The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianium

Abstract

A mechanism for the reduction and oxidadtion of methyl viologen by Clostridium pasteurianium hydrogenase (hydrogen:ferredoxin oxidoreductase, EC 1.12.7.1) is proposed. Double reciprocal plots for methyl viologen reduction and oxidation at pH values 7.0–9.85 are linear, and the plots for reduction and oxidation are intersecting. Such data are consistent with a mechanism in which the H 2 and one methyl viologen bind (either in order or randomly) with subsequent reduction and release of the methyl viologen. A second methyl viologen then is bound, reduced and released. Comparison of the calculated K eq ′ with the Haldane expression in which both methyl viologens react at the same rate show a large difference. This difference indicates that the two methyl viologens react at different rates. Addition of oxidized electron carriers inhibits the hydrogen-deuterium exhcnage reaction (i.e., the exchange of protons between H 2 and 2H 2O). CO reversibly inhibits methyl viologen reduction and is competitive vs. H 2. O 2 acts as an irreversible inhibitor.