Energy conservation by the H2:heterodisulfide oxidoreductase from Methanosarcina mazei Gö1: identification of two proton-translocating segments - PubMed (original) (raw)

Energy conservation by the H2:heterodisulfide oxidoreductase from Methanosarcina mazei Gö1: identification of two proton-translocating segments

T Ide et al. J Bacteriol. 1999 Jul.

Abstract

The membrane-bound H2:heterodisulfide oxidoreductase system of the methanogenic archaeon Methanosarcina mazei Gö1 catalyzed the H2-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 (DeltamicroH+) thereby generated was shown to drive ATP synthesis from ADP plus Pi, exhibiting stoichiometries of 0.25 ATP synthesized per two electrons transported for both partial reactions. ATP synthesis and the generation of DeltamicroH+ 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 H2:heterodisulfide oxidoreductase system; the first involves the 2-hydroxyphenazine-dependent hydrogenase, and the second involves the heterodisulfide reductase.

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Figures

FIG. 1

FIG. 1

Proton uptake by washed inverted vesicles from M. mazei Gö1. The experiments were performed as described in Materials and Methods. The amount of translocated protons was calculated from the difference between maximal alkalinization and the final baseline after reacidification. The reaction was started by pulses of 2-OH-phenazine or dihydro-2-OH-phenazine as indicated. SF 6847 was added as an ethanolic solution to a final concentration of 15 nmol/mg of protein. (A) H2-dependent reduction of 2-OH-phenazine under an atmosphere of molecular hydrogen; (B) dihydro-2-OH-phenazine-dependent reduction of CoM-S-S-CoB under an atmosphere of molecular nitrogen.

FIG. 2

FIG. 2

Redox-driven ATP synthesis as catalyzed by washed inverted vesicles from M. mazei Gö1. The experiments were performed as described in Materials and Methods. The concentrations of ADP, SF 6847, and DCCD were 0.16 mM, 25 μM, and 250 nmol/mg of protein, respectively. (A) ATP synthesis in the course of H2-dependent-2-OH-phenazine reduction. The glass cuvette was gassed with H2 and contained 600 μl of buffer A, 1.6 mM AMP, and 125 μM 2-OH-phenazine. The reaction was started by the addition of washed vesicles (14 μg of protein). The following additions were made: ADP (■), ADP and SF 6847 (▵), ADP and DCCD (▴), ADP, SF 6847, and DCCD (●), ADP under N2 (○). □, ADP omitted. (B) ATP synthesis coupled to dihydro-2-OH-phenazine-dependent heterodisulfide reduction. Details of the experiment and symbols are as described for panel A except that (i) the reaction mixture contained dihydro-2-OH-phenazine (instead of 2-OH-phenazine) and 240 nmol of CoM-S-S-CoB and (ii) open circles represent assays with ADP added and CoM-S-S-CoB omitted. The atmosphere was N2.

FIG. 3

FIG. 3

Tentative scheme of membrane-bound electron transfer coupled to proton translocation in M. mazei Gö1. Mphen, methanophenazine; MphenH2, dihydromethanophenazine; VhoG, 40-kDa subunit of the F420-nonreducing hydrogenase; VhoA, 60-kDa subunit of the F420-nonreducing hydrogenase; VhoC, cytochrome _b_1 (Cytb1) encoded by the third gene (vhoC) of the hydrogenase operon; HdrDE, subunits of the heterodisulfide reductase; FeS, iron-sulfur clusters; Ni, nickel-iron center of the F420-nonreducing hydrogenase.

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