Protein complexing in a methanogen suggests electron bifurcation and electron delivery from formate to heterodisulfide reductase

Abstract

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(420)-nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electron-bifurcation model of energy conservation, the composition of the complex also suggests that either H(2) or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H(2) via F(420)-nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H(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(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(2), is closely integrated into the methanogenic pathway.

Fig. 1.

The methanogenic pathway with hypothesized roles for the heterodisulfide reductase complex. Electron flow from formate or hydrogen to Hdr may drive the reduction of the CoM-S-S-CoB heterodisulfide, as well as the reduction of ferredoxin via flavin-mediated electron bifurcation as outlined by Thauer et al. (1). Eha, energy-conserving hydrogenase; Fdh, formate dehydrogenase; Fru, F₄₂₀-reducing hydrogenase; Fwd, formyl-MFR dehydrogenase; Hdr, heterodisulfide reductase; Hmd, H₂-dependent methylene-H₄MPT dehydrogenase; Mcr, methyl-CoM reductase; Mer, methylene-H₄MPT reductase; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT-CoM methyltransferase; Vhu, F₄₂₀-nonreducing hydrogenase.

Fig. 2.

Growth of Δvhu Δvhc strain vs. wild-type strain on formate or H₂. OD₆₆₀, optical density at 660 nm. (■) MM1272 (Δvhu Δvhc) grown on formate; (◆) MM901 (wild type) grown on formate; (●) MM1272 grown on H₂; (▲) MM901 grown on H₂. Data are from three independent cultures and error bars represent one SD around the mean.

Fig. 3.

Model of complex of proteins that interact with Hdr. When H₂ is used as the electron donor for methanogenesis, electrons are transferred to Hdr via Vhu. Flavin-mediated electron bifurcation at HdrA then results in reduction of the CoM-S-S-CoB heterodisulfide and a ferredoxin that is used by Fwd for the first step in methanogenesis. When hydrogen is limiting or is replaced by formate, Fdh is highly expressed (10) and incorporates into the complex. When formate is used as the electron donor for methanogenesis, electrons are transferred to Hdr from formate via Fdh. Fd(red), reduced ferredoxin; Fd(ox), oxidized ferredoxin.