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Asymmetry in the Ligand Coordination Sphere of the [FeFe] Hydrogenase Active Site Is Reflected in the Magnetic Spin Interactions of the Aza-propanedithiolate Ligand
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The Journal of Physical Chemistry Letters

Cite this: J. Phys. Chem. Lett. 2019, 10, 21, 6794–6799
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https://doi.org/10.1021/acs.jpclett.9b02354
Published October 3, 2019

Copyright © 2019 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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[FeFe] hydrogenases are very active enzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. Their active site, the H-cluster, contains a unique binuclear iron complex, [2Fe]H, with CN– and CO ligands as well as an aza-propane-dithiolate (ADT) moiety featuring a central amine functionality that mediates proton transfer during catalysis. We present a pulsed 13C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with 13C. We observed that the corresponding two 13C hyperfine interactions are of opposite sign and corroborated this finding using density functional theory calculations. The spin polarization in the ADT ligand is shown to be linked to the asymmetric coordination of the distal iron site with its terminal CN– and CO ligands. We propose that this asymmetry is relevant for the enzyme reactivity and is related to the (optimal) stabilization of the iron-hydride intermediate in the catalytic cycle.

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The active site of [FeFe] hydrogenases, called the “H-cluster”, features a two-iron subcluster that is coordinated by CN– and CO ligands as well as a unique 2-aza-propane 1,3-dithiolate (ADT) bridging ligand. (1) The [2Fe]H subcluster is linked to a generic [4Fe–4S] cubane cluster through a cysteine thiolate bridge (Figures 1 and 2). It is assumed that the amine moiety of the ADT bridge functions as a proton shuttle to and from the open coordination site at the iron site (Fed) distal to the [4Fe–4S]H subcluster. The catalytically active oxidized state (Hox) is characterized by a mixed valence redox configuration [Fe(II)Fe(I)] in the [2Fe]H subcluster. This state features a so-called “frustrated Lewis pair” (FLP), (2) with the electrophilic Fed center adjacent to the Brønsted basic amine of the ADT. (3) The FLP splits H2 heterolytically placing a proton on the amine and a hydride on Fed. This Fed–H species is immediately oxidized through the electron transport chain connected to the iron core of the H-cluster. This oxidation converts the hydridic Fed–H to an acidic Fed–H species. The subsequent stages in the catalytic cycle (Figure 1) feature several protonation, reduction, and proton coupled electron transfer (PCET) steps. (4,5) This multistep sequence results in a very flat energy landscape, which explains the very high catalytic rates observed in [FeFe] hydrogenases (up to 10,000 s–1). (6,7)

Figure 1

Figure 1. Structure of the H-cluster in [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) and the proposed catalytic cycle.

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Figure 2

Figure 2. Left: Schematic structure of the H-cluster in its Hox state, triply isotope labeled with 57Fe, as well as 13C and 2H in the aza-propane-dithiolate (ADT) ligand. Right: Q-band EPR spectrum (pseudo modulated FID detected) of our Hox preparation. Fitted g-values: (2.1008, 2.0398, 1.9966). A small contribution (<5%) from the Hox-CO state is evident from the feature at g = 2.006 and is marked by an asterisk. Full analysis of the EPR spectrum is presented in Figure S2.

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The electronic structure of the H-cluster is strongly affected by the spin exchange interactions between the two subclusters and within the [4Fe–4S]H subcluster as monitored by pulsed EPR and paramagnetic NMR studies. (8−11) These effects certainly contribute to the facile, virtually barrier-free, electron transport over the H-cluster. The protonation of the bridging ADT ligand amine group appears to play a central role in modulating the electron transport between the two subclusters. (4) Therefore, the electronic structure, as reflected by the magnetic spin parameters of the ADT ligand, may show features that can be related to the overall catalytic properties of the H-cluster. Indeed, paramagnetic NMR of Hox showed spin polarization of the methylene protons, i.e., positive and negative spin densities. (11) In this communication, we investigate the 13C-labeled methylene carbons of the ADT ligand using electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) spectroscopy and show that spin-polarization also occurs for these nuclei. This phenomenon is explored using density functional theory (DFT) quantum chemical calculations and is found to be dependent on the asymmetry in the ligand coordination at Fed.

Isotope Labeling

For the 13C-ENDOR experiments described herein, we took advantage of a triply labeled (2 × 57Fe, 2 × 13C, 4 × 2H) synthetic precursor of [2Fe]H, initially prepared for ongoing nuclear resonance vibrational spectroscopy (NRVS) experiments. The new synthetic route for this compound is described in the Supporting Information (SI) section A and Scheme S1. The [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) was produced using artificial maturation (12,13) of the apoenzyme expressed in E. coli with the triply labeled [2Fe]H precursor (see Figure 2). The enzyme was allowed to oxidize under an N2 atmosphere (so-called “auto-oxidation”) to produce the Hox state. This is characterized by a mixed valence Fe(II)Fe(I) binuclear subcluster showing a characteristic rhombic S = 1/2 EPR spectrum with g-parameters (2.1008, 2.0398, 1.9966) (see Figures 2 and S2). (1,14) The EPR and FTIR spectra (Figure S1) are consistent with those obtained previously for the native Hox state. (1)

Mims-ENDOR

Orientation-selective 13C Mims ENDOR (see Experimental Section in the SI and Scheme 1) was recorded at Q-band (34 GHz) at field positions corresponding to the range from g = 2.0 to g = 2.1 (Figure 3). In order to facilitate spectral fitting, the ENDOR spectra were normalized to the same amplitude and symmetrized with respect to the Larmor frequency (indicated by the arrows in Figure 3).

Scheme 1

Scheme 1. Pulse Sequence of the 13C Mims ENDOR Experiment
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Figure 3

Figure 3. (a–i) Orientation-selective 13C Mims Q-band ENDOR (34 GHz) spectra of CrHydA1 in the Hox state recorded at 15 K. The indicated field positions are given in mT. π/2 pulses were 20 ns. The waiting time τ was set to 200 ns, while the 400W RF pulse had a length of 60 μs (see Scheme 1). The red traces represent spectral fits obtained using a home-written MATLAB script making use of first order perturbation theory to calculate the ENDOR transition frequencies (details in SI). For the raw (normalized) ENDOR traces (unsymmetrized) along with the number of scans, see Figure S3.

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At the g = 2.0 and g = 2.1 positions (magnetic field values of 1219 and 1158.7 mT, respectively), only molecules with one of their g-axes (x or z, respectively) oriented along the magnetic field contribute to the ENDOR spectrum. These positions are called “pseudo single crystal positions”. In these spectra, clearly two doublets centered at the 13C nuclear Larmor frequency (12.85 MHz at 1200 mT) are observed corresponding to the nuclear spin transitions of the two 13C nuclei in the ADT ligand. The different splitting and line-width of the two doublets indicate that the two 13C nuclei have significantly different hyperfine interaction (HFI) tensors differing in magnitudes and orientations. ENDOR spectra recorded at intermediate field positions contain contributions from molecules with multiple orientations giving rise to an EPR transition at the effective g-value corresponding to the field position. These multiple orientations lead to a broadening of the ENDOR spectra showing “powder-type” features. The line-shapes at, e.g., 1165.8 and 1205.4 G are reminiscent of “near axial hyperfine anisotropy” for the 13C nuclei. Therefore, in order to reduce the number of free variables, spectral fitting was started assuming axial HFI tensors for both 13C nuclei. First order perturbation theory was employed to calculate the ENDOR frequencies. The (1 – cos(2πAτ)) dependence of the ENDOR amplitude was taken into account. This procedure provided a fair but nonoptimal fit to the experimental ENDOR spectra. Subsequently, the rhombicity parameter was relaxed, and individual scaling of the two ENDOR components was applied. The final fitted parameters are listed in Table 1 and Figure S4. Given the local mirror symmetry of the ADT ligand (Figure 2), it is surprising that the two 13C HFI-tensors have significantly different magnitudes (1.9 vs 1.2 MHz for the isotropic component).
Table 1. Experimental and DFT-Predicted 13C HFI Anisotropic and Isotropic (Fermi Contact) Spin Couplings (MHz) for the Two Methylene Carbons A1 and A2 in the ADT Ligand in the Hox Statea
 A1 (MHz)A2 (MHz)
XYZisoXYZiso
exptl1.001.303.301.87–1.49–1.75–0.45–1.23
Anative1.201.463.081.91–1.34–1.09–0.26–0.90
Enative0.460.802.191.15–1.51–1.23–0.30–1.02
Ad-iso–1.67–1.38–0.50–1.181.311.593.332.07
Ap-iso1.231.503.201.97–1.71–1.41–0.57–1.23
Apd-iso–1.41–1.12–0.30–0.941.161.423.051.88
Ad-rot0.841.062.741.55–2.31–2.09–0.38–1.60
Ap-(CN)2–1.44–1.080.17–0.78–1.37–1.020.22–0.72
Ad-(CN)2–0.460.070.400.01–0.130.380.780.34
a

The experimental (exptl) and representative DFT (Anative) values are in bold. Other values are from alternative isomeric DFT models shown in Figure S7.

