Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.