.28 Å) revealed Bu isocyanate bound to the active site cluster and identified a novel type of Ni-C bond in CODHs. The structure suggests the occurrence of tetrahedral in addn. to square-planar nickel complexes in product-bound states of this enzyme. Furthermore, we discovered a mol. of Bu isocyanide in a hydrophobic channel leading to the active site, revealing a unique architecture for the substrate channel of CODH-II compared with the bifunctional CODHs.
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  39. 39
    Lemaire, O. N.; Wagner, T. Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum. Biochim. Biophys. Acta, Bioenerg. 2021, 1862, 148330  DOI: 10.1016/j.bbabio.2020.148330
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    39
    Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum
    Lemaire, Olivier N.; Wagner, Tristan
    Biochimica et Biophysica Acta, Bioenergetics (2021), 1862 (1), 148330CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
    Clostridium autoethanogenum, the bacterial model for biol. conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concns. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient mol. machineries for CO-conversion. Here, we structurally and biochem. characterized the key enzyme of the CO-converting metab.: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2β2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystn. artifact. Therefore, the internal CO-channeling system, crit. to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidn. and acetyl-CoA prodn. Biochem. expts. showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidn. and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.
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    Kung, Y.; Doukov, T. I.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. Crystallographic snapshots of cyanide- and water-bound C-clusters from bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Biochemistry 2009, 48, 7432– 7440,  DOI: 10.1021/bi900574h
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    Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase
    Kung, Yan; Doukov, Tzanko I.; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L.
    Biochemistry (2009), 48 (31), 7432-7440CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Nickel-contg. carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidn. of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallog. characterized as either a NiFe4S4 or a NiFe4S5 metal center, the latter contg. a fifth, addnl. sulfide that bridges Ni and a unique Fe site. To det. whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H2O/OH- mol. and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a soln. contg. potassium cyanide. In both structures, the substrate H2O/OH- mol. exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H2O/OH- mol. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidn. by the C-cluster, the catalytic center of an environmentally important enzyme.
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    Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states
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    Journal of Biological Chemistry (1998), 273 (7), 4059-4064CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent redn. of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.
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    Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-contg. carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265 exhibits hydroxylamine reductase activity. Both Cys531 and His265 are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.
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    A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold
    Jeoung, Jae-Hun; Fesseler, Jochen; Domnik, Lilith; Klemke, Friederike; Sinnreich, Malte; Teutloff, Christian; Dobbek, Holger
    Angewandte Chemie, International Edition (2022), 61 (18), e202117000CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
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    Carbon Monoxide Dehydrogenase Reaction Mechanism: A Likely Case of Abnormal CO2 Insertion to a Ni-H- Bond
    Amara, Patricia; Mouesca, Jean-Marie; Volbeda, Anne; Fontecilla-Camps, Juan C.
    Inorganic Chemistry (2011), 50 (5), 1868-1878CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    Ni-contg. carbon monoxide dehydrogenases (CODH), present in many anaerobic microorganisms, catalyze the reversible oxidn. of CO to CO2 at the so-called C-cluster. This atypical active site is composed of a [NiFe3S4] cluster and a single unusual iron ion called ferrous component II or Feu that is bridged to the cluster via one sulfide ion. After addnl. refinement of recently published high-resoln. structures of COOHx-, OHx-, and CN-bound CODH from Carboxydothermus hydrogenoformans, we have used computational methods on the predominant resulting structures to investigate the spectroscopically well-characterized catalytic intermediates, Cred1 and the two-electron more-reduced Cred2. Several models were geometry-optimized for both states using hybrid quantum mech./mol. mech. potentials. The comparison of calcd. Mossbauer parameters of these active site models with exptl. data allows us to propose that the Cred1 state has a Feu-Ni2+ bridging hydroxide ligand and the Cred2 state has a hydride terminally bound to Ni2+. Using our combined structural and theor. data, we put forward a revised version of an earlier proposal for the catalytic cycle of Ni-contg. CODH that agrees with available spectroscopic and structural data. This mechanism involves an abnormal CO2 insertion into the Ni2+-H- bond.
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This article is cited by 2 publications.

  1. Yudhajeet Basak, Jae‐Hun Jeoung, Lilith Domnik, Holger Dobbek. Stepwise O 2 ‐Induced Rearrangement and Disassembly of the [NiFe 4 (OH)(μ 3 ‐S) 4 ] Active Site Cluster of CO Dehydrogenase. Angewandte Chemie 2023, 135 (32) https://doi.org/10.1002/ange.202305341
  2. Yudhajeet Basak, Jae‐Hun Jeoung, Lilith Domnik, Holger Dobbek. Stepwise O 2 ‐Induced Rearrangement and Disassembly of the [NiFe 4 (OH)(μ 3 ‐S) 4 ] Active Site Cluster of CO Dehydrogenase. Angewandte Chemie International Edition 2023, 62 (32) https://doi.org/10.1002/anie.202305341
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  • Abstract

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    Figure 1

    Figure 1. The active site structures of CODH show (A) water bound to cluster C (PDB ID: 3B53) and (B) CO2 bound to cluster C (PDB ID: 4UDX). Bold-labeled amino acids have been exchanged and studied in this work. Cys residues coordinating the [3Fe4S] site are not shown for clarity. The alternative Fe1 position is shown as a transparent gray sphere.

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    Figure 2

    Figure 2. Crystal structures of H96D-CODH-IICh. A stereo view of cluster C with its outer sphere environment. 2Fobs – Fcalc electron density map (gray mesh) is contoured at 1.5 σ. H-bond interactions are shown in blue lines. Atoms are color-coded: green for C, blue for N, red for O, gold for S, brown for Fe, light blue for Ni. The alternative Fe1 position is shown as a transparent gray sphere. Waters (w1–w4) are numbered as in WT-CODH-IICh (Figure S1), whereas wa indicates the newly observed water.

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    Figure 3

    Figure 3. Crystal structures of the CODH-IICh I567 variants. Structures of cluster C with the 2Fobs – Fcalc electron density map (gray mesh) contoured at 1.5 σ for (A) I567L-CODH-IICh, (B) I567T-CODH-IICh, and (C) I567A-CODH-IICh are shown as stereo view. Atoms are color-coded as in Figure 2. Wild-type Leu is shown as transparent sticks with red outline, and the second minor conformation of H93 in C is shown in the atom-colored transparent stick model. H-bond interactions are indicated by blue lines. While conserved waters of WT are numbered in the same order, newly observed waters are shown in alphabetic order and given equivalent names when the shift of position is less than 1.1 Å between variants.

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    Figure 4

    Figure 4. Restricting access to the active site. (A) WT-CODH-IICh. (12) (B) I567L-CODH-IICh. (C) I567T-CODH-IICh. (D) I567A-CODH-IICh. Residues above the Ni ion delineating the gas channel are shown with their van der Waals spheres including hydrogen atoms (white spheres). Cluster C of the WT is shown to indicate the Ni position (blue sphere). Atoms are color-coded as in Figure S1.

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    Figure 5

    Figure 5. Crystal structures of CODH-IICh variants at the CO2 binding site. Structures of (A) H93A-CODH-IICh and (B) K563A-CODH-IICh are shown in stereo view with the 2Fobs – Fcalc electron density map (gray mesh) contoured at 1.5 σ. Transparent, red-outlined sticks indicate superimposed wild-type residues. Waters conserved in WT-CODH-IICh are numbered, additional waters (wf and we) are also observed in I567T-CODH-IICh (Figure 3B). The second minor conformations of K563 in (A) and H93 in (B) are shown as transparent sticks. Atoms are color-coded as in Figure 2. H-bond interactions are indicated by blue lines.

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    Figure 6

    Figure 6. Kinetic parameters of CODH-IICh variants. Only exchanged side chains are shown with the corresponding kinetic parameters, Km and kcat, and transition state energy difference ΔΔGES⧧ (42) for K563A.

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    Figure 7

    Figure 7. Transformed cluster of (A) K563H-CODH-IICh and (B) C295D-CODH-IICh shown in stereo view. 2Fobs – Fcalc electron density maps (gray mesh) were contoured at 1.5 σ. Anomalous difference Fourier maps are shown in transparent surface for Fe (cyan) with a contour of 6.0 σ and for S (purple) with a contour of 2.5 σ. Atoms are color-coded as in Figure 2. Fe atoms are numbered. Alternative positions are labeled (a) and (b). H-bonds are shown in blue lines.

