The purified H2-uptake hydrogenase of Bradyrhizobium japonicum, containing no cytochrome b, catalyzed efficient H2-ubiquinone oxidoreductase activity. Hydrogen-oxidizing membranes also catalyzed H2-ubiquinone oxidoreductase activity, and the site of ubiquinone reduction was localized to the H2-quinone oxidoreductase complex based on comparative antimycin A and HQNO titrations of both H2-ubiquinone-1 oxidoreductase and ubiquinol-1 oxidase activities. A variety of quinones could function as electron acceptors of both pure or membrane-bound hydrogenase, including ubiquinone-0 Q0), ubiquinone-1 (Q1), duroquinone and menadione, indicating relatively loose substrate specificity with regard to the quinone head group. Both the redox potential and the quinone structure determined the efficiency of hydrogenase turnover. Among short-chain ubiquinones, the isoprenoid chain length had a profound affect on Km, with each additional isoprenoid unit resulting in the Km of the membrane-bound enzyme to decrease more than an order of magnitude. For pure enzyme, the Km values for Q0, Q1 and Q2 were 1.97 mM, 68.8 μM and 3.1 μM, respectively. Vmax was also influenced by the substrate isoprenoid chain length for the pure enzyme. The inhibition patterns of H2-dependent Q1 versus MB reduction by the quinone analogs (2-n-heptyl-4-hydroxyquinoline N-oxide and Antimycin A) were significantly different, and clear differences in pH optima for the two activities were observed. In addition, the two hydrogen-dependent electron acceptor activities (Q1 and MB) exhibited different time-dependent inactivation patterns by the chemical modification reagent diazobenzene sulfonate. Ubiquinone and MB therefore react by different mechanisms (perhaps at different sites) within the hydrogenase complex in situ. The inhibition pattern of hydrogen-ubiquinone oxidoreductase activity by antimycin A was clearly different than antimycin A inhibition of ubiquinol oxidation at the bc1 complex. This is, to our knowledge, the first report of antimycin A inhibition of a hydrogenase complex, and also of a quinone reducing site of a primary dehydrogenase. When pure hydrogenase is assayed in the absence of dithionite, a delay (lag phase) is observed prior to attainment of full activity. The length of this lag period (in minutes) was inversely dependent on ubiquinone concentration, and was greatly reduced (but not eliminated) at saturating ubiquinone levels. These effects were obtained with both Q1 and MB as electron acceptor, and the lag phases with Q1 were significantly longer than with MB. Electron acceptor binding to hydrogenase is thus required for reductive activation of hydrogenase during turnover. © 1995.
Ferber, D. M., Moy, B., & Maier, R. J. (1995). Bradhyrhizobium japonicum hydrogen-ubiquinone oxidoreductase activity: quinone specificity, inhibition by quinone analogs, and evidence for separate sites of electron acceptor reactivity. BBA - Bioenergetics, 1229(3), 334–346. https://doi.org/10.1016/0005-2728(95)00012-8