Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO
            <sub>2</sub>
            Assimilation in the Archaeon
            <i>Methanococcus maripaludis</i>

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170

183

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 electronbifurcation 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.energy conservation | Archaea | formate dehydrogenase | formylmethanofuran dehydrogenase | F 420 -nonreducing hydrogenase T he biochemical steps in methanogenesis from CO 2 are well known, but the interactions that lead to net energy conservation are not well understood. The steps in the pathway are diagrammed in Fig. 1 (1). The first step involves the reduction of CO 2 and covalent attachment to a unique cofactor, methanofuran (MFR), via the action of formylmethanofuran dehydrogenase (Fwd) to generate formyl-MFR. This represents an energy-consuming step in the pathway and is dependent on reduced ferredoxin, thought to be produced at the expense of a chemiosmotic membrane potential via the energy-conserving hydrogenase, Eha. Next, the formyl group is transferred to another carrier, tetrahydromethanopterin (H 4 MPT), and is then reduced to generate methyl-H 4 MPT. The methyl group is then transferred to yet another carrier, coenzyme M (HS-CoM), by methyl-H 4 MPT-CoM methyltransferase (Mtr) to generate methyl-S-CoM. At this point, Na + ions are translocated across the cell membrane. The final step involves reduction of the methyl group to CH 4 and capture of HS-CoM by coenzyme B (HS-CoB) to form a CoM-S-S-CoB heterodisulfide. To regenerate HS-CoM and HS-CoB, another enzyme is used, heterodisulfid...

Section: Discussionmentioning

confidence: 93%

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

Costa

Wong

Wang

et al. 2010

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170

183

“…The hydrogenase Hmd catalyzes the second reductive step directly with H 2 , but its function is redundant with Mtd, which uses reduced F 420 for the same purpose (11). Finally, the anabolic energy-converting hydrogenase Ehb is nonessential in the presence of fixed carbon and is not required for methanogenesis (8,9). Only Eha remains as possibly essential.…”

Section: Resultsmentioning

confidence: 99%

“…Two of these reactions are those mentioned above, the concomitant reduction of Fd and CoM-S-SCoB that occurs in electron bifurcation, and the H 2 -dependent reduction of Fd by the energy-converting hydrogenase Eha, both of which are proposed to lead to the endergonic reduction of CO 2 to formyl-MFR. A third such reaction, which reduces a Fd with another energy-converting hydrogenase, Ehb, functions in anabolic CO 2 fixation reactions and does not appear to be involved in methanogenesis (8,9). Here we present an analysis of electron flow in methanogens without cytochromes, focusing on the role of H 2 when formate is the electron donor for methanogenesis.…”

mentioning

confidence: 99%

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

Lie

Costa

Lupa

et al. 2012

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108

114

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...

“…Genes for the biosynthetic energyconserving hydrogenase B (Ehb; Mmp1621-1629), carbon monoxide dehydrogenase/acetyl CoA synthase (Mmp0979-0985), and pyruvate oxidoreductase (Por; Mmp1502-1507), which use ferredoxins as electron carriers, had only moderately increased mRNA levels under H 2 limitation. We previously observed a similar increase in mRNA for these genes when Ehb was mutationally inactivated (8,19), suggesting a response to maintain flux through this pathway when the electron donor is limiting. Genes for the step of the methanogenic pathway that uses ferredoxin [the energy-conserving hydrogenase A (Eha)] were not significantly affected by H 2 limitation, nor could an effect of H 2 limitation be discerned for the methylreductase (Mcr), the non-F 420 -reducing hydrogenases (Vhu or Vhc), or the heterodisulfide reductases (Hdrs).…”

Section: Distinct Effect Of H2 Limitation On Genes Encoding F420-depementioning

confidence: 93%

Functionally distinct genes regulated by hydrogen limitation and growth rate in methanogenic Archaea

Hendrickson

Haydock

Moore

et al. 2007

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The use of molecular hydrogen as electron donor for energy generation is a defining characteristic of the hydrogenotrophic methanogens, an ancient group that dominates the phylum Euryarchaeota. We present here a global study of changes in mRNA abundance in response to hydrogen availability for a hydrogenotrophic methanogen. Cells of Methanococcus maripaludis were grown by using continuous culture to deconvolute the effects of hydrogen limitation and growth rate, and microarray analyses were conducted. Hydrogen limitation markedly increased mRNA levels for genes encoding enzymes of the methanogenic pathway that reduce or oxidize the electron-carrying deazaflavin, coenzyme F420. F420-dependent redox functions in energy-generating metabolism are characteristic of the methanogenic Archaea, and the results show that their regulation is distinct from other redox processes in the cell. Rapid growth increased mRNA levels of the gene for an unusual hydrogenase, the hydrogen-dependent methylenetetrahydromethanopterin dehydrogenase.coenzyme F420 ͉ microarrays ͉ Methanococcus maripaludis