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  • References

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      A review. There are 6 known pathways by which CO2 is fixed, with the Calvin cycle and photosynthesis providing most of the fixed carbon. Under anaerobic conditions, the Wood-Ljungdahl pathway is a predominant CO2 sink, and the Ni-contg. metalloenzymes, carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), are the key enzymes in this pathway. Here, the structure, function, and catalytic mechanism of these 2 metalloenzymes, that have defined novel biochem. mechanisms involving organometallic chem. to catalyze their reactions, are discussed. CODH, esp. coupled to ACS and other enzymes of the Wood-Ljungdahl pathway, offers great potential for biotechnol. through the conversion of simple abundant compds. into needed chems. and fuels. Developing an industrial process that efficiently couples CO2 redn. to CO with a carbonylation reaction would be an important advance to the chem. industry because C-C bond formation by reactions with CO is instrumental in many industrial processes. CODH/ACS catalyze such a coupled process as an important component of the biol. carbon cycle. If fuels could be made from CO2, these C-C bond-forming reactions will be of even more importance in energy generation.
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      A review on characterization, x-ray structure, and spectroscopic and redox properties of the Ni-contg. CO dehydrogenase (CODH) family of enzymes. The complexity and functional unity of the metal center structures, tunnels, and catalytic mechanisms employed by the CODH family of enzymes are discussed.
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      Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes
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      Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabs. for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally assocd. with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large no. of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geol. times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary anal. of the WL Me branch will help resolve this issue.
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      Henstra, Anne M.; Dijkema, Cor; Stams, Alfons J. M.
      Environmental Microbiology (2007), 9 (7), 1836-1841CODEN: ENMIFM; ISSN:1462-2912. (Blackwell Publishing Ltd.)
      The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here, the authors explore the capacity of A. fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate, the culture OD660 increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the obsd. concns. of acetate (18 mM) and formate (8.2 mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the Me group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4 μmol formate oxidized min-1 mg-1) was detected in cell-free exts. and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A. fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compds. as electron acceptor.
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    6. 6
      Techtmann, S. M.; Colman, A. S.; Robb, F. T. ‘That which does not kill us only makes us stronger’: The role of carbon monoxide in thermophilic microbial consortia. Environ. Microbiol. 2009, 11, 1027– 1037,  DOI: 10.1111/j.1462-2920.2009.01865.x
      6
      'That which does not kill us only makes us stronger': the role of carbon monoxide in thermophilic microbial consortia
      Techtmann, Stephen M.; Colman, Albert S.; Robb, Frank T.
      Environmental Microbiology (2009), 11 (5), 1027-1037CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
      A review. Carbon monoxide (CO), while a potent toxin, is also a key intermediate in major autotrophic pathways such as methanogenesis and acetogenesis. The ability of purple sulfur bacteria to use CO as an energy source was first described by Uffen in 1976. The prototype extremely thermophilic carboxydotroph Carboxydothermus hydrogenoformans was described in 1991. Eight bacteria and one archaeon that utilize CO have since been isolated and described from diverse geothermal environments. They derive energy from the oxidn. of CO with water to form CO2 and H2. Most of these isolates thrive with headspace CO partial pressures around 1 atm, which is grossly elevated relative to CO concns. in geothermal effluents. To account for this, we suggest that under consortial growth conditions the carboxydotrophs occupy microniches in which biogenic CO accumulates locally to high concns. CO oxidizers dissipate these potentially toxic CO hot spots with the prodn. of H2, CO2 and acetate whose subsequent oxidn. fuels other thermophiles. The identification of genes related to anaerobic CO oxidn. in many metagenomic databases attests to widespread distribution of carboxydotrophs. Current evidence suggests that CO-oxidizing bacteria and archaea hold a vital niche in thermophilic ecosystems.
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    7. 7
      Techtmann, S. M.; Lebedinsky, A. V.; Colman, A. S.; Sokolova, T. G.; Woyke, T.; Goodwin, L.; Robb, F. T. Evidence for horizontal gene transfer of anaerobic carbon monoxide dehydrogenases. Front. Microbiol. 2012, 3, 132,  DOI: 10.3389/fmicb.2012.00132
      7
      Evidence for horizontal gene transfer of anaerobic carbon monoxide dehydrogenases
      Techtmann Stephen M; Lebedinsky Alexander V; Colman Albert S; Sokolova Tatyana G; Woyke Tanja; Goodwin Lynne; Robb Frank T
      Frontiers in microbiology (2012), 3 (), 132 ISSN:.
      Carbon monoxide (CO) is commonly known as a toxic gas, yet both cultivation studies and emerging genome sequences of bacteria and archaea establish that CO is a widely utilized microbial growth substrate. In this study, we determined the prevalence of anaerobic carbon monoxide dehydrogenases ([Ni,Fe]-CODHs) in currently available genomic sequence databases. Currently, 185 out of 2887, or 6% of sequenced bacterial and archaeal genomes possess at least one gene encoding [Ni,Fe]-CODH, the key enzyme for anaerobic CO utilization. Many genomes encode multiple copies of [Ni,Fe]-CODH genes whose functions and regulation are correlated with their associated gene clusters. The phylogenetic analysis of this extended protein family revealed six distinct clades; many clades consisted of [Ni,Fe]-CODHs that were encoded by microbes from disparate phylogenetic lineages, based on 16S rRNA sequences, and widely ranging physiology. To more clearly define if the branching patterns observed in the [Ni,Fe]-CODH trees are due to functional conservation vs. evolutionary lineage, the genomic context of the [Ni,Fe]-CODH gene clusters was examined, and superimposed on the phylogenetic trees. On the whole, there was a correlation between genomic contexts and the tree topology, but several functionally similar [Ni,Fe]-CODHs were found in different clades. In addition, some distantly related organisms have similar [Ni,Fe]-CODH genes. Thermosinus carboxydivorans was used to observe horizontal gene transfer (HGT) of [Ni,Fe]-CODH gene clusters by applying Kullback-Leibler divergence analysis methods. Divergent tetranucleotide frequency and codon usage showed that the gene cluster of T. carboxydivorans that encodes a [Ni,Fe]-CODH and an energy-converting hydrogenase is dissimilar to its whole genome but is similar to the genome of the phylogenetically distant Firmicute, Carboxydothermus hydrogenoformans. These results imply that T carboxydivorans acquired this gene cluster via HGT from a relative of C. hydrogenoformans.
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    8. 8
      Dobbek, H.; Svetlitchnyi, V.; Gremer, L.; Huber, R.; Meyer, O. Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster. Science 2001, 293, 1281– 1285,  DOI: 10.1126/science.1061500
      8
      Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster
      Dobbek, Holger; Svetlitchnyi, Vitali; Gremer, Lothar; Huber, Robert; Meyer, Ortwin
      Science (Washington, DC, United States) (2001), 293 (5533), 1281-1285CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The homodimeric Ni-contg. carbon monoxide dehydrogenase (I) from the anaerobic bacterium, Carboxydothermus hydrogenoformans, catalyzes the oxidn. of CO to CO2. Here, the crystal structure of reduced I was solved at 1.6 Å resoln. This structure represents the prototype for Ni-contg. I from anaerobic bacteria and archae. I contained 5 metal clusters, of which clusters B, B', and a subunit-bridging, surface-exposed cluster D, were cubane-type [4Fe-4S] clusters. I active site clusters C and C' were novel, asym. [Ni-4Fe-5S] clusters. The integral Ni ion, which is the likely site of CO oxidn., was coordinated by 4 S ligands with square planar geometry.
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    9. 9
      Drennan, C. L.; Heo, J.; Sintchak, M. D.; Schreiter, E.; Ludden, P. W. Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 11973– 11978,  DOI: 10.1073/pnas.211429998
      9
      Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase
      Drennan, Catherine L.; Heo, Jongyun; Sintchak, Michael D.; Schreiter, Eric; Ludden, Paul W.
      Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (21), 11973-11978CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from R. rubrum was detd. to 2.8-Å resoln. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biol. oxidn. of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a 4-metal cubane. Surprisingly, the results of anomalous dispersion data presented here suggest that the mononuclear site contains Fe and not Ni, and that the 4-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the 4-metal cluster are bridged by means of Cys-531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the 2 subunits. Each monomer is comprised of 3 domains: a helical domain at the N-terminus, an α/β (Rossmann-like) domain in the middle, and an α/β (Rossmann-like) domain at the C-terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The 2 Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biol. CO oxidn. and has broader significance for the roles of Ni and Fe in biol. systems.
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    10. 10
      Doukov, T. I.; Iverson, T. M.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/ acetyl-CoA synthase. Science 2002, 298, 567– 572,  DOI: 10.1126/science.1075843
      10
      A Ni-Fe-Cu Center in a Bifunctional Carbon Monoxide Dehydrogenase/ Acetyl-CoA Synthase
      Doukov, Tzanko I.; Iverson, Tina M.; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L.
      Science (Washington, DC, United States) (2002), 298 (5593), 567-572CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A metallocofactor contg. iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (CoA) synthase from Moorella thermoacetica (f. Clostridium thermoaceticum). Our structure at 2.2 angstrom resoln. reveals that the cofactor responsible for the assembly of acetyl-CoA contains a [Fe4S4] cubane bridged to a copper-nickel binuclear site. The presence of these three metals together in one cluster was unanticipated and suggests a newly discovered role for copper in biol. The different active sites of this bifunctional enzyme complex are connected via a channel, 138 angstroms long, that provides a conduit for carbon monoxide generated at the C-cluster on one subunit to be incorporated into acetyl-CoA at the A-cluster on the other subunit.
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    11. 11
      Darnault, C.; Volbeda, A.; Kim, E. J.; Legrand, P.; Vernède, X.; Lindahl, P. A.; Fontecilla-Camps, J. C. Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase. Nat. Struct. Mol. Biol. 2003, 10, 271– 279,  DOI: 10.1038/nsb912
      11
      Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase
      Darnault, Claudine; Volbeda, Anne; Kim, Eun Jin; Legrand, Pierre; Vernede, Xavier; Lindahl, Paul A.; Fontecilla-Camps, Juan C.
      Nature Structural Biology (2003), 10 (4), 271-279CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Group)
      The crystal structure of the tetrameric α2β2 acetyl-CoA synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 Å resoln. Surprisingly, the two α subunits display different (open and closed) conformations. Furthermore, x-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the α subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been obsd. in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters contg. two Ni ions are catalytically active.
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    12. 12
      Jeoung, J.-H.; Dobbek, H. Carbon dioxide activation at the Ni,Fe-cluster of anaerobic carbon monoxide dehydrogenase. Science 2007, 318, 1461– 1464,  DOI: 10.1126/science.1148481
      12
      Carbon Dioxide Activation at the Ni,Fe-Cluster of Anaerobic Carbon Monoxide Dehydrogenase
      Jeoung, Jae-Hun; Dobbek, Holger
      Science (Washington, DC, United States) (2007), 318 (5855), 1461-1464CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Anaerobic CO dehydrogenases catalyze the reversible oxidn. of CO to CO2 at a complex Ni-, Fe-, and S-contg. metal center called cluster C. We report crystal structures of CO dehydrogenase II from Carboxydothermus hydrogenoformans in three different states. In a reduced state, exogenous CO2 supplied in soln. is bound and reductively activated by cluster C. In the intermediate structure, CO2 acts as a bridging ligand between Ni and the asym. coordinated Fe, where it completes the square-planar coordination of the Ni ion. It replaces a water/hydroxo ligand bound to the Fe ion in the other two states. The structures define the mechanism of CO oxidn. and CO2 redn. at the Ni-Fe site of cluster C.
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    13. 13
      Gong, W.; Hao, B.; Wei, Z.; Ferguson, D. J.; Tallant, T.; Krzycki, J. A.; Chan, M. K. Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9558– 9563,  DOI: 10.1073/pnas.0800415105
      13
      Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex
      Gong, Weimin; Hao, Bing; Wei, Zhiyi; Ferguson, Donald J., Jr.; Tallant, Thomas; Krzycki, Joseph A.; Chan, Michael K.
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (28), 9558-9563CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallog. of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidn. has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α2ε2 component from the α8β8γ8δ8ε8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane prodn. on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, resp., and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
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    14. 14
      Gencic, S.; Duin, E. C.; Grahame, D. A. Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila. J. Biol. Chem. 2010, 285, 15450– 15463,  DOI: 10.1074/jbc.M109.080994
      14
      Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila: Acetyl c-c bond fragmentation at the a cluster promoted by protein conformational changes
      Gencic, Simonida; Duin, Evert C.; Grahame, David A.
      Journal of Biological Chemistry (2010), 285 (20), 15450-15463CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria and by the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex in Archaea. In both systems, a nickel- and Fe/S-contg. active site metal center, the A cluster, catalyzes acetyl C-C bond formation/breakdown. Carbonyl group exchange of [1-14C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO2 was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidn. to CO2 at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by anal. of three recombinant A cluster proteins, ACDS β subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACSCh), and truncated ACSCh lacking its 317-amino acid N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni+-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C-C bond fragmentation. Protein conformational changes, related to "open/closed" states previously identified crystallog., were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni2+-acetyl intermediate, controlling Ni2+-acetyl fragmentation and Ni2+(CO)(CH3) condensation. EPR spectral changes likely reflect variations in the Ni+-CO equatorial coordination environment in closed buried hydrophobic and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, vs. interdomain contacts in ACS, ensures that CO is not released from the ACDS β subunit in the absence of appropriate interactions with the α2ε2 CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO2.
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    15. 15
      Staples, C. R.; Heo, J.; Spangler, N. J.; Kerby, R. L.; Roberts, G. P.; Ludden, P. W. Rhodospirillum rubrum CO-dehydrogenase. Part 1. Spectroscopic studies of CODH variant C531A indicate the presence of a binuclear [FeNi] cluster. J. Am. Chem. Soc. 1999, 121, 11034– 11044,  DOI: 10.1021/ja990396i
      15
      Rhodospirillum rubrum CO-Dehydrogenase. Part 1. Spectroscopic Studies of CODH Variant C531A Indicate the Presence of a Binuclear [FeNi] Cluster
      Staples, Christopher R.; Heo, Jongyun; Spangler, Nathan J.; Kerby, Robert L.; Roberts, Gary P.; Ludden, Paul W.
      Journal of the American Chemical Society (1999), 121 (48), 11034-11044CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A variant of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum was constructed by site-directed mutagenesis of the cooS gene to yield a CODH with ala in place of cys-531. This variant form of CODH (C531A) has a metal content identical to that of wild-type CODH but has an extremely slow turnover rate. Cys-531 is not essential for construction of the [Fe4S4] clusters or for incorporation of nickel. The Km for Me viologen is identical to that of wild-type CODH, but the Km for CO is approx. 30% that of wild-type CODH. The data suggest that in C531A CODH a rate-limiting step has been introduced at the point of electron transfer from the Ni site to an assocd. [Fe4S4]C cluster. Examn. of indigo carmine-poised, CO-pretreated C531A CODH revealed the presence of a paramagnetic species (g = 2.33, 2.10, 2.03; gave = 2.16), which was also obsd. in dithionite-treated samples. This species was shown to represent as much as 0.90±0.10 spins/mol., yet prodn. of the species from fully oxidized C531A CODH did not involve a concurrent decrease in the molar extinction coeff. at 420 nm, indicating that the [Fe4S4] clusters remained in the 2+ oxidn. state. 61Ni-substituted CO-pretreated C531A CODH, when poised with indigo carmine, showed no broadening of the resonances, indicating that no detectable spin d. resides upon Ni. Comparisons of the EPR spectrum of the gave = 2.16 species to Ni-C(CO) and Ni-C of Alcaligenes eutrophus [NiFe] hydrogenase are presented. On the basis of these comparisons and on the lack of 61Ni broadening, the gave = 2.16 resonance is interpreted as arising from a [(COL)Fe3+-Ni2+-H-]4+ (S = 1/2) system, where COL is an activating nonsubstrate CO ligand. On the basis of the absence of spectroscopic features present in wild-type CODH, and representing coupled forms of the putative [FeNi] cluster with a [Fe4S4], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the assocd. [Fe4S4] cluster.
