An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase

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 β-CH₂ protons of the nickel-coordinating cysteine 549, and a third proton (a _iso=8.9 MHz) is assigned to a β-CH₂ proton of cysteine 546. Using D₂O 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 $ {\left( {3d_{{z^{2} }} } \right)} $ orbital. Nitrogen signals that belong to the nitrogen N_ε 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.