Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum

Abstract

Cell-free extracts of the homoacetate-fermenting bacterium C. thermoaceticum catalyze the H-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5-9 and was extremely sensitive to aeration. EDTA did not significantly reduce the lability of the enzymic activity to oxidation (aeration). At 50.degree. C, when methyl viologen and H were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts was .apprx. 4 .mu.mol of H2 oxidized/min per mg protein; 4-fold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55.degree. C, but was slowly inactivated at 70.degree. C. NAD, NADP, FAD, FMN and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cytochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide and CO inhibit catalysis. A kinetic study is presented and the possible physiological roles for hydrogenase in C. thermoaceticum are discussed.