Mims TRIPLE

In order to determine the relative signs of the two fitted 13C HFIs, a triple resonance experiment was conducted at a field position corresponding to g = 2.02 (magnetic field position near 1218 mT, see Figure 3) where the two ENDOR doublet features are nicely separated and still relatively sharp (Figure 4 and Scheme 2). Adjusting the radiofrequency pumping pulse (RF1) to one of the ENDOR transitions inverts the spin state populations corresponding to this transition. This also affects (reduces) the population difference of the other nuclear spin transition in the same mS manifold resulting in a reduction of the ENDOR amplitude of the corresponding signal (see SI for more details). (15,16) Pumping the high frequency line of the sharp 13C doublet leads to a reduction in amplitude of the low frequency line of the broad 13C doublet (upper spectra in Figure 4). Likewise, pumping the low frequency line of the sharp doublet causes a reduction in amplitude of the high frequency line of the broad doublet. We must, therefore, conclude that the signs of the two 13C hyperfine interactions are opposite (Tables 1 and S1). This implies that at one of the ADT carbon nuclei a negative (β) spin density is localized.

Scheme 2

Scheme 2. Pulse Sequence of the 13C Mims Triple Resonance Experimenta
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aSee Scheme S2 for extra details.

Figure 4

Figure 4. Mims TRIPLE resonance experiment (see pulse sequence in Scheme 2) at B = 1218 mT. The black traces represent the (unsymmetrized) 13C TRIPLE experiment with the pumping frequency RF1 off resonance at the 13C Larmor frequency, i.e., equivalent to the 13C ENDOR spectrum at B = 1218 mT (see Figure 3). The red traces represent the 13C Mims TRIPLE experiment with the RF1 pumping frequency tuned to one of the sharp ENDOR transitions. The “TRIPLE effect” is indicated by the red asterisks.

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DFT Calculations

In order to obtain more insight into the underlying mechanism of this spin polarization phenomenon, we conducted DFT calculations using a [2Fe]H subcluster model in which the (oxidized) [4Fe–4S]H subcluster (S = 0) is modeled by a protonated thiol (ethyl-SH) group (Figure S5). The representative model is called “A” due to the “axial” conformation of the ADT–NH– moiety, with its proton pointing toward Fed. Using this modeling level, previously referred to as “S (small)”, (17,18) the magnitude and sign of the experimental 13C-ADT HFIs for both carbon nuclei could be best reproduced. In Figure S6 the spin-density distribution of the (i) corresponding (see the DFT methods described in the SI) singly occupied molecular orbital (SOMO), (ii) formally doubly occupied MOs (Total-SOMO), as well as (iii) that of all MOs (Total) of the binuclear subsite are displayed with two contour values. A summary of these DFT results is presented in Figure 5.

Figure 5

Figure 5. Top (a): Isosurfaces of total spin density at the 5 × 10–4 a.u. cutoff (corresponding to the Fermi contact HFI term Aiso(13C) = 0.6 MHz) for the representative HoxS = 1/2 DFT model Anative, showing positive (blue) density on the “right” 13C and negative (green) spin density on the “left” 13C nuclei. The model itself is shown in thin wire. Bottom (b): View of DFT model Anative along the Fep–Fed axis displaying the orientation of the calculated methylene 13C hyperfine tensors (Table S1). For extended information, see Figures S6 and S7.

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Notably, the corresponding SOMO has 74% contribution from the Fed 3d shell. It turns out that the “left” methylene carbon, i.e., the carbon on the same side as the distal terminal CO ligand, has negative spin density due to spin polarization since the SOMO has only a vanishing contribution on this atom. The “right” carbon nucleus (on the same side as the distal CN– ligand) has positive spin density due to delocalization of the SOMO onto this center. No other MOs noticeably contribute to the spin densities at the two 13C nuclei. Although very small in their magnitude, the calculated atomic spin populations at the two ADT carbon nuclei (Table S3) are nevertheless consistent with the observed 13C HFI values. It is, therefore, tempting to assume that this asymmetric spin distribution is associated with the asymmetric ligand configuration at the distal iron center Fed. To verify this hypothesis, two isomeric DFT models were investigated in which the positions of the CN– and CO ligands were swapped at either Fep (“Ap-iso”) or Fed (“Ad-iso”), see Figure S7. Obviously, swapping CN– ligands at both Fe sites creates a pseudomirror image of the native [2Fe]H isomer (“Apd-iso”). A full mirror image can only be obtained if the protonation position of the ethyl-SH ligand is also swapped. Exchanging the CN– and CO ligand at the proximal iron Fep did not have an effect on the signs of the methylene 13C HFI. However, swapping the terminal CN– and CO ligands at the distal iron Fed changes the spin-polarization in the ADT ligand (Table 1). The behavior described is rationalized by the [Fe(II)pFe(I)d] electronic structure of the [2Fe]H subcluster in the Hox state with its S = 1/2 spin localization at the Fe(I)d distal iron site, while the Fe(II)p proximal site remains low-spin. (19,20) Other investigated DFT models are “Enative”, having the −NH– proton in its alternative “equatorial” position pointing toward Fep, (21) and “Ad-rot” with CN– at Fed rotated to the position trans to the bridging CO ligand; (22) these two models represent [2Fe]H subcluster structural alternatives often discussed in the literature. While the two alternatives Enative and Ad-rot predict the same orientation of the 13C-ADT HFI tensors as seen in Anative, they produce an inferior match to the experimental HFI values (Figure S7, Tables 1 and S1). Interestingly, all the above-described ‘A’ (axial) isomeric models are found essentially equi-energetic within 1 kcal/mol, while the ‘E’ (equatorial) isomer is calculated +5 kcal/mol higher in its energy. Two additional intersite ligand swap isomers studied have both CN– ligands at either Fep (‘Ap-(CN)2’) or Fed (‘Ad-(CN)2’); these two structures are predicted to have significantly higher (+123/129 kcal/mol) relative energies. Notably, placing two negative CN– ligands at Fed in the Ad-(CN)2 isomer produces an inverted [Fe(I)pFe(II)d] oxidation pattern for the iron sites, having Fep as the spin center (Table S2).
The local CN–/CO ligand asymmetry at the distal iron site, which features the open coordination in the Hox state, seems to be the key to inducing the spin-polarization in the ADT ligand. A further result of this asymmetry is the spin polarization of the methylene protons as was observed in our paramagnetic NMR studies. (11) Since in our [2Fe]H precursor the methylene protons have been labeled with 2H, we also performed 2H Mims ENDOR experiments at the three canonical field positions (gx, gy, gz) (see Figure S8). Signals of up to three 2H nuclei could be observed and show hyperfine couplings up to 0.6 MHz. The signals are, however, much weaker than those of the 13C nuclei, which, currently, precludes the application of a triple 2H ENDOR experiment.
To address the question of the relevance of the asymmetry in coordination of the distal iron atom for the catalytic cycle, we investigated how it influences the stabilization of the hydride-bound Hhyd-E state, which is formed from the 2e–-reduced (vs Hox), protonated HsredH+ state (see Figure 1). We calculated the energetics for this process for three isomers: the native configuration ‘A’ with one terminal CN– on each [2Fe]H iron, ‘p-(CN)2’ with both CN– ligands coordinated at the proximal iron, and ‘d-(CN)2’ with both CN– ligands coordinating the distal iron (see Figures S7 and S9). Clearly, the non-native isomeric configurations are of academic interest only since they do not occur in [FeFe] hydrogenase and cannot be synthesized. Moreover, the relative energies of the non-native isomers are around 130 kcal/mol higher than that of the native configuration (Figure S9). Since in our truncated model the [4Fe–4S]H subcluster is absent, HsredH+ is indistinguishable from HredH+. This also means that the models are diamagnetic, and no magnetic effects can be modeled. Nevertheless, it can be assumed that the thermodynamics of the Fed–H– hydride formation (i.e., the HsredH+-to-Hhyd-E intramolecular proton transfer from the ADT–NH2+– bridgehead to the distal iron) is well represented since the [4Fe–4S]H subcluster is unlikely to have a major impact on this process. It should be noted that the hydride formation can involve a transition from terminal CO in the H(s)redH+ state to bridging CO in Hhyd (see Figures 1 and S9). It is not clear if this ligand rearrangement also occurs to its full extent in the enzyme on the time scale of catalysis. For the ‘A’ native and p-(CN)2 isomers, the Hhyd-E hydride formation is predicted exothermic: ΔHnative = −3.4 kcal/mol, ΔHp-(CN)2 = −7.8 kcal/mol. For the d-(CN)2 isomer, the hydride formation is endothermic, ΔHd-(CN)2 = +16.8 kcal/mol. For the p-(CN)2 model, the distal iron Fed has a lack of charge density (in the absence of CN– coordination), providing extra stabilization of the hydride. However, model d-(CN)2 has excess charge density (with two CN– coordinating) at the distal iron, which prevents stabilization of the hydride at Fed. The charge density is more equally distributed over the two-iron core of [2Fe]H in the native configuration and seems to provide the perfect compromise to sufficiently stabilize the hydride state for fast catalysis yet not overstabilizing it, in order to avoid formation of a thermodynamic sink, which would strongly slow down the catalysis. We, therefore, conclude that the asymmetry of the ligand coordination at Fed ensures a flat energy landscape for the hydride formation and transfer during the reversible catalytic cycle of [FeFe] hydrogenase. This finding significantly advances the design strategies for artificial hydrogen conversion catalysts: Apart from the ligand geometry, also the charge balance at the catalytic iron center needs to be carefully controlled.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.9b02354.

