Essential anaplerotic role for the energy-converting SEE COMMENTARY hydrogenase Eha in hydrogenotrophic methanogenesis Thomas J. Liea,1, Kyle C. Costaa,1, Boguslaw Lupab, Suresh Korpolec, William B. Whitmanb, and John A. Leigha,2 aDepartment of Microbiology, University of Washington, Seattle WA 98195; bDepartment of Microbiology, University of Georgia, Athens, GA 30602; and cMicrobial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Sector 39A, Chandigarh, India Edited by Ralph S. Wolfe, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 11, 2012 (received for review May 24, 2012) Despite decades of study, electron flow and energy conservation provided by the final step of methanogenesis, which involves an in methanogenic Archaea are still not thoroughly understood. For exergonic reduction of a heterodisulfide of two methanogenic methanogens without cytochromes, flavin-based electron bifurcation cofactors (CoM-S-S-CoB) by heterodisulfide reductase (Hdr). has been proposed as an essential energy-conserving mechanism that Methanogens with cytochromes harvest the energy yielded in couples exergonic and endergonic reactions of methanogenesis. How- heterodisulfide reduction with a proton-exporting electron trans- ever, an alternative hypothesis posits that the energy-converting hy- port chain. However, methanogens without cytochromes lack this drogenase Eha provides a chemiosmosis-driven electron input to the electron transport chain and an alternative explanation is required. endergonic reaction. In vivo evidence for both hypotheses is incom- Here we present results supporting an emerging view of meth- plete. By genetically eliminating all nonessential pathways of H2 me- anogenesis without cytochromes. The emerging model diverges Methanococcus maripaludis tabolism in the model methanogen and from the conventional picture of a linear pathway of CO2 reduction using formate as an additional electron donor, we isolate electron flow to methane. Instead, a cyclical pathway involving electron bifurca- for methanogenesis from flux through Eha. We find that Eha does not tion has been proposed (1) (Fig. 1). The reductions of the hetero- function stoichiometrically for methanogenesis, implying that electron disulfide and CO2 are coupled in the flavin-containing enzyme bifurcation must operate in vivo. We show that Eha is nevertheless complex centered around Hdr. For each pair of electrons accepted, essential, and a substoichiometric requirement for H2 suggests that one electron is used for the exergonic reduction of CoM-S-S-CoB, its role is anaplerotic. Indeed, H2 via Eha stimulates methanogenesis and one is used to reduce a low-potential ferredoxin that in turn from formate when intermediates are not otherwise replenished. donates electrons for the reduction of CO to formyl-MFR. Hence, fi 2 These results t the model for electron bifurcation, which renders electron bifurcation, a nonchemiosmotic form of energy conserva- the methanogenic pathway cyclic, and as such requires the replen- tion, couples the exergonic and endergonic steps of methanogenesis fi ishment of intermediates. De ning a role for Eha and verifying and allows for the net availability of chemiosmotic energy for ATP electron bifurcation provide a complete model of methanogenesis synthesis. The electron bifurcation model renders methanogenesis where all necessary electron inputs are accounted for. a cyclic process, in which late steps are coupled by electron flow to the initial step, and explains why in cell extracts, CH4 production hydrogenotrophs | H2:F420 oxidoreductase | ferredoxin | from CO2 requires an input of C-1 intermediates (4). Electron bi- MICROBIOLOGY formate dehydrogenase furcation is supported by experiments with whole cells (5), with purified enzymes (6), and by the characterization of an enzyme ethanogenesis is an anaerobic respiration carried out by a complex in which it could take place (7). However, these studies do Mphylogenetically related group of Archaea within the phy- not explain the presence in most methanogens without cytochromes lum Euryarchaeota. Methanogens are divided into two metabolic of the energy-converting hydrogenase Eha that is apparently linked types, those without and those with cytochromes (1). Methanogens to the first step (2). Electron flux from this hydrogenase would without cytochromes use H2 as an electron donor and are termed appear to compete with flux from electron bifurcation as well as to hydrogenotrophic. Some species can substitute H2 with formate, consume chemiosmotic energy, leaving a deficit for ATP synthesis. and a few can use secondary alcohols. CO2 is the electron acceptor Whatever the correct model for energy conservation, it likely and is reduced to methane. Methanogens with cytochromes re- centers around reactions that reduce low-potential ferredoxins. duce certain methyl compounds or the methyl carbon of acetate Three such reactions are proposed to occur in methanogens to methane and are called methylotrophic. Many can also use H2 without cytochromes. Two of these reactions are those men- and