Purification and properties of the hydrogenase of Desulfovibrio desulfuricans

Abstract

Hydrogenase from Desulfovibrio desulfuricans has been purified about fifty-fold. At the highest level of purity 1 mg of protein N catalyses the oxidation of 2.6·10⁶ μl of H₂ per hour by methylene blue at 34°. Purified hydrogenase was found to be activated by FeCl₂ or FeCl₃, the increase in activity being the same with benzyl viologen or methylene blue as oxidant. A thermostable cytochrome-like component was obtained during the purification, which has no hydrogenase activity, but could be reduced by hydrogen and hydrogenase and reoxidised by O₂, methylene blue or FMN. Methylene blue and benzyl viologen were reduced rapidly and at the same rate by hydrogen in the presence of purified hydrogenase; FMN, FAD and riboflavin were reduced at about one-hundredth of this rate, while DPN, TPN, sulfate and ferricyanide were not reduced. The optimum pH for methylene blue reduction was 8.4, cysteine or glutathione was essential for enzyme activity and serum albumin had a protective action at high enzyme dilutions. Hydrogenase was inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine or glutathione. Cyanide and other metal-binding agents, hydroxylamine and nitrite were also inhibitory.