  • New synthetic route for the 57Fe labeled ADT precursor, experimental and computational procedures, supplementary figures and tables (PDF)

  • Cartesian coordinates of structurally optimized DFT models (ZIP)

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Author Information

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  • Corresponding Authors
  • Authors
    • James A. Birrell - Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany;  Orcidhttp://orcid.org/0000-0002-0939-0573
    • Casseday P. Richers - School of Chemical Sciences, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
    • Martin Kaupp - Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany;  Orcidhttp://orcid.org/0000-0003-1582-2819
    • Thomas B. Rauchfuss - School of Chemical Sciences, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, United States;  Orcidhttp://orcid.org/0000-0003-2547-5128
    • Stephen P. Cramer - SETI Institute, Mountain View, California 94043, United States
    • Wolfgang Lubitz - Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany;  Orcidhttp://orcid.org/0000-0001-7059-5327
  • Author Contributions

    E.J.R. and V.P. contributed equally to this work.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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E.J.R, J.A.B., and W.L. would like to thank the Max Planck Society for continuous financial support. V.P. and M.K. acknowledge funding by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC 2008/1 (UniSysCat)–390540038. J.A.B. acknowledges funding from the DFG SPP 1927 “Iron–Sulfur for Life” project (Project No. BI 2198/1-1). The contributions of T.B.R. and C.P.R. were funded by the U.S. National Institutes of Health through GM61153. SPC was funded by NIH GM65440.

