Contents 1. Introduction 4331 2. Structure of [NiFe] and [FeFe] Hydrogenases 4333 2.1. [NiFe] Hydrogenases 4333 2.2. [FeFe] Hydrogenases 4335 3. Redox States of Hydrogenases 4336 3.1. [NiFe] Hydrogenase 4336 3.2. [FeFe] Hydrogenase 4337 4. A Survey of Structural Methods Used in Hydrogenase Research 4338 5. Magnetic Resonance Studies of [NiFe] Hydrogenases 4339 5.1. Overview of EPR Spectra in the Various Redox States of the Enzyme 4339 5.2. The Presence of Ni and Fe in the Active Site 4340 5.3. The Oxidized (As-Isolated) States 4341 5.3.1. g Tensor Analysis and the Ligand Field 4341 5.3.2. Hyperfine Interactions 4342 5.3.3. Activation and Inactivation Studies 4343 5.3.4. The Fe−S Clusters 4344 5.4. The Active Intermediate State 4344 5.4.1. g Tensor Analysis and the Ligand Field 4344 5.4.2. Hyperfine Interactions 4345 5.4.3. The Fe−S Clusters 4345 5.5. Inhibition of the Enzyme 4346 5.5.1. Inhibition by O 2 4346 5.5.2. Inhibition by CO 4346 5.5.3. Other Inhibitory Agents 4346 5.6. Light Sensitivity of the Enzyme 4346 5.7. EPR-Silent States 4347 5.8. Other Hydrogenases Containing Nickel 4347 5.9. DFT Calculations and Electronic Structure 4347 5.10. The Catalytic Cycle 4349 6. Magnetic Resonance Studies of [FeFe] Hydrogenases 4350 6.1. Overview of EPR Spectra in Various Redox States of the Enzyme 4350 6.2. The Oxidized (As Isolated) State 4350 6.3. The Intermediate States 4351 6.4. The H 2 -Reduced State 4352 6.5. The CO-Inhibited State 4353 6.6. Light Sensitivity of the CO-Inhibited State 4354 6.7. Electronic Structure of the H-Cluster 4354 6.7.1. Origin of the 57 Fe hyperfine couplings in the H-Cluster 4354 6.7.2. Redox States of the Iron Atoms in the Binuclear Cluster 4354 6.8. Possible Mechanisms for the Catalytic Cycle 4355 7. Concluding Remarks 4356 8. List of Abbreviations 4357 9. Acknowledgments 4358 10. Appendix I. Advanced EPR Methods Used in Hydrogenase Research 4358 10.1. FID-Detected EPR 4358 10.2. ESE-Detected EPR 4358 10.3. Three-Pulse Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy 4359 10.4. Four-Pulse ESEEM (HYSCORE) 4359 10.5. Pulse Electron−Nuclear Double Resonance (ENDOR) Spectroscopy 4359 10.6. Pulse Electron−Nuclear−Nuclear Triple Resonance 4359 10.7. Pulse Electron−Electron Double Resonance (PELDOR/DEER) Spectroscopy 4359 10.8. ELDOR-Detected NMR 4360 11. Appendix II. Crystal Field Considerations for Ni III and Ni I in a Square Pyramidal Crystal Field 4360 12. Appendix III. The Spin Exchange Model of the H-cluster in [FeFe] Hydrogenase 4360 13. References 4361

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83-93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a(iso)=13.7, 11.8 MHz) are assigned to the beta-CH(2) protons of the nickel-coordinating cysteine 549, and a third proton (a(iso)=8.9 MHz) is assigned to a beta-CH(2) proton of cysteine 546. Using D(2)O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron-recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075-13083)-is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a(iso)=-3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni (3d(z)(2)) orbital. Nitrogen signals that belong to the nitrogen N(epsilon) of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.

The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labeled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F (DvMF) [NiFe]-hydrogenase. A unique ‘wagging’ mode involving H− motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations. Upon Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.