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

H L Drake
1982 Journal of Bacteriology  
Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to,aeration. EDTA did not significantly reduce the lability of the enzymic activity to oxidation (aeration). At 50°C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the
more » ... drogenase, the specific activity of cell-free extracts approximated 4 p,mol of H2 oxidized per min per mg of protein; fourfold 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°C but was slowly inactivated at 700C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cyctochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiological roles for hydrogenase in C. thermoaceticum are discussed. yeast extract, 5.0; tryptone, 5.0; (NH42SO4, 0.5; MgSO4-7H2O, 0.5; Fe(NH4)2(SO4)2, 0.04; H2SeO2, 702 on May 9, 2020 by guest
doi:10.1128/jb.150.2.702-709.1982 fatcat:vanf6tapmbff5a2l3oopykt6zu