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    Intercluster Redox Coupling Influences Protonation at the H-cluster in [FeFe] Hydrogenases
    Rodriguez-Macia, Patricia; Pawlak, Krzysztof; Ruediger, Olaf; Reijerse, Edward J.; Lubitz, Wolfgang; Birrell, James A.
    Journal of the American Chemical Society (2017), 139 (42), 15122-15134CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    [FeFe] hydrogenases catalyze proton redn. and hydrogen oxidn. displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] sub-cluster ([2Fe]H) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S]H). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S]F clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic mol. catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S]F cluster and the [4Fe-4S]H sub-cluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the pKa of its one electron reduced state. This may help to sustain H2 prodn. at high pH values. These results may apply to all [FeFe] hydrogenases contg. accessory clusters.
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  6. 6
    Glick, B. R.; Martin, W. G.; Martin, S. M. Purification and Properties of the Periplasmic Hydrogenase from Desulfovibrio desulfuricans. Can. J. Microbiol. 1980, 26, 1214– 1223,  DOI: 10.1139/m80-203
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    Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans
    Glick, Bernard R.; Martin, William G.; Martin, Stanley M.
    Canadian Journal of Microbiology (1980), 26 (10), 1214-23CODEN: CJMIAZ; ISSN:0008-4166.
    The periplasmic hydrogenase (I) of D. desulfuricans was isolated and purified. Cells were washed with Tris-EDTA and the enzyme pptd. from the wash with (NH4)2SO4. Absorption chromatog. on DEAE-Sephacel and hydroxylapatite yielded I at >95% purity as judged by gel electrophoresis. I catalyzed the prodn. of >9000 μmol H2/min/mg protein from reduced Me viologen at 37°. It is very stable and resisted inactivation by heat (50% activity remained after 5 min in air at 65°) and by enzyme inhibitors (except N-ethylmaleimide and K ferricyanide). After storage in air at 4° for 1 mo, no activity was lost. I activity was sensitive to ionic environmental changes. With Me viologen the optimum pH was 5.5, but with p-xylene polymeric viologen the optimum was about pH 7 but less sharp. The mol. wt. was 47 × 103, 52 × 103, and 56 × 103 by SDS-gel electrophoresis, gel chromatog., and sedimentation equil., resp., and the isoelec. point was at pH 6.0. I consisted of 1 polypeptide chain with proline at the N-terminal end. I may be useful in the prodn. of H2 from water and solar energy.
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  7. 7
    Hatchikian, E. C.; Forget, N.; Fernández, V. M.; Williams, R.; Cammack, R. Further Characterization of the [Fe]-Hydrogenase from Desulfovibrio desulfuricans ATCC 7757. Eur. J. Biochem. 1992, 209, 357– 365,  DOI: 10.1111/j.1432-1033.1992.tb17297.x
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    7
    Further characterization of the [iron]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757
    Hatchikian, E. Claude; Forget, Nicole; Fernandez, Victor M.; Williams, Ruth; Cammack, Richard
    European Journal of Biochemistry (1992), 209 (1), 357-65CODEN: EJBCAI; ISSN:0014-2956.
    The properties of the periplasmic hydrogenase from D. desulfuricans ATCC 7757 were reinvestigated. The pure enzyme exhibited a mol. mass of 53.5 kDa as measured by anal. ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, resp. The N-terminal amino acid sequences of its large and small subunits, detd. up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/mol. and had ε400, 52.5 mM-1·cm-1. The purified hydrogenase showed a specific activity of 62 kV/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its max. activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approx. 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addn., an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Mol. properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.
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  8. 8
    Silakov, A.; Reijerse, E. J.; Albracht, S. P. J.; Hatchikian, E. C.; Lubitz, W. The Electronic Structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: A Q-band 57Fe ENDOR and HYSCORE Study. J. Am. Chem. Soc. 2007, 129, 11447– 11458,  DOI: 10.1021/ja072592s
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    8
    The Electronic Structure of the H-Cluster in the [FeFe]-Hydrogenase from Desulfovibrio desulfuricans: A Q-band 57Fe-ENDOR and HYSCORE Study
    Silakov, Alexey; Reijerse, Eduard J.; Albracht, Simon P. J.; Hatchikian, E. Claude; Lubitz, Wolfgang
    Journal of the American Chemical Society (2007), 129 (37), 11447-11458CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The active site of the 57Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examd. using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (Hox) and the CO inhibited form (Hox-CO) all six 57Fe hyperfine couplings were detected. The anal. shows that the apparent spin d. extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six 57Fe nuclei in the H-cluster of the Hox-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S]H (cubane) subcluster. Two weak 57Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe]H subcluster. For the Hox state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An 57Fe line broadening anal. of the X-band CW EPR spectrum indicated, however, that all six 57Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe]H assumes a [FeIFeII] redox configuration where the paramagnetic FeI atom is attached to the [4Fe-4S]H subcluster. The 57Fe hyperfine interactions of the formally diamagnetic [4Fe-4S]H are due to an exchange interaction between the two subclusters as has been discussed earlier by C. V. Popescu and E. Muenck (1999). This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the obsd. modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.
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  9. 9
    Silakov, A.; Reijerse, E. J.; Lubitz, W. Unraveling the Electronic Properties of the Photoinduced States of the H-Cluster in the [FeFe] Hydrogenase from D. desulfuricans. Eur. J. Inorg. Chem. 2011, 2011, 1056– 1066,  DOI: 10.1002/ejic.201001080
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  10. 10
    Silakov, A.; Wenk, B.; Reijerse, E.; Albracht, S. P. J.; Lubitz, W. Spin Distribution of the H-cluster in the Hox-CO state of the [FeFe] Hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR Study of 14N and 13C Nuclear Interactions. J. Biol. Inorg. Chem. 2009, 14, 301– 313,  DOI: 10.1007/s00775-008-0449-5
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    10
    Spin distribution of the H-cluster in the Hox-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions
    Silakov, Alexey; Wenk, Brian; Reijerse, Eduard; Albracht, Simon P. J.; Lubitz, Wolfgang
    JBIC, Journal of Biological Inorganic Chemistry (2009), 14 (2), 301-313CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)
    Hydrogenases are enzymes which catalyze the reversible cleavage of mol. hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster.". The H-cluster consists of the redox-active binuclear subcluster ([2Fe]H) coordinated by CN- and CO ligands and the cubane-like [4Fe-4S]H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the Hox-CO state the open coordination site at the [2Fe]H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepd. with 13CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three 13C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were obsd. and assigned to 13CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, resp. No 13CO exchange of the CO ligand to the proximal iron was obsd. The hyperfine interactions detected indicate a rather large distribution of the spin d. over the terminal and bridging CO ligands attached to the distal iron. Furthermore, 14N nuclear spin interactions were measured. On the basis of the obsd. 14N hyperfine couplings, which result from the CN- ligands of the [2Fe]H subcluster, it has been concluded that there is very little unpaired spin d. on the cyanides of the binuclear subcluster.
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  11. 11
    Rumpel, S.; Ravera, E.; Sommer, C.; Reijerse, E.; Fares, C.; Luchinat, C.; Lubitz, W. 1H NMR Spectroscopy of [FeFe] Hydrogenase: Insight into the Electronic Structure of the Active Site. J. Am. Chem. Soc. 2018, 140, 131– 134,  DOI: 10.1021/jacs.7b11196
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    11
    1H NMR spectroscopy of [FeFe] hydrogenase: Insight into the electronic structure of the active site
    Rumpel, Sigrun; Ravera, Enrico; Sommer, Constanze; Reijerse, Edward; Fares, Christophe; Luchinat, Claudio; Lubitz, Wolfgang
    Journal of the American Chemical Society (2018), 140 (1), 131-134CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii was studied using 1H NMR spectroscopy to identify the paramagnetically shifted 1H resonances assocd. with both the [4Fe-4S]H and the [2Fe]H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 contg. only [4Fe-4S]H was reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active Hox and the CO-inhibited Hox-CO state, revealed addnl. upfield and downfield shifted 1H resonances originating from the 4 methylene protons of the azadithiolate ligand in the [2Fe]H subsite. The two axial protons were affected by neg. spin d., while the 2 equatorial protons experienced pos. spin d. These protons could be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.
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  12. 12
    Berggren, G.; Adamska, A.; Lambertz, C.; Simmons, T. R.; Esselborn, J.; Atta, M.; Gambarelli, S.; Mouesca, J. M.; Reijerse, E. J.; Lubitz, W.; Happe, T.; Artero, V.; Fontecave, M. Biomimetic Assembly and Activation of (FeFe)-Hydrogenases. Nature 2013, 499, 66– 69,  DOI: 10.1038/nature12239
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    12
    Biomimetic assembly and activation of [FeFe]-hydrogenases
    Berggren, G.; Adamska, A.; Lambertz, C.; Simmons, T. R.; Esselborn, J.; Atta, M.; Gambarelli, S.; Mouesca, J.-M.; Reijerse, E.; Lubitz, W.; Happe, T.; Artero, V.; Fontecave, M.
    Nature (London, United Kingdom) (2013), 499 (7456), 66-69CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Hydrogenases are the most active mol. catalysts for hydrogen prodn. and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron center (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN- ligands. Through a complex multienzymic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chem. has been used to prep. remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymic activities thus far. Here we show that three synthetic mimics (contg. different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein contg. the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogs. This simple methodol. provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.
