Flavin-based Electron Bifurcation Research Articles

The Wolfe cycle comes full circle

In 1988, Rouviere and Wolfe (1) suggested that methane formation from H2 and CO2 by methanogenic archaea could be a cyclical process. Indirect evidence indicated that the first step, the reduction of CO2 to formylmethanofuran, was somehow coupled to the last step, the reduction of the heterodisulfide (CoM-S-S-CoB) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Over 2 decades passed until the coupling mechanism was unraveled in 2011: Via flavin-based electron bifurcation, the reduction of CoM-S-S-CoB with H2 provides the reduced ferredoxin (Fig. 1h) required for CO2 reduction to formylmethanofuran (2) (Fig. 1a). However, one question still remained unanswered: How are the intermediates replenished that are removed for the biosynthesis of cell components from CO2 (orange arrows in Fig. 1)? This anaplerotic (replenishing) reaction has recently been identified by Lie et al. (3) as the sodium motive force-driven reduction of ferredoxin with H2 catalyzed by the energy-converting hydrogenase EhaA-T (green arrow in Fig. 1).

Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

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 H(2) 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 H(2) suggests that its role is anaplerotic. Indeed, H(2) 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.

NADP+Reduction with Reduced Ferredoxin and NADP+Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex inClostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred2-+NADH+2 NADP++H+=Fdox+NAD++2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.

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

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.

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.