title: Pre-steady-state kinetics of the reactions of [NiFe]-hydrogenase from Chromatium vinosum with H2 and CO.
abstract: Results are presented of the first rapid-mixing/rapid-freezing studies with a [NiFe]-hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia-SR state) to the Nia-C* state was completed in less than 8 ms, a rate consistent with the H2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia-SR state did not react with CO. It was discovered, contrary to expectations, that the Nia-C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia-C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia-C* state decayed within 40 ms, due to the generation of the Nia-S.CO state (the EPR-silent state of the enzyme with bound CO). The Nia-C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Nia*.CO) by illumination at 30 K (evoking the Nia-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia-L* and the Nia*.CO states is formally monovalent.
authors: E.C. Slater Institute, University of Amsterdam, The Netherlands.
authors: R P
authors: RP
authors: Happe
authors: W
authors: W
authors: Roseboom
authors: S P
authors: SP
authors: Albracht
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001594:Benzyl Viologen / Q000737:chemistry; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry*; D002844:Chromatium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000737:chemistry*; D006864:Hydrogenase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010269:Paraquat / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D001594:Benzyl Viologen; D002248:Carbon Monoxide; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010269:Paraquat; D010100:Oxygen