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    16. 16
      Fraser, D. M.; Lindahl, P. A. Evidence for a proposed intermediate redox state in the CO/CO2 active site of acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermoaceticum. Biochemistry 1999, 38, 15706– 15711,  DOI: 10.1021/bi990398f
      16
      Evidence for a Proposed Intermediate Redox State in the CO/CO2 Active Site of Acetyl-CoA Synthase (Carbon Monoxide Dehydrogenase) from Clostridium thermoaceticum
      Fraser, Daniel M.; Lindahl, Paul A.
      Biochemistry (1999), 38 (48), 15706-15711CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      When samples of the enzyme in the Cred1 state were reduced with Ti3+ citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded Cred2. The EPR-silent state formed within 1 min of adding Ti3+ citrate, while Cred2 formed after 60 min. Ti3+ citrate appeared to slow the rate by which Cred2 formed from Cred1 and stabilize the C-cluster in the previously proposed Cint state. This is the first strong evidence for Cint, and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti3+ citrate had a different effect on the enzyme in a CO2 atmosphere; it shifted redn. potentials of metal centers (relative to those obtained using CO) and did not stabilize Cint. Different redox behavior was also obsd. when Me viologen and benzyl viologen were used as reductants. This variability was exploited to prep. enzyme samples in which EPR from Cred2 was present without interfering signals from Bred. The satn. properties of Bred depended upon the redox state of the enzyme. Three satn. "modes", called Sat1-Sat3, were obsd. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2.04 and 1.90 resonances, and was obsd. in samples poised at slightly neg. potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly assocd. with intermediate redox states and the presence of CO2. Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was obsd. at the most neg. potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.
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    17. 17
      Lindahl, P. A. Implications of a carboxylate-bound C-cluster structure of carbon monoxide dehydrogenase. Angew. Chem., Int. Ed. 2008, 47, 4054– 4056,  DOI: 10.1002/anie.200800223
      17
      Implications of a carboxylate-bound C-cluster structure of carbon monoxide dehydrogenase
      Lindahl, Paul A.
      Angewandte Chemie, International Edition (2008), 47 (22), 4054-4056CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Seeing the C: Nickel-contg. carbon monoxide dehydrogenases reversibly oxidize CO to CO2 at a {[Fe3S4]:[Ni···Fea]} active site known as the C-cluster. Recently reported structures of the enzyme by Jeoung and Dobbek, including those of CO2-bound and OH-bound intermediates, shed new light on the enzyme's catalytic mechanism. This highlight describes these developments and their implications.
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    18. 18
      Grahame, D. A.; DeMoll, E. Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri. Biochemistry 1995, 34, 4617– 4624,  DOI: 10.1021/bi00014a015
      18
      Substrate and Accessory Protein Requirements and Thermodynamics of Acetyl-CoA Synthesis and Cleavage in Methanosarcina barkeri
      Grahame, David A.; DeMoll, Edward
      Biochemistry (1995), 34 (14), 4617-24CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Enzymol. studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted to identify and characterize physiol. relevant substrates and reactions in acetyl-CoA synthesis and decompn. in methanogens. Whereas previous investigations employed carbon monoxide as substrate and reducing agent for acetyl-CoA synthesis, the authors discovered that bicarbonate (or CO2) acts as a highly efficient carbonyl group precursor substrate in the presence of either hydrogen or Ti3+·EDTA as reducing agent. In reactions with Ti3+·EDTA, synthesis of acetyl-CoA was strongly dependent on ferredoxin, and in reactions with H2, dependence on ferredoxin was abs. Two major hydrogenases were resolved from the enzyme complex prepn. by HPLC gel filtration. One of these hydrogenases was shown to be active in reconstitution of acetyl-CoA synthesis in CO2-contg. reactions with H2 as reducing agent. The hydrogenase active in reconstitution was capable of reducing ferredoxin, but was unreactive toward the 8-hydroxy-5-deazaflavin deriv. coenzyme F420. In contrast, the hydrogenase that did not reconstitute acetyl-CoA synthesis was reactive with F420 but was unable to reduce ferredoxin. Further expts. were performed in which the value of the equil. const. (Keq) was detd. for the reaction: H2 + CO2 + CH3-H4SPt + CoASH .dblharw. acetyl-CoA + H4SPt + H2O (where CH-HSPt and HSPt stand for N-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, resp.). Keq for this reaction was 2.09×106 M-1ATMH2-1 at 37°. Calcns. of thermodn. values for addnl., related reactions were made and are discussed. The findings indicate that the hydrogen partial pressure is crit. in detg. whether net synthesis or cleavage of acetyl-CoA is favored. As partial pressures of H2 drop below approx. 10-3 atm, acetyl-CoA synthesis becomes more and more unfavorable. The results support the theory that redox potential inside the cell or hydrogen availability may regulate carbon flow through the ACDS complex in methanogens.
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    19. 19
      Thauer, R. K. Energy metabolism of methanogenic bacteria. Biochim. Biophys. Acta, Bioenerg. 1990, 1018, 256– 259,  DOI: 10.1016/0005-2728(90)90261-2
      19
      Energy metabolism of methanogenic bacteria
      Thauer, Rudolf K.
      Biochimica et Biophysica Acta, Bioenergetics (1990), 1018 (2-3), 256-9CODEN: BBBEB4; ISSN:0005-2728.
      A review with 41 refs.
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    20. 20
      Lindahl, P. A.; Münck, E.; Ragsdale, S. W. CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres. J. Biol. Chem. 1990, 265, 3873– 3879,  DOI: 10.1016/S0021-9258(19)39675-9
      20
      Carbon monoxide dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in carbon dioxide and argon atmospheres
      Lindahl, Paul A.; Munck, Eckard; Ragsdale, Stephen W.
      Journal of Biological Chemistry (1990), 265 (7), 3873-9CODEN: JBCHA3; ISSN:0021-9258.
      The EPR and redox properties of the metal clusters in carbon monoxide dehydrogenase (I) from C. thermoaceticum were studied. Controlled potential coulometric reductive titrns. of I were performed under Ar and CO2 atmospheres. In the titrns. performed under Ar, 5-8 electrons/dimer were required for redn., and 4 distinct EPR signals appeared. These included a signal with gave = 1.82 (Em ∼ -220 mV), 2 signals with the same g values but different linewidths at gave = 1.94 (Em ∼ -440 mV), and a signal at gave = 1.86 (Em ∼ -530 mV). All of the S = 1/2 EPR signals had low spin concns.; values of 0.2-0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also obsd., and these may account, at least to some degree, for the low spin concn. values. Under CO2, and at neg. potentials, I served as an electrocatalyst in the redn. of CO2 to CO. The apparent half-maximal activity for this redn. at pH 6.3 occurred at -430 mV, a potential near the thermodn. value. An EPR signal, arising from a complex contg. Ni, Fe, and the C from CO/CO2 developed along with this activity. The redn. of this complex is probably the last step to occur prior to the catalysis of CO2 redn.
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    21. 21
      Lindahl, P. A.; Ragsdale, S. W.; Münck, E. Mössbauer study of CO dehydrogenase from Clostridium thermoaceticum. J. Biol. Chem. 1990, 265, 3880– 3888,  DOI: 10.1016/S0021-9258(19)39676-0
      21
      Moessbauer study of carbon monoxide dehydrogenase from Clostridium thermoaceticum
      Lindahl, Paul A.; Ragsdale, Stephen W.; Munck, Eckard
      Journal of Biological Chemistry (1990), 265 (7), 3880-8CODEN: JBCHA3; ISSN:0021-9258.
      Moessbauer spectroscopy was used to study the metal clusters of carbon monoxide dehydrogenase from C. thermoaceticum. At potentials of >-200 mV, all of the ∼12 Fe atoms were found to reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials, a variety of components were obsd. About 40% of the Fe appeared to belong to 1 [Fe4S4]1+ cluster. The Moessbauer spectrum (∼1.8% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65 was also obsd. Also present was a doublet (9% of Fe) with ΔEQ = 2.90 mm/s and δ = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component appeared to interact with a Ni site to form an EPR-silent complex with half-integral electronic spin. The Fe environments of the S = 1/2 NiFeC complex were also characterized. This complex contributed ∼20% of the total Moessbauer absorption when the EPR signal had ∼0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, it is speculated that the NiFeC complex may consist of a Ni site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the Moessbauer and EPR data, taken together, led to the conclusion that current prepns., whereas homogeneous according to purifications stds., are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compns. in this enzyme premature.
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    22. 22
      Spangler, N. J.; Lindahl, P. A.; Bandarian, V.; Ludden, P. W. Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum. J. Biol. Chem. 1996, 271, 7973– 7977,  DOI: 10.1074/jbc.271.14.7973
      22
      Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum
      Spangler, Nathan J.; Lindahl, Paul A.; Bandarian, Vahe; Ludden, Paul W.
      Journal of Biological Chemistry (1996), 271 (14), 7973-7CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe4S4]2+/1+ cluster (C-center) that serves as the CO-oxidn. site and a std. [Fe4S4]2+/1+ cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in ESR (EPR) and Moessbauer studies. In this report, EPR-redox titrns. demonstrate that the fully oxidized, diamagnetic form of the C-center (Cox) undergoes a one-electron redn. to the Cred1 state (gav = 1.87) with a midpoint potential of -110 mV. The redn. of Cox to Cred1 is shown to coincide with the redn. of an [Fe4S4]2+/1+ cluster in redox-titrn. expts. monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe4S4]2+/1+ clusters, does not exhibit EPR-active states or reduced Fe4S4 clusters at potentials more pos. than -350 mV.
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    23. 23
      DeRose, V. J.; Telser, J.; Anderson, M. E.; Lindahl, P. A.; Hoffman, B. M. A multinuclear ENDOR study of the C-cluster in CO dehydrogenase from Clostridium thermoaceticum: Evidence for HxO and histidine coordination to the [Fe4S4] center. J. Am. Chem. Soc. 1998, 120, 8767– 8776,  DOI: 10.1021/ja9731480
      23
      A Multinuclear ENDOR Study of the C-Cluster in CO Dehydrogenase from Clostridium thermoaceticum: Evidence for HxO and Histidine Coordination to the [Fe4S4] Center
      DeRose, Victoria J.; Telser, Joshua; Anderson, Mark E.; Lindahl, Paul A.; Hoffman, Brian M.
      Journal of the American Chemical Society (1998), 120 (34), 8767-8776CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The C-cluster of carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidn. of CO to form CO2. This study reports electron nuclear double resonance (ENDOR) spectroscopy of the one-electron reduced (Cred1), the CN--inhibited, and the CO (or dithionite)-reduced (Cred2) forms of the C-cluster from Clostridium thermoaceticum CODH (CODHCt). The obsd. hyperfine interactions of 1,2H, 14N, 13C, and 57Fe support and extend the current Ni-X-[Fe4S4] C-cluster model in which a [Fe4S4] center is linked to a Ni ion through a unique iron, FCII. The unpaired electron spin apparently is localized on the [Fe4S4] component of the cluster, and thus the hyperfine interactions obsd. by ENDOR most probably reflect species assocd. with that component. A solvent-exchangeable proton with a max. hyperfine coupling of A(1H) = 16 MHz is detected in the Cred1 form, but not in the CN--inhibited or Cred2 forms. The exchangeable proton is assigned to a probable solvent-derived (HxO, x = 1, 2) ligand to FCII of the Cred1 [Fe4S4]1+ center and is predicted to be a substrate in CO/CO2 catalysis. For both Cred1 and Cred2, we find ENDOR features in the region expected for a nitrogen-donor ligand which likely arise from a histidine ligand to the [Fe4S4] center. 57Fe ENDOR detects at least two classes of Fe in Cred1 that most likely arise from the (Fe2.5+)2 mixed-valence pair. Their large max. couplings of A(57Fe) > 40 MHz support the unusual nature of the cluster; these do not change dramatically between the Cred1 and Cred2 forms of the enzyme. Cred2 formed by redn. with 13CO reveals no new 13C features, strongly suggesting that neither CO nor its oxidized products are bound to the [Fe4S4] center in Cred2. Taken together, these ENDOR assignments suggest that in the Cred1 state, the unique Fe ion of the CODH C-cluster has an available coordination site that stably binds HxO or CN- and that redn. of the C-cluster results in rearrangement at that site, causing loss of the bound aq. ligand.
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    24. 24
      Macgregor, S. A.; Lu, Z.; Eisenstein, O.; Crabtree, R. H. Why nickel (II) binds CO best in trigonal bipyramidal and square pyramidal geometries and possible consequences for CO dehydrogenase. Inorg. Chem. 1994, 33, 3616– 3618,  DOI: 10.1021/ic00094a030
      24
      Why Nickel(II) Binds CO Best in Trigonal Bipyramidal and Square Pyramidal Geometries and Possible Consequences for CO Dehydrogenase
      Macgregor, Stuart A.; Lu, Zheng; Eisenstein, Odile; Crabtree, Robert H.
      Inorganic Chemistry (1994), 33 (16), 3616-18CODEN: INOCAJ; ISSN:0020-1669.
      Ni(II) is normally too weak a π-donor to bind CO, but rare examples of such species are known such as TBP [NiCl2(PMe3)2(CO)] (I). By means of Extended Huckel calcns., the authors show why (I) is able to bind CO. In contrast to other geometries typical of Ni(II), TBP has a filled high-lying M(dπ) b2 orbital and so encourages π-back donation. The distortion from ideal TBP by closing an equatorial X-Ni-X angle, due to the presence of π-donor chlorides, causes an addnl. destabilization of the filled metal dπ orbital (b2) and makes this Ni(II) an even better π-donor and capable of binding CO. These ideas suggest likely geometries and ligand types for the active site structure of the Ni enzyme, CO Dehydrogenase.
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    25. 25
      Terranova, U. Residues surrounding the active centre of carbon monoxide dehydrogenase are key in converting CO2 to CO. J. Biol. Inorg. Chem. 2021, 26, 617– 624,  DOI: 10.1007/s00775-021-01878-4
      25
      Residues surrounding the active center of carbon monoxide dehydrogenase are key in converting CO2 to CO
      Terranova, Umberto
      JBIC, Journal of Biological Inorganic Chemistry (2021), 26 (5), 617-624CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      The enzyme carbon monoxide dehydrogenase is capable of efficiently converting CO2 to CO and, therefore, can enable an affordable CO2 recycling strategy. The redn. of CO2 occurs at a peculiar nickel-iron-sulfur cluster, following a mechanism that remains little understood. In this study, we have used ab initio mol. dynamics simulations to explore the free energy landscape of the reaction. We predict the existence of a COOH ligand that strongly interacts with the surrounding protein residues and favors a mechanism where a H2O mol. is eliminated before CO. We have taken advantages of the insights offered by our simulations to revisit the catalytic mechanism and the role of the residues surrounding the active center in particular, thus assisting in the design of inorg. catalysts that mimic the enzyme.
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    26. 26
      Fesseler, J.; Jeoung, J.-H.; Dobbek, H. How the [NiFe4S4] cluster of CO dehydrogenase activates CO2 and NCO–. Angew. Chem., Int. Ed. 2015, 54, 8560– 8564,  DOI: 10.1002/anie.201501778
      26
      How the [NiFe4S4] Cluster of CO Dehydrogenase Activates CO2 and NCO-
      Fesseler, Jochen; Jeoung, Jae-Hun; Dobbek, Holger
      Angewandte Chemie, International Edition (2015), 54 (29), 8560-8564CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Ni,Fe-contg. CO dehydrogenases (CODHs) use a [NiFe4S4] cluster, termed cluster C, to reversibly reduce CO2 to CO with high turnover no. Binding to Ni and Fe activates CO2, but current crystal structures have insufficient resoln. to analyze the geometry of bound CO2 and reveal the extent and nature of its activation. The crystal structures of CODH in complex with CO2 and the isoelectronic inhibitor NCO- are reported at true at. resoln. (dmin≤1.1 Å). Like CO2, NCO- is a μ2,η2 ligand of the cluster and acts as a mechanism-based inhibitor. While bound CO2 has the geometry of a carboxylate group, NCO- is transformed into a carbamoyl group, thus indicating that both mols. undergo a formal two-electron redn. after binding and are stabilized by substantial π backbonding. The structures reveal the combination of stable μ2,η2 coordination by Ni and Fe2 with reductive activation as the basis for both the turnover of CO2 and inhibition by NCO-.
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    27. 27
      Parkin, A.; Seravalli, J.; Vincent, K. A.; Ragsdale, S. W.; Armstrong, F. A. Rapid and efficient electrocatalytic CO2/CO interconversions by Carboxydothermus hydrogenoformans CO dehydrogenase I on an electrode. J. Am. Chem. Soc. 2007, 129, 10328– 10329,  DOI: 10.1021/ja073643o
      27
      Rapid and Efficient Electrocatalytic CO2/CO Interconversions by Carboxydothermus hydrogenoformans CO Dehydrogenase I on an Electrode
      Parkin, Alison; Seravalli, Javier; Vincent, Kylie A.; Ragsdale, Stephen W.; Armstrong, Fraser A.
      Journal of the American Chemical Society (2007), 129 (34), 10328-10329CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The Ni-contg. carbon monoxide dehydrogenase I from Carboxydothermus hydrogenoformans adsorbed on a pyrolytic graphite "edge" electrode catalyzes rapid CO2/CO interconversions at the thermodn. potential.
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    28. 28
      Lazarus, O.; Woolerton, T. W.; Parkin, A.; Lukey, M. J.; Reisner, E.; Seravalli, J.; Pierce, E.; Ragsdale, S. W.; Sargent, F.; Armstrong, F. A. Water–gas shift reaction catalyzed by redox enzymes on conducting graphite platelets. J. Am. Chem. Soc. 2009, 131, 14154– 14155,  DOI: 10.1021/ja905797w
