title: A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.
abstract: The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the beta-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH- bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the "ready" state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the "unready" state of the enzyme.
authors.affiliation: Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany.
authors.forename: Maurice
authors.initials: M
authors.lastname: van Gastel
authors.forename: Matthias
authors.initials: M
authors.lastname: Stein
authors.forename: Marc
authors.initials: M
authors.lastname: Brecht
authors.forename: Olga
authors.initials: O
authors.lastname: Schröder
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Lendzian
authors.forename: Robert
authors.initials: R
authors.lastname: Bittl
authors.forename: Hideaki
authors.initials: H
authors.lastname: Ogata
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D003545:Cysteine / Q000737:chemistry; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011522:Protons; D013455:Sulfur / Q000737:chemistry
chemical_list: D008024:Ligands; D011522:Protons; D013455:Sulfur; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine