Solubilization and Properties of a Particulate Hydrogenase from Methanobacterium Strain G2R

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Abstract
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. About 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69-85% 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 solubiized and soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by O2 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.degree. but not 77.degree. C. A number of nonspecific dyes, FAD, and riboflavin 5''-phosphate were effective electron acceptors; NAD, NADP and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform and chloramphenicol. The MW of the solubilized enzyme was 900,000, with subunits of MW 38,500, 50,700 and approximately 80,000. In intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

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