      28
      Water-Gas Shift Reaction Catalyzed by Redox Enzymes on Conducting Graphite Platelets
      Lazarus, Oliver; Woolerton, Thomas W.; Parkin, Alison; Lukey, Michael J.; Reisner, Erwin; Seravalli, Javier; Pierce, Elizabeth; Ragsdale, Stephen W.; Sargent, Frank; Armstrong, Fraser A.
      Journal of the American Chemical Society (2009), 131 (40), 14154-14155CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The water-gas shift (WGS) reaction (CO + H2O ← CO2 + H2) is of major industrial significance in the prodn. of H2 from hydrocarbon sources. High temps. are required, typically in excess of 200 °C, using d-metal catalysts on oxide supports. In our study the WGS process is sepd. into two half-cell electrochem. reactions (H+ redn. and CO oxidn.), catalyzed by enzymes attached to a conducting particle. The H+ redn. reaction is catalyzed by a hydrogenase, Hyd-2, from Escherichia coli, and CO oxidn. is catalyzed by a carbon monoxide dehydrogenase (CODH I) from Carboxydothermus hydrogenoformans. This results in a highly efficient heterogeneous catalyst with a turnover frequency, at 30 °C, of at least 2.5 s-1 per min. functional unit (a CODH/Hyd-2 pair) which is comparable to conventional high-temp. catalysts.
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    29. 29
      Panda, R.; Zhang, Y.; McLauchlan, C. C.; Venkateswara Rao, P.; Tiago de Oliveira, F. A.; Münck, E.; Holm, R. H. Initial structure modification of tetrahedral to planar nickel(II) in a nickel–iron–sulfur cluster related to the C-cluster of carbon monoxide dehydrogenase. J. Am. Chem. Soc. 2004, 126, 6448– 6459,  DOI: 10.1021/ja030627s
      29
      Initial Structure Modification of Tetrahedral to Planar Nickel(II) in a Nickel-Iron-Sulfur Cluster Related to the C-Cluster of Carbon Monoxide Dehydrogenase
      Panda, Rashmishree; Zhang, Yugen; McLauchlan, Craig C.; Rao, P. Venkateswara; Tiago de Oliveira, F. A.; Muenck, E.; Holm, R. H.
      Journal of the American Chemical Society (2004), 126 (20), 6448-6459CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A method was devised that creates a planar NiII site from a tetrahedral site in a NiFe3S4 cubane-type cluster. Reaction of [(Ph3P)NiFe3S4(LS3)]2- (2) with 1,2-bis(dimethylphosphino)ethane affords [(dmpe)NiFe3S4(LS3)]2- (3) (dmpe = 1,2-bis(dimethylphosphino)ethane, LS3 = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-)), isolated in ∼45% yield as (Et4N)2[3a]·2.5MeCN and (Et4N)2[3b]·0.25MeCN, both of which occur in triclinic space group P‾1. Each cryst. form contains two crystallog. inequivalent clusters with the same overall structure but slightly different dimensions. The cluster is bound by three thiolate terminal ligands to semirigid cavitand ligand LS3. The NiFe3S4 core contains three tetrahedral sites, one Fe(μ3-S)3(SR) and two Fe(μ3-S)2(μ2-S)(SR) with normal metric features, and one distorted square planar Ni(μ3-S)2P2 site in a Ni(μ3-S)2Fe face with mean bond lengths Ni-P = 2.147(9) Å and Ni-S = 2.29(2) Å. The opposite Fe2(μ3-S)(μ2-S) face places the μ2-S atom at nonbonding and variable distances (2.60-2.90 Å) above the Ni atom. Binding of the strong-field ligand dmpe results in a planar NiII site and deconstruction of the full cubane geometry. The structure approximates that established crystallog. in the C-cluster of C. Hydrogenoformans CO dehydrogenase whose NiFe4S4 core contains a planar NiS4 site and three tetrahedral FeS4 sites in a fragment that is bridged by sulfide atoms to an exo Fe atom. Mossbauer studies of polycryst. samples contg. both clusters 3a and 3b reveal at least two cluster types. The spectroscopically best defined cluster accounts for ∼54% of total Fe and exhibits hyperfine interactions quite similar to those reported for the S = 5/2 state of the protein-bound cubane-type cluster [ZnFe3S4]1+, whose Mossbauer spectrum revealed a high-spin Fe2+ site and a delocalized Fe2.5+Fe2.5+ pair. Development of reactions leading to a planar Ni and a sulfide-bridged Fe atom is requisite to attainment of a synthetic analog of this complex protein-bound cluster. This work demonstrates a tetrahedral (2) → planar (3) NiII stereochem. conversion can be effected by binding of ligands that generate a sufficiently strong in-plane ligand field.
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    30. 30
      Sun, J.; Tessier, C.; Holm, R. H. Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase. Inorg. Chem. 2007, 46, 2691– 2699,  DOI: 10.1021/ic062362z
      30
      Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase
      Sun, Jibin; Tessier, Christian; Holm, R. H.
      Inorganic Chemistry (2007), 46 (7), 2691-2699CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      Substitution reactions at the Ni site of the cubane-type cluster [(Ph3P)NiFe3S4(LS3)]2- (2; LS3 = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-)) were studied in a synthetic approach to the C-clusters of CODH (CO dehydrogenase). Reaction of 2 with RS- or toluene-3,4-dithiolate (tdt) affords [(RS)NiFe3S4(LS3)]3- (R = Et (5), H (6)) or [(tdt)NiFe3S4(LS3)]3- (7), demonstrating that anionic S ligands can be bound at the NiII site. Clusters 5 and 6 contain tetrahedral Ni(μ3-S)3(SR) sites. Cluster 7 is of particular interest because it includes a cubanoid NiFe3(μ2-S)(μ3-S)3 core and an approx. planar Ni(tdt)(μ3-S)2 unit. The cubanoid structure is found in all C-clusters, and an NiS4-type unit is reported in C-hydrogenoformans CODH. Clusters 5/6 are formulated to contain the core [NiFe3S4]1+ ≡ Ni2+ (S = 1) + [Fe3S4]1- (S = 5/2) and 7 the core [NiFe3S4]2+ ≡ Ni2+ (S = 0) + [Fe3S4]0 (S = 2) from structure, 57Fe isomer shifts, and 1H NMR isotropic shifts. Also reported are [(EtS)CuFe3S4(LS3)]3- (9) and [Fe4S4(LS3)(tdt)]3- (11). The structures of 5-7, 9, and 11 are presented. Cluster 11, with a five-coordinate Fe(tdt)(μ3-S)3 site, provides a clear structural contrast with 7, which is currently the closest approach to a C-cluster but lacks the exo Fe atom found in the NiFe4S4,5 cores of the native clusters.
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    31. 31
      Kim, E. J.; Feng, J.; Bramlett, M. R.; Lindahl, P. A. Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases. Biochemistry 2004, 43, 5728– 5734,  DOI: 10.1021/bi036062u
      31
      Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases
      Kim, Eun Jin; Feng, Jian; Bramlett, Matthew R.; Lindahl, Paul A.
      Biochemistry (2004), 43 (19), 5728-5734CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidn. of CO to CO2 at a nickel-iron-sulfur active site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semiconserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. The activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or position 123. Activity was also rescued by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no ESR signals originating from the C-cluster. Electronic absorption and metal anal. suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or the stability of the C-cluster, and possibly for eliciting the redox chem. of the C-cluster required for catalytic activity.
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    32. 32
      Inoue, T.; Takao, K.; Yoshida, T.; Wada, K.; Daifuku, T.; Yoneda, Y.; Fukuyama, K.; Sako, Y. Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster. Biochem. Biophys. Res. Commun. 2013, 441, 13– 17,  DOI: 10.1016/j.bbrc.2013.09.143
      32
      Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster
      Inoue, Takahiro; Takao, Kyosuke; Yoshida, Takashi; Wada, Kei; Daifuku, Takashi; Yoneda, Yasuko; Fukuyama, Keiichi; Sako, Yoshihiko
      Biochemical and Biophysical Research Communications (2013), 441 (1), 13-17CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
      A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-contg. carbon monoxide dehydrogenases (CODHs). His261, which coordinates one of the Fe atoms with Cys295, is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys295, we constructed CODH-II variants. Ala substitution for the Cys295 substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addn., the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys295 indirectly and His261 together affect Ni-coordination in the C-cluster.
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    33. 33
      Wittenborn, E. C.; Cohen, S. E.; Merrouch, M.; Léger, C.; Fourmond, V.; Dementin, S.; Drennan, C. L. Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase. J. Biol. Chem. 2019, 294, 13017– 13026,  DOI: 10.1074/jbc.RA119.009610
      33
      Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase
      Wittenborn, Elizabeth C.; Cohen, Steven E.; Merrouch, Meriem; Leger, Christophe; Fourmond, Vincent; Dementin, Sebastien; Drennan, Catherine L.