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  13. 13
    Esselborn, J.; Lambertz, C.; Adamska-Venkatesh, A.; Simmons, T.; Berggren, G.; Noth, J.; Siebel, J. F.; Hemschemeier, A.; Artero, V.; Reijerse, E. J.; Fontecave, M.; Lubitz, W.; Happe, T. Spontaneous Activation of [FeFe]-Hydrogenases by an Inorganic [2Fe] Active Site Mimic. Nat. Chem. Biol. 2013, 9, 607– 609,  DOI: 10.1038/nchembio.1311
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    13
    Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic
    Esselborn, Julian; Lambertz, Camilla; Adamska-Venkatesh, Agnieszka; Simmons, Trevor; Berggren, Gustav; Noth, Jens; Siebel, Judith; Hemschemeier, Anja; Artero, Vincent; Reijerse, Edward; Fontecave, Marc; Lubitz, Wolfgang; Happe, Thomas
    Nature Chemical Biology (2013), 9 (10), 607-609_CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Hydrogenases catalyze the formation of hydrogen. The cofactor (H-cluster) of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] sub-cluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here it is shown that a chem. mimic of the [2Fe] sub-cluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H2-producing catalysts.
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  14. 14
    Lubitz, W.; Reijerse, E.; van Gastel, M. [NiFe] and [FeFe] Hydrogenases Studied by Advanced Magnetic Resonance Techniques. Chem. Rev. 2007, 107, 4331– 4365,  DOI: 10.1021/cr050186q
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    14
    [NiFe] and [FeFe] Hydrogenases Studied by Advanced Magnetic Resonance Techniques
    Lubitz, Wolfgang; Reijerse, Eduard; van Gastel, Maurice
    Chemical Reviews (Washington, DC, United States) (2007), 107 (10), 4331-4365CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The understanding of the basic principles of hydrogen prodn. and utilization in microbes is a goal of major importance both for basic research and possible applications. A mechanistic knowledge of the hydrogen conversion and consumption process would allow us to use the organisms or the isolated enzymes in biotechnol. hydrogen prodn. processes. Furthermore, this would provide the necessary fundamental knowledge for designing biomimetic or bioinspired artificial "hydrogenase catalysts" for large-scale hydrogen prodn. in the future. Hydrogenases are ancient enzymes that facilitate the uptake and oxidn. of dihydrogen to protons and release of electrons and also the reverse reaction in a true equil. process. The enzyme hydrogenase is found in many microorganisms from bacteria and archae to eukarya. The review describes in detail the structure and mechanism of action of [NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques.
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  15. 15
    Schweiger, A.; Jeschke, G. Principles of Pulse Electron Paramagnetic Resonance; Oxford University Press: Oxford, 2001.
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    There is no corresponding record for this reference.
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    Harmer, J. R. Hyperfine Spectroscopy - ENDOR. Emagres 2016, 5, 1493– 1514,  DOI: 10.1002/9780470034590.emrstm1515
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    There is no corresponding record for this reference.
  17. 17
    Pham, C. C.; Mulder, D. W.; Pelmenschikov, V.; King, P. W.; Ratzloff, M. W.; Wang, H.; Mishra, N.; Alp, E. E.; Zhao, J.; Hu, M. Y.; Tamasaku, K.; Yoda, Y.; Cramer, S. P. Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment. Angew. Chem., Int. Ed. 2018, 57, 10605– 10609,  DOI: 10.1002/anie.201805144
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    17
    Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment
    Pham, Cindy C.; Mulder, David W.; Pelmenschikov, Vladimir; King, Paul W.; Ratzloff, Michael W.; Wang, Hongxin; Mishra, Nakul; Alp, Esen E.; Zhao, Jiyong; Hu, Michael Y.; Tamasaku, Kenji; Yoda, Yoshitaka; Cramer, Stephen P.
    Angewandte Chemie, International Edition (2018), 57 (33), 10605-10609CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calcns. was used to observe and characterize Fe-H/D bending modes in Chlamydomonas reinhardi [FeFe]-hydrogenase (CrHydA1) Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We obsd. a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calcns., we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.
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  18. 18
    Reijerse, E. J.; Pham, C. C.; Pelmenschikov, V.; Gilbert-Wilson, R.; Adamska-Venkatesh, A.; Siebel, J. F.; Gee, L. B.; Yoda, Y.; Tamasaku, K.; Lubitz, W.; Rauchfuss, T. B.; Cramer, S. P. Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy. J. Am. Chem. Soc. 2017, 139, 4306– 4309,  DOI: 10.1021/jacs.7b00686
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    18
    Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy
    Reijerse, Edward J.; Pham, Cindy C.; Pelmenschikov, Vladimir; Gilbert-Wilson, Ryan; Adamska-Venkatesh, Agnieszka; Siebel, Judith F.; Gee, Leland B.; Yoda, Yoshitaka; Tamasaku, Kenji; Lubitz, Wolfgang; Rauchfuss, Thomas B.; Cramer, Stephen P.
    Journal of the American Chemical Society (2017), 139 (12), 4306-4309CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    [FeFe]-hydrogenases catalyze the reversible redn. of protons to mol. hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging Cys residue to a [2Fe]H subsite coordinated by CN- and CO ligands featuring a dithiol-amine moiety which serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly obsd. exptl. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) expts. in conjunction with DFT calcns. on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally showed the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.
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  19. 19
    Fiedler, A. T.; Brunold, T. C. Computational Studies of the H-Cluster of Fe-Only Hydrogenases: Geometric, Electronic, and Magnetic Properties and Their Dependence on the [Fe4S4 ] Cubane. Inorg. Chem. 2005, 44, 9322– 9334,  DOI: 10.1021/ic050946f
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    19
    Computational Studies of the H-Cluster of Fe-Only Hydrogenases: Geometric, Electronic, and Magnetic Properties and Their Dependence on the [Fe4S4] Cubane
    Fiedler, Adam T.; Brunold, Thomas C.
    Inorganic Chemistry (2005), 44 (25), 9322-9334CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The active sites of Fe-only hydrogenases (FeHases) feature an unusual polynuclear iron-sulfur cluster, known as the H-cluster, that consists of a [Fe4S4] cubane linked to a di-iron subunit (the [2Fe]H component) via a bridging cysteine ligand (SCys). While previous computational studies of FeHases employed H-cluster models that only included the [2Fe]H component, we have utilized d. functional theory (DFT), in conjunction with the broken-symmetry (BS) approach, to explore the geometric, electronic, and magnetic properties of the entire H-cluster. These calcns. have allowed us to evaluate, for the first time, the influence of the [Fe4S4] cubane on the [2Fe]H component of the H-cluster in its active (Hox) and CO-inhibited (Hox-CO) states, both of which are paramagnetic (S = 1/2). Our results reveal that the presence of the cubane tunes both the position and the donor strength of the SCys ligand, which, in turn, modulates the internal geometric and electronic structures of the [2Fe]H subcluster. Importantly, the BS methodol. provides an accurate description of the exchange interactions within the H-cluster, permitting insight into the electronic origin of the changes in magnetic properties obsd. exptl. upon conversion of Hox to Hox-CO. Specifically, while the unpaired spin d. in the Hox state is localized on the distal Fe center, in the Hox-CO state, it is delocalized over the [2Fe]H component, such that the proximal Fe center acquires significant spin d. (where distal and proximal refer to the positions of the Fe centers relative to the cubane). To validate our H-cluster models on the basis of exptl. data, two DFT-based approaches and the semiempirical INDO/S method have been employed to compute ESR parameters for the H-cluster states. While most computations yield reasonably accurate g values and ligand hyperfine coupling consts. (i.e., A values) for the Hox and Hox-CO states, they fail to reproduce the isotropic 57Fe A tensors found exptl. Finally, extension of the computational methodol. employed successfully for the Hox and Hox-CO states to the metastable Hoxphoto state, generated by irradn. of the Hox-CO state at cryogenic temps., has allowed us to discriminate between proposed structural models for this species.
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  20. 20
    Greco, C.; Silakov, A.; Bruschi, M.; Ryde, U.; De Gioia, L.; Lubitz, W. Magnetic Properties of [FeFe]-Hydrogenases: A Theoretical Investigation Based on Extended QM and QM/MM Models of the H-Cluster and Its Surroundings. Eur. J. Inorg. Chem. 2011, 2011, 1043– 1049,  DOI: 10.1002/ejic.201001058
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    Pelmenschikov, V.; Birrell, J. A.; Pham, C. C.; Mishra, N.; Wang, H.; Sommer, C.; Reijerse, E.; Richers, C. P.; Tamasaku, K.; Yoda, Y.; Rauchfuss, T. B.; Lubitz, W.; Cramer, S. P. Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory. J. Am. Chem. Soc. 2017, 139, 16894– 16902,  DOI: 10.1021/jacs.7b09751
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    Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory
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    [FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to mol. hydrogen at an exceptionally high rate. We have characterized the catalytically competent hydride state in the [FeFe]-hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) and D. Functional Theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calcns. showed that these spectral features result from an iron-bound terminal hydride, with the Fe-H vibrational frequencies highly dependent on interactions between the hydride and the pendant amine base of the adt cofactor as well as the conserved cysteine terminating the proton transfer chain to the active site. The results are consistent with a conformation of the active site cofactor representing a catalytic state one step before H2 formation. The obsd. motions, therefore, provide mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.
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    A review. A twisted mimic: Upon oxidn. of [(μ-SCH2C(CH3)2CH2S-) {FeI(CO)2PMe3}2], rearrangement yields the mixed-valent FeIFeII cation in a square-pyramid/inverted square-pyramid geometry with a semibridging CO ligand, closely mimicking the [FeFe] hydrogenase enzyme active site. According to de novo design principles, the steric effect of bridgehead bulk in the S-S bridging ligand stabilizes this structure in the absence of the protein matrix.
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Cite this: J. Phys. Chem. Lett. 2019, 10, 21, 6794–6799
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  • Abstract

