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

Abstract

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. M ethanogenesis is an anaerobic respiration carried out by a phylogenetically related group of Archaea within the phylum Euryarchaeota. Methanogens are divided into two metabolic types, those without and those with cytochromes (1). Methanogens without cytochromes use H 2 as an electron donor and are termed hydrogenotrophic. Some species can substitute H 2 with formate, and a few can use secondary alcohols. CO 2 is the electron acceptor and is reduced to methane. Methanogens with cytochromes reduce certain methyl compounds or the methyl carbon of acetate to methane and are called methylotrophic. Many can also use H 2 and CO 2 , as can hydrogenotrophic methanogens.Although the pathways of methanogenesis have long been known, an understanding of energy conservation has been slower to emerge. Methanogens with and without cytochromes both export Na + when a methyl group is transferred from the carrier tetrahydromethanopterin (H 4 MPT) to coenzyme M (CoM) (Fig. 1). The Na + gradient across the membrane is used directly for ATP synthesis or is converted by an antiporter to a proton gradient. However, for methanogenesis from CO 2 , the initial reduction of CO 2 to a formyl group attached to methanofuran (MFR) is endergonic. How energy is provided to drive this reaction is not well understood. Methanogens with and without cytochromes have membrane-associated energy-converting hydrogenases that couple the reduction of low-potential ferredoxins (Fd) to a chemiosmotic membrane gradient (2). If such a Fd donates electrons for CO 2 reduction, an energy-converting hydrogenase is the conduit of ener...

Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis

“…For 62 of these genes, the EI correctly predicted whether the gene was required for growth under the conditions used here. For example, independent genetic evidence demonstrated that three genes of the Energy conserving hydrogenase a (ehaHIJ), which encode the cation translocator of this enzyme complex, were essential (11). Their low EIs in the current studies were consistent with this conclusion.…”

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

“…Methanococcus maripaludis (Lie et al, 2012;Porat et al, 2006) Diversity and distribution of microbial H 2 metabolism C Greening et al A description of the hydrogenase subgroups/subtypes defined here is provided in Table 1.…”

Discovery of a novel methanogen prevalent in thawing permafrost

“…stordalenmirensis' genome is the lack of discernable Ech or Eha hydrogenases that provide sub-stoichiometric amounts of the reduced ferredoxin necessary for the reduction of carbon dioxide (Fig. 2) 14,15 . A distantly related putative hydrogenase with no known archaeal orthologues was identified, which may fulfil this function (Supplementary Table 3), although we cannot rule out that Eha or Ech hydrogenases may be encoded in the small unassembled portions of the genome.…”