Energy conservation by the H2:Heterodisulfide oxidoreductase from Methanosarcina mazei Go1:: Identification of two proton-translocating segments

The membrane-bound H-2:heterodisulfide oxidoreductase system of the methanogenic archaeon Methanosarcina mazei Go1 catalyzed the H-2-dependent reduction of 2-hydroxyphenazine and the dihydro-2-hydroxyphenazine-dependent reduction of the heterodisulfide of HS-CoM and HS-CoB (CoM-S-S-CoB). Washed inverted vesicles of this organism were found to couple both processes with the transfer of protons across the cytoplasmic membrane. The maximal H+/2e(-) ratio was 0.9 for each reaction. The electrochemical proton gradient (Delta mu(H+)) thereby generated was shown to drive ATP synthesis from ADP plus P-i, exhibiting stoichiometries of 0.25 ATP synthesized per two electrons transported for both partial reactions. ATP synthesis and the generation of Delta mu(H+) were abolished by the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847), The ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide did not affect H+ translocation but led to an almost complete inhibition of ATP synthesis and decreased the electron transport rates. The latter effect was relieved by the addition of SF 6847. Thus, the energy-conserving systems showed a stringent coupling which resembles the phenomenon of respiratory control. The results indicate that two different proton-translocating segments are present in the H-2:heterodisulfide oxidoreductase system; the first involves the 2-hydroxyphenazine-dependent hydrogenase, and the second involves the heterodisulfide reductase.