      Journal of Biological Chemistry (2019), 294 (35), 13017-13026CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel-iron-sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2. Like other complex metalloenzymes, CODH requires dedicated assembly machinery to form the fully intact and functional C-cluster. In particular, nickel incorporation into the C-cluster depends on the maturation factor CooC; however, the mechanism of nickel insertion remains poorly understood. Here, we compare X-ray structures (1.50-2.48 Å resoln.) of CODH from Desulfovibrio vulgaris (DvCODH) heterologously expressed in either the absence (DvCODH-CooC) or presence (DvCODH+CooC) of co-expressed CooC. We find that the C-cluster of DvCODH-CooC is fully loaded with iron but does not contain any nickel. Interestingly, the so-called unique iron ion (Feu) occupies both its canonical site (80% occupancy) and the nickel site (20% occupancy), with addn. of reductant causing further mismetallation of the nickel site (60% iron occupancy). We also demonstrate that a DvCODH variant that lacks a surface-accessible iron-sulfur cluster (the D-cluster) has a C-cluster that is also replete in iron but lacks nickel, despite co-expression with CooC. In this variant, all Feu is in its canonical location, and the nickel site is empty. This D-cluster-deficient CODH is inactive despite attempts to reconstitute it with nickel. Taken together, these results suggest that an empty nickel site is not sufficient for nickel incorporation. Based on our findings, we propose a model for C-cluster assembly that requires both CooC and a functioning D-cluster, involves precise redox-state control, and includes a two-step nickel-binding process.
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    34. 34
      Ciaccafava, A.; Tombolelli, D.; Domnik, L.; Fesseler, J.; Jeoung, J.-H.; Dobbek, H.; Mroginski, M. A.; Zebger, I.; Hildebrandt, P. When the inhibitor tells more than the substrate: The cyanide-bound state of a carbon monoxide dehydrogenase. Chem. Sci. 2016, 7, 3162– 3171,  DOI: 10.1039/C5SC04554A
      34
      When the inhibitor tells more than the substrate: the cyanide-bound state of a carbon monoxide dehydrogenase
      Ciaccafava, Alexandre; Tombolelli, Daria; Domnik, Lilith; Fesseler, Jochen; Jeoung, Jae-Hun; Dobbek, Holger; Mroginski, Maria Andrea; Zebger, Ingo; Hildebrandt, Peter
      Chemical Science (2016), 7 (5), 3162-3171CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Carbon monoxide dehydrogenase (CODH) is a key enzyme for reversible CO interconversion. To elucidate structural and mechanistic details of CO binding at the CODH active site (C-cluster), cyanide is frequently used as an iso-electronic substitute and inhibitor. However, previous studies revealed conflicting results on the structure of the cyanide-bound complex and the mechanism of cyanide-inhibition. To address this issue in this work, we have employed IR spectroscopy, crystallog., site directed mutagenesis, and theor. methods to analyze the cyanide complex of the CODH from Carboxydothermus hydrogenoformans (CODHIICh). IR spectroscopy demonstrates that a single cyanide binds to the Ni ion. Whereas the inhibitor could be partially removed at elevated temp., irreversible degrdn. of the C-cluster occurred in the presence of an excess of cyanide on the long-minute time scale, eventually leading to the formation of [Fe(CN)6]4- and [Ni(CN)4]2- complexes. Theor. calcns. based on a new high-resoln. structure of the cyanide-bound CODHIICh indicated that cyanide binding to the Ni ion occurs upon dissocn. of the hydroxyl ligand from the Fe1 subsite of the C-cluster. The hydroxyl group is presumably protonated by Lys563 which, unlike to His93, does not form a hydrogen bond with the cyanide ligand. A stable deprotonated ε-amino group of Lys563 in the cyanide complex is consistent with the nearly unchanged C-N stretching in the Lys563Ala variant of CODHIICh. These findings support the view that the proton channel connecting the soln. phase with the active site displays a strict directionality, controlled by the oxidn. state of the C-cluster.
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    35. 35
      Svetlitchnyi, V.; Peschel, C.; Acker, G.; Meyer, O. Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans. J. Bacteriol. 2001, 183, 5134– 5144,  DOI: 10.1128/JB.183.17.5134-5144.2001
      35
      Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans
      Svetlitchnyi, Vitali; Peschel, Christine; Acker, Georg; Meyer, Ortwin
      Journal of Bacteriology (2001), 183 (17), 5134-5144CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit mol. masses, N-terminal sequences, peptide maps, and immunol. reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in assocn. with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H2O → CO2 + 2 e- + 2 H+. Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) μmol of CO oxidized min-1 mg-1 of protein (Me viologen, pH 8.0, 70°). The two enzymes oxidize CO very efficiently, as indicated by kcat/Km values at 70° of 1.3·109 M-1 CO s-1 (CODH I) and 1.7·109 M-1 CO s-1 (CODH II). The apparent Km values at pH 8.0 and 70° are 30 and 18 μM CO for CODH I and CODH II, resp. Acetyl CoA synthase activity is not assocd. with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers contg. 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, resp. ESR (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.
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    36. 36
      Domnik, L.; Merrouch, M.; Goetzl, S.; Jeoung, J.-H.; Léger, C.; Dementin, S.; Fourmond, V.; Dobbek, H. CODH-IV: A high-efficiency CO-scavenging CO dehydrogenase with resistance to O2. Angew. Chem., Int. Ed. 2017, 56, 15466– 15469,  DOI: 10.1002/anie.201709261
      36
      CODH-IV: A high efficiency CO-scavenging CO dehydrogenase with resistance to O2
      Domnik, Lilith; Merrouch, Meriem; Goetzl, Sebastian; Jeoung, Jae-Hun; Leger, Christophe; Dementin, Sebastien; Fourmond, Vincent; Dobbek, Holger
      Angewandte Chemie, International Edition (2017), 56 (48), 15466-15469CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Carbon monoxide dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO2. Genomic anal. previously indicated that the metabolic functions of CODHs vary. The genome of Carboxydothermus hydrogenoformans encodes 5 CODHs (CODH-I-V), of which CODH-IV is found in a gene cluster near a peroxide-reducing enzyme. Our kinetic and crystallog. expts. revealed that CODH-IV differs from other CODHs in several characteristic properties: it has a very high affinity for CO, oxidizes CO at a diffusion-limited rate over a wide range of temps., and is more tolerant to O2 than CODH-II. Thus, our observations support the idea that CODH-IV is a CO scavenger in defense against oxidative stress and highlight that CODHs are more diverse in terms of reactivity than expected.
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    37. 37
      Doukov, T. I.; Blasiak, L. C.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. Xenon in and at the end of the tunnel of bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Biochemistry 2008, 47, 3474– 3483,  DOI: 10.1021/bi702386t
      37
      Xenon in and at the End of the Tunnel of Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase
      Doukov, Tzanko I.; Blasiak, Leah C.; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L.
      Biochemistry (2008), 47 (11), 3474-3483CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a Me group, CoA, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermol. CO transport. Here, we report a 2.5 A resoln. structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calcn. program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid compn. surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.
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    38. 38
      Jeoung, J.-H.; Dobbek, H. n-Butyl isocyanide oxidation at the [NiFe4S4OHx] cluster of CO dehydrogenase. J. Biol. Inorg. Chem. 2012, 17, 167– 173,  DOI: 10.1007/s00775-011-0839-y
      38
      n-Butyl isocyanide oxidation at the [NiFe4S4OHx] cluster of CO dehydrogenase
      Jeoung, Jae-Hun; Dobbek, Holger
      JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (2), 167-173CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidn. of carbon monoxide by reaction with water to yield carbon dioxide, two protons, and two electrons. Two principal types of CODHs can be distinguished. Ni,Fe-contg. CODHs contain a [NiFe4S4OHx] cluster within their active site, to which the direct binding of the substrates water and carbon dioxide has been revealed by protein X-ray crystallog. N-Bu isocyanide is a slow-turnover substrate of CODHs, whose oxidn. at the active site shows several parallels to the oxidn. of carbon monoxide. Here, we report the crystal structure of CODH-II from Carboxydothermus hydrogenoformans resulting from the enzymic oxidn. of Bu isocyanide to Bu isocyanate at its active site cluster. The high resoln. of the structure (dmin = 1.28 Å) revealed Bu isocyanate bound to the active site cluster and identified a novel type of Ni-C bond in CODHs. The structure suggests the occurrence of tetrahedral in addn. to square-planar nickel complexes in product-bound states of this enzyme. Furthermore, we discovered a mol. of Bu isocyanide in a hydrophobic channel leading to the active site, revealing a unique architecture for the substrate channel of CODH-II compared with the bifunctional CODHs.
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    39. 39
      Lemaire, O. N.; Wagner, T. Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum. Biochim. Biophys. Acta, Bioenerg. 2021, 1862, 148330  DOI: 10.1016/j.bbabio.2020.148330
      39
      Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum
      Lemaire, Olivier N.; Wagner, Tristan
      Biochimica et Biophysica Acta, Bioenergetics (2021), 1862 (1), 148330CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