CO2, as can hydrogenotrophic methanogens. tioned above, the concomitant reduction of Fd and CoM-S-S- Although the pathways of methanogenesis have long been CoB that occurs in electron bifurcation, and the H2-dependent known, an understanding of energy conservation has been slower reduction of Fd by the energy-converting hydrogenase Eha, both to emerge. Methanogens with and without cytochromes both of which are proposed to lead to the endergonic reduction of + export Na when a methyl group is transferred from the carrier CO2 to formyl-MFR. A third such reaction, which reduces a Fd tetrahydromethanopterin (H4MPT) to coenzyme M (CoM) (Fig. 1). with another energy-converting hydrogenase, Ehb, functions in + The Na gradient across the membrane is used directly for ATP anabolic CO2 fixation reactions and does not appear to be in- synthesis or is converted by an antiporter to a proton gradient. volved in methanogenesis (8, 9). However, for methanogenesis from CO2, the initial reduction of CO2 to a formyl group attached to methanofuran (MFR) is end- ergonic. How energy is provided to drive this reaction is not well Author contributions: T.J.L., K.C.C., B.L., W.B.W., and J.A.L. designed research; T.J.L., K.C.C., understood. Methanogens with and without cytochromes have B.L., and S.K. performed research; T.J.L., K.C.C., B.L., S.K., W.B.W., and J.A.L. analyzed membrane-associated energy-converting hydrogenases that couple data; and T.J.L., K.C.C., B.L., W.B.W., and J.A.L. wrote the paper. the reduction of low-potential ferredoxins (Fd) to a chemiosmotic The authors declare no conflict of interest. membrane gradient (2). If such a Fd donates electrons for CO2 This article is a PNAS Direct Submission. reduction, an energy-converting hydrogenase is the conduit of en- See Commentary on page 15084. ergy for this reaction. Indeed, for methanogens with cytochromes, 1T.J.L. and K.C.C. contributed equally to this work. an energy-converting hydrogenase is required for CO2 reduction (3). 2To whom correspondence should be addressed. E-mail: [email protected]. fi However, the energy requirement for the rststepinthepathway This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. results in a need for additional energy conservation. This could be 1073/pnas.1208779109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208779109 PNAS | September 18, 2012 | vol. 109 | no. 38 | 15473–15478 Downloaded by guest on September 23, 2021 H + Na+ translocation 2 potentially the first step via electron bifurcation (1, 6, 7), can be CO2 + MFR CH4 Fd(ox) substituted by formate dehydrogenase (Fdh) during growth on Fwd Eha CoM-S-S-CoB Fd(red) formate (7). The F420-reducing hydrogenases (Fru and Frc) Fwd generate F420H2 for the second and third reductive steps of Hdr Formyl-MFR H4MPT methanogenesis, but the Fdh is also F420 reducing (12, 13). The Mcr Fdh hydrogenase Hmd catalyzes the second reductive step directly Vhu/ HS-CoB Vhc Ftr MFR with H2, but its function is redundant with Mtd, which uses re- Formate duced F420 for the same purpose (11). Finally, the anabolic en- CO H 2 ergy-converting hydrogenase Ehb is nonessential in the presence CH3-CoM 2 Formyl-H4MPT Fdh F420 fi H+ of xed carbon and is not required for methanogenesis (8, 9). Mtr HS-CoM Mch Only Eha remains as possibly essential. Na+ F H 420 2 H2O translocation Based on the above considerations, we expected that H2 would Methenyl-H MPT not be needed as an intermediate for methanogenesis from CH -H MPT F H 4 3 4 420 2 formate. Indeed, experiments with cell suspensions have already Fru/Frc H2 F F420H2 shown that rates of methanogenesis can substantially exceed 420 Mer Hmd H+ rates of H2 production from formate (5). As a further test, our F420H2 F420H2 approach here was to genetically remove formate-hydrogen lyase Mtd activity, so that H2 would not be produced from formate. If Methylene-H4MPT F420 growth still occurred on formate without added H2, then H2 was not a required intermediate. Because Fdh is F420 reducing, re- Fig. 1. The methanogenic pathway. Eha, energy-converting hydrogenase moval of formate-hydrogen lyase activity amounts to the removal A; Fdh, formate dehydrogenase; Fru and Frc, F420-reducing hydrogenases; of F420:H2 oxidoreductase activity. Two such activities are Ftr, formyl-MFR:H4MPT formyltransferase; Fwd, formyl-MFR dehydrogenase; fi known, the direct Fru or Frc activity and the Hmd-Mtd cycle Hdr, heterodisul de reductase; Hmd, H2-dependent methylene-H4MPT de- fru frc hmd hydrogenase; Mch, methenyl-H4MPT cyclohydrolase; Mcr, methyl-CoM re- (Fig. 1) (11). Therefore, deletion of , , and should ductase; Mer, methylene-H4MPT reductase; Mtd, F420-dependent methylene- eliminate both modes of formate-hydrogen lyase activities. H4MPT dehydrogenase; Mtr, methyl-H4MPT-CoM methyltransferase; Vhu However, cell suspensions of a ΔfruΔfrcΔhmd mutant (MM1290, and Vhc, F420-nonreducing (Hdr-associated) hydrogenases. henceforth designated Δ3H2ase) still produced substantial H2 from formate (Fig. 2). Furthermore, the mutant grew not only on formate as predicted, but also on H , albeit poorly (Fig.