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In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579-591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, [F.sub.420]-nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electronbifurcation model of energy conservation, the composition of the complex also suggests that either [H.sub.2] or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-[H.sub.2] via [F.sub.420]-nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heteroclisulfide rather than the use of [H.sub.2] as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking [F.sub.420]-nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like [H.sub.2], is closely integrated into the methanogenic pathway. energy conservation | Archaea | formate dehydrogenase | forrnylrnethanofuran dehydrogenase | [F.sub.420]-nonreducing hydrogenase doi/ 10.1073/pnas.1003653107

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H2 metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H2 suggests that its role is anaplerotic. Indeed, H2 via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for. [ABSTRACT FROM AUTHOR]