Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.
In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J. Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H(-)) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(I) oxidation state is assumed for this species with a proton (H(+)) removed from the bridge between the nickel and the iron atom.
Foerster
Stefanie
S
Max-Volmer-Laboratorium für Biophysikalische Chemie, Fakultät für Mathematik und Naturwissenschaften, Technische Universität Berlin, D-10623 Berlin, Germany.
Stein
Matthias
M
Brecht
Marc
M
Ogata
Hideaki
H
Higuchi
Yoshiki
Y
Lubitz
Wolfgang
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation
D001426:Bacterial Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase