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

Hydrogenases are enzymes catalyzing the reversible heterolytic splitting of molecular hydrogen. Despite extensive investigations of this class of enzymes its catalytic mechanism is not yet well understood. In this paper spectroscopic investigations of the active site of [FeFe] hydrogenase are presented. The so-called H-cluster consists of a bi-nuclear catalytically active subcluster connected to a [4Fe4S] ferredoxin-like unit via a Cys-thiol bridge. An important feature of the H-cluster is that both irons in the bi-nuclear subcluster are coordinated by CN and CO ligands. The bi-nuclear site also carries a dithiol bridge, whose central atom has not yet been identified. Nitrogen and oxygen are the most probable candidates from a mechanistic point of view. Here we present a study of the (14)N nuclear quadrupole and hyperfine interactions of the active oxidized state of the H-cluster using advanced EPR methods. In total three (14)N nuclei with quadrupole couplings of 0.95 MHz, 0.35 MHz and 1.23 MHz were detected using hyperfine sublevel correlation spectroscopy (HYSCORE). The assignment of the signals is based on their (14)N quadrupole couplings in combination with DFT calculations. One signal is assigned to the CN ligand of the distal iron, one to a Lys side chain nitrogen and one to the putative nitrogen of the dithiol bridge. Hence, these results provide the first experimental evidence for a di-(thiomethyl)amine ligand (-S-CH(2)-NH-CH(2)-S-) in the bi-nuclear subcluster. This finding is important for understanding the mechanism of [FeFe] hydrogenases, since the nitrogen is likely to act as an internal base facilitating the heterolytic splitting/formation of H(2).

The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-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 (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe 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 [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe 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 Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. 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 observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.