Affordable Access

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

  • Foerster, Stefanie
  • van Gastel, Maurice
  • Brecht, Marc
  • Lubitz, Wolfgang
Published Article
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
Publication Date
Jan 01, 2005
PMID: 15611882


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.

Report this publication


Seen <100 times