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    Figure 1

    Figure 1. Structure of the H-cluster in [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) and the proposed catalytic cycle.

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    Figure 2

    Figure 2. Left: Schematic structure of the H-cluster in its Hox state, triply isotope labeled with 57Fe, as well as 13C and 2H in the aza-propane-dithiolate (ADT) ligand. Right: Q-band EPR spectrum (pseudo modulated FID detected) of our Hox preparation. Fitted g-values: (2.1008, 2.0398, 1.9966). A small contribution (<5%) from the Hox-CO state is evident from the feature at g = 2.006 and is marked by an asterisk. Full analysis of the EPR spectrum is presented in Figure S2.

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    Scheme 1

    Scheme 1. Pulse Sequence of the 13C Mims ENDOR Experiment
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    Figure 3

    Figure 3. (a–i) Orientation-selective 13C Mims Q-band ENDOR (34 GHz) spectra of CrHydA1 in the Hox state recorded at 15 K. The indicated field positions are given in mT. π/2 pulses were 20 ns. The waiting time τ was set to 200 ns, while the 400W RF pulse had a length of 60 μs (see Scheme 1). The red traces represent spectral fits obtained using a home-written MATLAB script making use of first order perturbation theory to calculate the ENDOR transition frequencies (details in SI). For the raw (normalized) ENDOR traces (unsymmetrized) along with the number of scans, see Figure S3.

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    Scheme 2

    Scheme 2. Pulse Sequence of the 13C Mims Triple Resonance Experimenta
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    aSee Scheme S2 for extra details.

    Figure 4

    Figure 4. Mims TRIPLE resonance experiment (see pulse sequence in Scheme 2) at B = 1218 mT. The black traces represent the (unsymmetrized) 13C TRIPLE experiment with the pumping frequency RF1 off resonance at the 13C Larmor frequency, i.e., equivalent to the 13C ENDOR spectrum at B = 1218 mT (see Figure 3). The red traces represent the 13C Mims TRIPLE experiment with the RF1 pumping frequency tuned to one of the sharp ENDOR transitions. The “TRIPLE effect” is indicated by the red asterisks.

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    Figure 5

    Figure 5. Top (a): Isosurfaces of total spin density at the 5 × 10–4 a.u. cutoff (corresponding to the Fermi contact HFI term Aiso(13C) = 0.6 MHz) for the representative HoxS = 1/2 DFT model Anative, showing positive (blue) density on the “right” 13C and negative (green) spin density on the “left” 13C nuclei. The model itself is shown in thin wire. Bottom (b): View of DFT model Anative along the Fep–Fed axis displaying the orientation of the calculated methylene 13C hyperfine tensors (Table S1). For extended information, see Figures S6 and S7.

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  • References

    This article references 22 other publications.

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      A review. The current state of knowledge on hydrogenases, esp. recent advances made in understanding the detailed structure and function of these important enzymes. The authors provide an overview of important previous achievements with the main focus on [NiFe] and [FeFe] hydrogenases, and in part also on [Fe] hydrogenases. Recent progress on biomimetic model systems for hydrogenases and devices using hydrogenases both in fuel cells and for H2 prodn. are presented with emphasis on functional aspects. The great progress made in synthesizing model systems for hydrogenases that are functionally active is promising for the future employment of such catalysts in hydrogen technologies.
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      A review. Frustrated Lewis pairs (FLPs) are combinations of Lewis acids and Lewis bases in soln. that are deterred from strong adduct formation by steric and/or electronic factors. This opens pathways to novel cooperative reactions with added substrates. Small-mol. binding and activation by FLPs has led to the discovery of a variety of new reactions through unprecedented pathways. Hydrogen activation and subsequent manipulation in metal-free catalytic hydrogenations is a frequently obsd. feature of many FLPs. The current state of this young but rapidly expanding field is outlined in this Review and the future directions for its broadening sphere of impact are considered.
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      Birrell, J. A.; Rüdiger, O.; Reijerse, E. J.; Lubitz, W. Semisynthetic Hydrogenases Propel Biological Energy Research into a New Era. Joule 2017, 1, 61– 76,  DOI: 10.1016/j.joule.2017.07.009
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      Semisynthetic Hydrogenases Propel Biological Energy Research into a New Era
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      Joule (2017), 1 (1), 61-76CODEN: JOULBR; ISSN:2542-4351. (Cell Press)
      Hydrogenases are enzymes that catalyze the reversible conversion of hydrogen into protons and electrons with high activity and efficiency. Recently, it was discovered that semisynthetic [FeFe] hydrogenases could be created simply by mixing recombinantly produced enzymes, lacking a fully assembled active-site H-cluster, with chem. synthesized di-iron precursor complexes. By using a variety of chem. distinct or isotopically labeled cofactors, crucial insight has been garnered in our understanding of the mechanism of [FeFe] hydrogenases. In particular, the study of proton-coupled events at the H-cluster and stabilization of a terminal hydride bound state have benefited from this approach. This new information could guide the development of novel mol. catalysts with activity and efficiency similar to those of the enzymes. In this Perspective, we review the discoveries attributed to the invention of semisynthetic [FeFe] hydrogenases and discuss the future implications of this technol. for research on hydrogenases, mol. catalysts, and beyond.
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      Sommer, C.; Adamska-Venkatesh, A.; Pawlak, K.; Birrell, J. A.; Rüdiger, O.; Reijerse, E. J.; Lubitz, W. Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of FeFe Hydrogenases. J. Am. Chem. Soc. 2017, 139, 1440– 1443,  DOI: 10.1021/jacs.6b12636
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      Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases
      Sommer, Constanze; Adamska-Venkatesh, Agnieszka; Pawlak, Krzysztof; Birrell, James A.; Ruediger, Olaf; Reijerse, Edward J.; Lubitz, Wolfgang
      Journal of the American Chemical Society (2017), 139 (4), 1440-1443CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN- ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidn. states: [4Fe-4S]2+H2+H ↹ [4Fe-4S]+H and [Fe(I)Fe(II)]H ↹ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H+ + 2e- ↹ H2. Using FTIR spectro-electrochem. on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and det. their intrinsic thermodn. parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]+H - [Fe(I)Fe(II)]H and HredH+ = [4Fe-4S]2+H - [Fe(I)Fe(I)]H (H+) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs. -418 mV); the protonation event (Hred/HredH+) has a pKa ≈ 7.2.
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      Rodrı́guez-Maciá, P.; Pawlak, K.; Rüdiger, O.; Reijerse, E. J.; Lubitz, W.; Birrell, J. A. Inter-cluster Redox Coupling Influences Protonation at the H-cluster in [FeFe] Hydrogenases. J. Am. Chem. Soc. 2017, 139, 15122– 15134,  DOI: 10.1021/jacs.7b08193
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      Intercluster Redox Coupling Influences Protonation at the H-cluster in [FeFe] Hydrogenases
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      Journal of the American Chemical Society (2017), 139 (42), 15122-15134CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      [FeFe] hydrogenases catalyze proton redn. and hydrogen oxidn. displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] sub-cluster ([2Fe]H) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S]H). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S]F clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic mol. catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S]F cluster and the [4Fe-4S]H sub-cluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the pKa of its one electron reduced state. This may help to sustain H2 prodn. at high pH values. These results may apply to all [FeFe] hydrogenases contg. accessory clusters.
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      Glick, B. R.; Martin, W. G.; Martin, S. M. Purification and Properties of the Periplasmic Hydrogenase from Desulfovibrio desulfuricans. Can. J. Microbiol. 1980, 26, 1214– 1223,  DOI: 10.1139/m80-203
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      Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans
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      The periplasmic hydrogenase (I) of D. desulfuricans was isolated and purified. Cells were washed with Tris-EDTA and the enzyme pptd. from the wash with (NH4)2SO4. Absorption chromatog. on DEAE-Sephacel and hydroxylapatite yielded I at >95% purity as judged by gel electrophoresis. I catalyzed the prodn. of >9000 μmol H2/min/mg protein from reduced Me viologen at 37°. It is very stable and resisted inactivation by heat (50% activity remained after 5 min in air at 65°) and by enzyme inhibitors (except N-ethylmaleimide and K ferricyanide). After storage in air at 4° for 1 mo, no activity was lost. I activity was sensitive to ionic environmental changes. With Me viologen the optimum pH was 5.5, but with p-xylene polymeric viologen the optimum was about pH 7 but less sharp. The mol. wt. was 47 × 103, 52 × 103, and 56 × 103 by SDS-gel electrophoresis, gel chromatog., and sedimentation equil., resp., and the isoelec. point was at pH 6.0. I consisted of 1 polypeptide chain with proline at the N-terminal end. I may be useful in the prodn. of H2 from water and solar energy.
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    7. 7
      Hatchikian, E. C.; Forget, N.; Fernández, V. M.; Williams, R.; Cammack, R. Further Characterization of the [Fe]-Hydrogenase from Desulfovibrio desulfuricans ATCC 7757. Eur. J. Biochem. 1992, 209, 357– 365,  DOI: 10.1111/j.1432-1033.1992.tb17297.x
      7
      Further characterization of the [iron]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757
      Hatchikian, E. Claude; Forget, Nicole; Fernandez, Victor M.; Williams, Ruth; Cammack, Richard
      European Journal of Biochemistry (1992), 209 (1), 357-65CODEN: EJBCAI; ISSN:0014-2956.
      The properties of the periplasmic hydrogenase from D. desulfuricans ATCC 7757 were reinvestigated. The pure enzyme exhibited a mol. mass of 53.5 kDa as measured by anal. ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, resp. The N-terminal amino acid sequences of its large and small subunits, detd. up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/mol. and had ε400, 52.5 mM-1·cm-1. The purified hydrogenase showed a specific activity of 62 kV/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its max. activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approx. 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addn., an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Mol. properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XmsVOms7w%253D&md5=bec09ba68d5f8077b364bf7f6f5196fb
    8. 8
      Silakov, A.; Reijerse, E. J.; Albracht, S. P. J.; Hatchikian, E. C.; Lubitz, W. The Electronic Structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: A Q-band 57Fe ENDOR and HYSCORE Study. J. Am. Chem. Soc. 2007, 129, 11447– 11458,  DOI: 10.1021/ja072592s
      8
      The Electronic Structure of the H-Cluster in the [FeFe]-Hydrogenase from Desulfovibrio desulfuricans: A Q-band 57Fe-ENDOR and HYSCORE Study
      Silakov, Alexey; Reijerse, Eduard J.; Albracht, Simon P. J.; Hatchikian, E. Claude; Lubitz, Wolfgang
      Journal of the American Chemical Society (2007), 129 (37), 11447-11458CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The active site of the 57Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examd. using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (Hox) and the CO inhibited form (Hox-CO) all six 57Fe hyperfine couplings were detected. The anal. shows that the apparent spin d. extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six 57Fe nuclei in the H-cluster of the Hox-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S]H (cubane) subcluster. Two weak 57Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe]H subcluster. For the Hox state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An 57Fe line broadening anal. of the X-band CW EPR spectrum indicated, however, that all six 57Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe]H assumes a [FeIFeII] redox configuration where the paramagnetic FeI atom is attached to the [4Fe-4S]H subcluster. The 57Fe hyperfine interactions of the formally diamagnetic [4Fe-4S]H are due to an exchange interaction between the two subclusters as has been discussed earlier by C. V. Popescu and E. Muenck (1999). This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the obsd. modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.
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    9. 9
      Silakov, A.; Reijerse, E. J.; Lubitz, W. Unraveling the Electronic Properties of the Photoinduced States of the H-Cluster in the [FeFe] Hydrogenase from D. desulfuricans. Eur. J. Inorg. Chem. 2011, 2011, 1056– 1066,  DOI: 10.1002/ejic.201001080
      There is no corresponding record for this reference.
    10. 10
      Silakov, A.; Wenk, B.; Reijerse, E.; Albracht, S. P. J.; Lubitz, W. Spin Distribution of the H-cluster in the Hox-CO state of the [FeFe] Hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR Study of 14N and 13C Nuclear Interactions. J. Biol. Inorg. Chem. 2009, 14, 301– 313,  DOI: 10.1007/s00775-008-0449-5
      10
      Spin distribution of the H-cluster in the Hox-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions
      Silakov, Alexey; Wenk, Brian; Reijerse, Eduard; Albracht, Simon P. J.; Lubitz, Wolfgang
      JBIC, Journal of Biological Inorganic Chemistry (2009), 14 (2), 301-313CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)
      Hydrogenases are enzymes which catalyze the reversible cleavage of mol. hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster.". The H-cluster consists of the redox-active binuclear subcluster ([2Fe]H) coordinated by CN- and CO ligands and the cubane-like [4Fe-4S]H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the Hox-CO state the open coordination site at the [2Fe]H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepd. with 13CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three 13C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were obsd. and assigned to 13CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, resp. No 13CO exchange of the CO ligand to the proximal iron was obsd. The hyperfine interactions detected indicate a rather large distribution of the spin d. over the terminal and bridging CO ligands attached to the distal iron. Furthermore, 14N nuclear spin interactions were measured. On the basis of the obsd. 14N hyperfine couplings, which result from the CN- ligands of the [2Fe]H subcluster, it has been concluded that there is very little unpaired spin d. on the cyanides of the binuclear subcluster.
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    11. 11
      Rumpel, S.; Ravera, E.; Sommer, C.; Reijerse, E.; Fares, C.; Luchinat, C.; Lubitz, W. 1H NMR Spectroscopy of [FeFe] Hydrogenase: Insight into the Electronic Structure of the Active Site. J. Am. Chem. Soc. 2018, 140, 131– 134,  DOI: 10.1021/jacs.7b11196
      11
      1H NMR spectroscopy of [FeFe] hydrogenase: Insight into the electronic structure of the active site
      Rumpel, Sigrun; Ravera, Enrico; Sommer, Constanze; Reijerse, Edward; Fares, Christophe; Luchinat, Claudio; Lubitz, Wolfgang