      Clostridium autoethanogenum, the bacterial model for biol. conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concns. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient mol. machineries for CO-conversion. Here, we structurally and biochem. characterized the key enzyme of the CO-converting metab.: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2β2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystn. artifact. Therefore, the internal CO-channeling system, crit. to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidn. and acetyl-CoA prodn. Biochem. expts. showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidn. and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.
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    40. 40
      Kung, Y.; Doukov, T. I.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. Crystallographic snapshots of cyanide- and water-bound C-clusters from bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Biochemistry 2009, 48, 7432– 7440,  DOI: 10.1021/bi900574h
      40
      Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase
      Kung, Yan; Doukov, Tzanko I.; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L.
      Biochemistry (2009), 48 (31), 7432-7440CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Nickel-contg. carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidn. of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallog. characterized as either a NiFe4S4 or a NiFe4S5 metal center, the latter contg. a fifth, addnl. sulfide that bridges Ni and a unique Fe site. To det. whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H2O/OH- mol. and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a soln. contg. potassium cyanide. In both structures, the substrate H2O/OH- mol. exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H2O/OH- mol. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidn. by the C-cluster, the catalytic center of an environmentally important enzyme.
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    41. 41
      Kung, Y.; Drennan, C. L. A role for nickel–iron cofactors in biological carbon monoxide and carbon dioxide utilization. Curr. Opin. Chem. Biol. 2011, 15, 276– 283,  DOI: 10.1016/j.cbpa.2010.11.005
      41
      A role for nickel-iron cofactors in biological carbon monoxide and carbon dioxide utilization
      Kung, Yan; Drennan, Catherine L.
      Current Opinion in Chemical Biology (2011), 15 (2), 276-283CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Ni-Fe contg. enzymes are involved in the biol. utilization of CO, CO2, and H2. Interest in these enzymes has increased in recent years due to H2 fuel initiatives and concerns over development of new methods for CO2 sequestration. One Ni-Fe enzyme called carbon monoxide dehydrogenase (CODH) is a key player in the global C cycle and carries out the interconversion of the environmental pollutant CO and the greenhouse gas CO2. The Ni-Fe center responsible for this important chem., the C-cluster, has been the source of much controversy, but several recent structural studies have helped to direct the field toward a unifying mechanism. Here, the authors summarize the current state of understanding of this fascinating metallocluster.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXksFKntLw%253D&md5=32b5b52a2f454ea5ce44c2c403822de6
    42. 42
      Copeland, R. A. Enzymes: A practical introduction to structure, mechanism, and data analysis, 2nd ed.; Wiley: New York, 2000, 122– 123.
      There is no corresponding record for this reference.
    43. 43
      Spangler, N. J.; Meyers, M. R.; Gierke, K. L.; Kerby, R. L.; Roberts, G. P.; Ludden, P. W. Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states. J. Biol. Chem. 1998, 273, 4059– 4064,  DOI: 10.1074/jbc.273.7.4059
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      Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states
      Spangler, Nathan J.; Meyers, Monica R.; Gierke, Karin L.; Kerby, Robert L.; Roberts, Gary P.; Ludden, Paul W.
      Journal of Biological Chemistry (1998), 273 (7), 4059-4064CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent redn. of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.
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    44. 44
      Heo, J.; Wolfe, M. T.; Staples, C. R.; Ludden, P. W. Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase. J. Bacteriol. 2002, 184, 5894– 5897,  DOI: 10.1128/JB.184.21.5894-5897.2002
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      Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase
      Heo, Jongyun; Wolfe, Marcus T.; Staples, Christopher R.; Ludden, Paul W.
      Journal of Bacteriology (2002), 184 (21), 5894-5897CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-contg. carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265 exhibits hydroxylamine reductase activity. Both Cys531 and His265 are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.
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    45. 45
      Jeoung, J. H.; Fesseler, J.; Domnik, L.; Klemke, F.; Sinnreich, M.; Teutloff, C.; Dobbek, H. A morphing [4Fe-3S-nO]-cluster within a carbon monoxide dehydrogenase scaffold. Angew. Chem., Int. Ed. 2022, 61, e202117000  DOI: 10.1002/anie.202117000
      45
      A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold
      Jeoung, Jae-Hun; Fesseler, Jochen; Domnik, Lilith; Klemke, Friederike; Sinnreich, Malte; Teutloff, Christian; Dobbek, Holger
      Angewandte Chemie, International Edition (2022), 61 (18), e202117000CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Ni,Fe-contg. carbon monoxide dehydrogenases (CODHs) catalyze the reversible redn. of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidn., nor CO2 redn. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xms1OhsrY%253D&md5=d2b0f84481f154172e7278ac7fa527d7
    46. 46
      Rebelein, J. G.; Stiebritz, M. T.; Lee, C. C.; Hu, Y. Activation and reduction of carbon dioxide by nitrogenase iron proteins. Nat. Chem. Biol. 2017, 13, 147– 149,  DOI: 10.1038/nchembio.2245
      46
      Activation and reduction of carbon dioxide by nitrogenase iron proteins
      Rebelein, Johannes G.; Stiebritz, Martin T.; Lee, Chi Chung; Hu, Yilin
      Nature Chemical Biology (2017), 13 (2), 147-149CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO2 and CO under ambient conditions. Catalytic redn. of CO2 to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-assocd. [Fe4S4] clusters. These observations establish the Fe protein as a model for investigation of CO2 activation while suggesting its biotechnol. adaptability for recycling the greenhouse gas into useful products.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFShtrfK&md5=9367ca8f74afff2277adc8a75c6d0bcf
    47. 47
      Amara, P.; Mouesca, J.-M.; Volbeda, A.; Fontecilla-Camps, J. C. Carbon monoxide dehydrogenase reaction mechanism: A likely case of abnormal CO2 insertion to a Ni–H – bond. Inorg. Chem. 2011, 50, 1868– 1878,  DOI: 10.1021/ic102304m
      47
      Carbon Monoxide Dehydrogenase Reaction Mechanism: A Likely Case of Abnormal CO2 Insertion to a Ni-H- Bond
      Amara, Patricia; Mouesca, Jean-Marie; Volbeda, Anne; Fontecilla-Camps, Juan C.
      Inorganic Chemistry (2011), 50 (5), 1868-1878CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      Ni-contg. carbon monoxide dehydrogenases (CODH), present in many anaerobic microorganisms, catalyze the reversible oxidn. of CO to CO2 at the so-called C-cluster. This atypical active site is composed of a [NiFe3S4] cluster and a single unusual iron ion called ferrous component II or Feu that is bridged to the cluster via one sulfide ion. After addnl. refinement of recently published high-resoln. structures of COOHx-, OHx-, and CN-bound CODH from Carboxydothermus hydrogenoformans, we have used computational methods on the predominant resulting structures to investigate the spectroscopically well-characterized catalytic intermediates, Cred1 and the two-electron more-reduced Cred2. Several models were geometry-optimized for both states using hybrid quantum mech./mol. mech. potentials. The comparison of calcd. Mossbauer parameters of these active site models with exptl. data allows us to propose that the Cred1 state has a Feu-Ni2+ bridging hydroxide ligand and the Cred2 state has a hydride terminally bound to Ni2+. Using our combined structural and theor. data, we put forward a revised version of an earlier proposal for the catalytic cycle of Ni-contg. CODH that agrees with available spectroscopic and structural data. This mechanism involves an abnormal CO2 insertion into the Ni2+-H- bond.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXosVyktw%253D%253D&md5=3b04ba13fd0ec5994ed4c03b890b22aa

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.2c02922.

    • Materials and methods, diffraction data collection, structure determination, structure refinement, statistics of data collection and structure refinement, Fe–S distances and angles of the [Fe4(μ3-S)4] cluster from C295D-CODH-IICh and K563H-CODH-IICh, stereo view of cluster C and its surroundings, steady state kinetics of CO oxidation, and sequence alignment (PDF)

    Accession Codes

    The coordinates and structure factor amplitudes of CODH-IICh variants were deposited in the Protein Data Bank under the accession names of 7ZX3 for C295D, 7ZX5 for I567T, 7ZX6 for I567L, 7ZXC for H96D, 7ZXJ for K563A, 7ZXL for H93A, 7ZXX for K563H, and 7ZY1 for I567A.


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