Intracellular localization of the hydrogenase in Desulfotomaculum orientis

Cell extracts of Desulfotomaculum orientis, grown with H2 plus sulfate as sole energy source, revealed hydrogenase activities between 0.3 and 2 μmol H2 per min and mg protein when methyl viologen was used as electron acceptor. With benzyl viologen, methylene blue, FAD or FMN, lower activities were found; NAD was not reduced. The hydrogenase activity was strongly inhibited by CuCl2; however, copper inhibition was not observed with whole cells, indicating that the hydrogenase is located intracellularly. After high-speed centrifugation of cell-free extracts, varying proportions, between 11 and 90%, of the hydrogenase were detected in the soluble fraction, the rest being associated with the membrane fraction.