      Journal of the American Chemical Society (2018), 140 (1), 131-134CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii was studied using 1H NMR spectroscopy to identify the paramagnetically shifted 1H resonances assocd. with both the [4Fe-4S]H and the [2Fe]H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 contg. only [4Fe-4S]H was reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active Hox and the CO-inhibited Hox-CO state, revealed addnl. upfield and downfield shifted 1H resonances originating from the 4 methylene protons of the azadithiolate ligand in the [2Fe]H subsite. The two axial protons were affected by neg. spin d., while the 2 equatorial protons experienced pos. spin d. These protons could be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.
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    12. 12
      Berggren, G.; Adamska, A.; Lambertz, C.; Simmons, T. R.; Esselborn, J.; Atta, M.; Gambarelli, S.; Mouesca, J. M.; Reijerse, E. J.; Lubitz, W.; Happe, T.; Artero, V.; Fontecave, M. Biomimetic Assembly and Activation of (FeFe)-Hydrogenases. Nature 2013, 499, 66– 69,  DOI: 10.1038/nature12239
      12
      Biomimetic assembly and activation of [FeFe]-hydrogenases
      Berggren, G.; Adamska, A.; Lambertz, C.; Simmons, T. R.; Esselborn, J.; Atta, M.; Gambarelli, S.; Mouesca, J.-M.; Reijerse, E.; Lubitz, W.; Happe, T.; Artero, V.; Fontecave, M.
      Nature (London, United Kingdom) (2013), 499 (7456), 66-69CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Hydrogenases are the most active mol. catalysts for hydrogen prodn. and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron center (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN- ligands. Through a complex multienzymic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chem. has been used to prep. remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymic activities thus far. Here we show that three synthetic mimics (contg. different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein contg. the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogs. This simple methodol. provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.
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    13. 13
      Esselborn, J.; Lambertz, C.; Adamska-Venkatesh, A.; Simmons, T.; Berggren, G.; Noth, J.; Siebel, J. F.; Hemschemeier, A.; Artero, V.; Reijerse, E. J.; Fontecave, M.; Lubitz, W.; Happe, T. Spontaneous Activation of [FeFe]-Hydrogenases by an Inorganic [2Fe] Active Site Mimic. Nat. Chem. Biol. 2013, 9, 607– 609,  DOI: 10.1038/nchembio.1311
      13
      Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic
      Esselborn, Julian; Lambertz, Camilla; Adamska-Venkatesh, Agnieszka; Simmons, Trevor; Berggren, Gustav; Noth, Jens; Siebel, Judith; Hemschemeier, Anja; Artero, Vincent; Reijerse, Edward; Fontecave, Marc; Lubitz, Wolfgang; Happe, Thomas
      Nature Chemical Biology (2013), 9 (10), 607-609_CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Hydrogenases catalyze the formation of hydrogen. The cofactor (H-cluster) of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] sub-cluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here it is shown that a chem. mimic of the [2Fe] sub-cluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H2-producing catalysts.
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    14. 14
      Lubitz, W.; Reijerse, E.; van Gastel, M. [NiFe] and [FeFe] Hydrogenases Studied by Advanced Magnetic Resonance Techniques. Chem. Rev. 2007, 107, 4331– 4365,  DOI: 10.1021/cr050186q
      14
      [NiFe] and [FeFe] Hydrogenases Studied by Advanced Magnetic Resonance Techniques
      Lubitz, Wolfgang; Reijerse, Eduard; van Gastel, Maurice
      Chemical Reviews (Washington, DC, United States) (2007), 107 (10), 4331-4365CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The understanding of the basic principles of hydrogen prodn. and utilization in microbes is a goal of major importance both for basic research and possible applications. A mechanistic knowledge of the hydrogen conversion and consumption process would allow us to use the organisms or the isolated enzymes in biotechnol. hydrogen prodn. processes. Furthermore, this would provide the necessary fundamental knowledge for designing biomimetic or bioinspired artificial "hydrogenase catalysts" for large-scale hydrogen prodn. in the future. Hydrogenases are ancient enzymes that facilitate the uptake and oxidn. of dihydrogen to protons and release of electrons and also the reverse reaction in a true equil. process. The enzyme hydrogenase is found in many microorganisms from bacteria and archae to eukarya. The review describes in detail the structure and mechanism of action of [NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques.
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    15. 15
      Schweiger, A.; Jeschke, G. Principles of Pulse Electron Paramagnetic Resonance; Oxford University Press: Oxford, 2001.
      There is no corresponding record for this reference.
    16. 16
      Harmer, J. R. Hyperfine Spectroscopy - ENDOR. Emagres 2016, 5, 1493– 1514,  DOI: 10.1002/9780470034590.emrstm1515
      There is no corresponding record for this reference.
    17. 17
      Pham, C. C.; Mulder, D. W.; Pelmenschikov, V.; King, P. W.; Ratzloff, M. W.; Wang, H.; Mishra, N.; Alp, E. E.; Zhao, J.; Hu, M. Y.; Tamasaku, K.; Yoda, Y.; Cramer, S. P. Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment. Angew. Chem., Int. Ed. 2018, 57, 10605– 10609,  DOI: 10.1002/anie.201805144
      17
      Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment
      Pham, Cindy C.; Mulder, David W.; Pelmenschikov, Vladimir; King, Paul W.; Ratzloff, Michael W.; Wang, Hongxin; Mishra, Nakul; Alp, Esen E.; Zhao, Jiyong; Hu, Michael Y.; Tamasaku, Kenji; Yoda, Yoshitaka; Cramer, Stephen P.
      Angewandte Chemie, International Edition (2018), 57 (33), 10605-10609CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calcns. was used to observe and characterize Fe-H/D bending modes in Chlamydomonas reinhardi [FeFe]-hydrogenase (CrHydA1) Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We obsd. a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calcns., we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.
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    18. 18
      Reijerse, E. J.; Pham, C. C.; Pelmenschikov, V.; Gilbert-Wilson, R.; Adamska-Venkatesh, A.; Siebel, J. F.; Gee, L. B.; Yoda, Y.; Tamasaku, K.; Lubitz, W.; Rauchfuss, T. B.; Cramer, S. P. Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy. J. Am. Chem. Soc. 2017, 139, 4306– 4309,  DOI: 10.1021/jacs.7b00686
      18
      Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy
      Reijerse, Edward J.; Pham, Cindy C.; Pelmenschikov, Vladimir; Gilbert-Wilson, Ryan; Adamska-Venkatesh, Agnieszka; Siebel, Judith F.; Gee, Leland B.; Yoda, Yoshitaka; Tamasaku, Kenji; Lubitz, Wolfgang; Rauchfuss, Thomas B.; Cramer, Stephen P.
      Journal of the American Chemical Society (2017), 139 (12), 4306-4309CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      [FeFe]-hydrogenases catalyze the reversible redn. of protons to mol. hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging Cys residue to a [2Fe]H subsite coordinated by CN- and CO ligands featuring a dithiol-amine moiety which serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly obsd. exptl. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) expts. in conjunction with DFT calcns. on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally showed the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.
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    19. 19
      Fiedler, A. T.; Brunold, T. C. Computational Studies of the H-Cluster of Fe-Only Hydrogenases: Geometric, Electronic, and Magnetic Properties and Their Dependence on the [Fe4S4 ] Cubane. Inorg. Chem. 2005, 44, 9322– 9334,  DOI: 10.1021/ic050946f
      19
      Computational Studies of the H-Cluster of Fe-Only Hydrogenases: Geometric, Electronic, and Magnetic Properties and Their Dependence on the [Fe4S4] Cubane
      Fiedler, Adam T.; Brunold, Thomas C.
      Inorganic Chemistry (2005), 44 (25), 9322-9334CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The active sites of Fe-only hydrogenases (FeHases) feature an unusual polynuclear iron-sulfur cluster, known as the H-cluster, that consists of a [Fe4S4] cubane linked to a di-iron subunit (the [2Fe]H component) via a bridging cysteine ligand (SCys). While previous computational studies of FeHases employed H-cluster models that only included the [2Fe]H component, we have utilized d. functional theory (DFT), in conjunction with the broken-symmetry (BS) approach, to explore the geometric, electronic, and magnetic properties of the entire H-cluster. These calcns. have allowed us to evaluate, for the first time, the influence of the [Fe4S4] cubane on the [2Fe]H component of the H-cluster in its active (Hox) and CO-inhibited (Hox-CO) states, both of which are paramagnetic (S = 1/2). Our results reveal that the presence of the cubane tunes both the position and the donor strength of the SCys ligand, which, in turn, modulates the internal geometric and electronic structures of the [2Fe]H subcluster. Importantly, the BS methodol. provides an accurate description of the exchange interactions within the H-cluster, permitting insight into the electronic origin of the changes in magnetic properties obsd. exptl. upon conversion of Hox to Hox-CO. Specifically, while the unpaired spin d. in the Hox state is localized on the distal Fe center, in the Hox-CO state, it is delocalized over the [2Fe]H component, such that the proximal Fe center acquires significant spin d. (where distal and proximal refer to the positions of the Fe centers relative to the cubane). To validate our H-cluster models on the basis of exptl. data, two DFT-based approaches and the semiempirical INDO/S method have been employed to compute ESR parameters for the H-cluster states. While most computations yield reasonably accurate g values and ligand hyperfine coupling consts. (i.e., A values) for the Hox and Hox-CO states, they fail to reproduce the isotropic 57Fe A tensors found exptl. Finally, extension of the computational methodol. employed successfully for the Hox and Hox-CO states to the metastable Hoxphoto state, generated by irradn. of the Hox-CO state at cryogenic temps., has allowed us to discriminate between proposed structural models for this species.
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    20. 20
      Greco, C.; Silakov, A.; Bruschi, M.; Ryde, U.; De Gioia, L.; Lubitz, W. Magnetic Properties of [FeFe]-Hydrogenases: A Theoretical Investigation Based on Extended QM and QM/MM Models of the H-Cluster and Its Surroundings. Eur. J. Inorg. Chem. 2011, 2011, 1043– 1049,  DOI: 10.1002/ejic.201001058
      There is no corresponding record for this reference.
    21. 21
      Pelmenschikov, V.; Birrell, J. A.; Pham, C. C.; Mishra, N.; Wang, H.; Sommer, C.; Reijerse, E.; Richers, C. P.; Tamasaku, K.; Yoda, Y.; Rauchfuss, T. B.; Lubitz, W.; Cramer, S. P. Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory. J. Am. Chem. Soc. 2017, 139, 16894– 16902,  DOI: 10.1021/jacs.7b09751
      21
      Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory
      Pelmenschikov, Vladimir; Birrell, James A.; Pham, Cindy C.; Mishra, Nakul; Wang, Hongxin; Sommer, Constanze; Reijerse, Edward; Richers, Casseday P.; Tamasaku, Kenji; Yoda, Yoshitaka; Rauchfuss, Thomas B.; Lubitz, Wolfgang; Cramer, Stephen P.
      Journal of the American Chemical Society (2017), 139 (46), 16894-16902CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      [FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to mol. hydrogen at an exceptionally high rate. We have characterized the catalytically competent hydride state in the [FeFe]-hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) and D. Functional Theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calcns. showed that these spectral features result from an iron-bound terminal hydride, with the Fe-H vibrational frequencies highly dependent on interactions between the hydride and the pendant amine base of the adt cofactor as well as the conserved cysteine terminating the proton transfer chain to the active site. The results are consistent with a conformation of the active site cofactor representing a catalytic state one step before H2 formation. The obsd. motions, therefore, provide mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.
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    22. 22
      Singleton, M. L.; Bhuvanesh, N.; Reibenspies, J. H.; Darensbourg, M. Y. Synthetic Support of De Novo Design: Sterically Bulky [FeFe]-Hydrogenase Models. Angew. Chem., Int. Ed. 2008, 47, 9492– 9495,  DOI: 10.1002/anie.200803939
      22
      Synthetic support of de novo design: sterically bulky [FeFe]-hydrogenase models
      Singleton, Michael L.; Bhuvanesh, Nattamai; Reibenspies, Joseph H.; Darensbourg, Marcetta Y.
      Angewandte Chemie, International Edition (2008), 47 (49), 9492-9495CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. A twisted mimic: Upon oxidn. of [(μ-SCH2C(CH3)2CH2S-) {FeI(CO)2PMe3}2], rearrangement yields the mixed-valent FeIFeII cation in a square-pyramid/inverted square-pyramid geometry with a semibridging CO ligand, closely mimicking the [FeFe] hydrogenase enzyme active site. According to de novo design principles, the steric effect of bridgehead bulk in the S-S bridging ligand stabilizes this structure in the absence of the protein matrix.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.9b02354.

    • New synthetic route for the 57Fe labeled ADT precursor, experimental and computational procedures, supplementary figures and tables (PDF)

    • Cartesian coordinates of structurally optimized DFT models (ZIP)


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