Kinetic Studies on Hydrogenase

The kinetics of paraH2-orthoH2 conversion and the H-D exchange reactions catalyzed by highly purified desulfovibrio hydrogenase [H2: ferricytochrome c3 oxidoreductase] were studied. These reactions were catalyzed by the enzyme in the absence of cytochrome c3, but were, in general, stimulated by the cytochrome. In the reaction system with paraH2 over D2O, the conversion of paraH2 to normal H2, as well as the isotope exchange reactions, were catalyzed by the enzyme, in contrast to earlier observations made with rather crude hydrogenase preparations from other species of bacteria. This reaction was not stimulated, or even inhibited, by cytochrome c3. Kinetic analyses of the rates of these reactions revealed that the two hydrogen atoms of the enzyme-bound hydrogen molecule have different exchange rates with hydrogen ions of the medium. All the kinetic constants for individual steps of the conversion and exchange reactions catalyzed by aqueous hydrogenase were calculated. A possible mechanism for the binding of a hydrogen molecule to the enzyme was proposed.