Electron Bifurcation and Confurcation in Methanogenesis and Reverse Methanogenesis

Reduction of the disulfide of coenzyme M and coenzyme B (CoMS–SCoB) by heterodisulfide reductases (HdrED and HdrABC) is the final step in all methanogenic pathways. Flavin-based electron bifurcation (FBEB) by soluble HdrABC homologs play additional roles in driving essential endergonic reactions at the expense of the exergonic reduction of CoMS–SCoM. In the first step of the CO2 reduction pathway, HdrABC complexed with hydrogenase or formate dehydrogenase generates reduced ferredoxin (Fdx2-) for the endergonic reduction of CO2 coupled to the exergonic reduction of CoMS–SCoB dependent on FBEB of electrons from H2 or formate. Roles for HdrABC:hydrogenase complexes are also proposed for pathways wherein the methyl group of methanol is reduced to methane with electrons from H2. The HdrABC complexes catalyze FBEB-dependent oxidation of H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- supplies electrons for reduction of the methyl group to methane. In H2- independent pathways, three-fourths of the methyl groups are oxidized producing Fdx2- and reduced coenzyme F420 (F420H2). The F420H2 donates electrons for reduction of the remaining methyl groups to methane requiring transfer of electrons from Fdx2- to F420. HdrA1B1C1 is proposed to catalyze FBEB-dependent oxidation of Fdx2- for the endergonic reduction of F420 driven by the exergonic reduction of CoMS–SCoB. In H2- independent acetotrophic pathways, the methyl group of acetate is reduced to methane with electrons derived from oxidation of the carbonyl group mediated by Fdx. Electron transport involves a membrane-bound complex (Rnf) that oxidizes Fdx2- and generates a Na+ gradient driving ATP synthesis. It is postulated that F420 is reduced by Rnf requiring HdrA2B2C2 catalyzing FBEB-dependent oxidation of F420H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- is recycled by Rnf and HdrA2B2C2 thereby conserving energy. The HdrA2B2C2 is also proposed to play a role in Fe(III)-dependent reverse methanogenesis. A flavin-based electron confurcating (FBEC) HdrABC complex is proposed for nitrate-dependent reverse methanogenesis in which the oxidation of CoM-SH/CoB-SH and Fdx2- is coupled to reduction of F420. The F420H2 donates electrons to a membrane complex that generates a proton gradient driving ATP synthesis.


INTRODUCTION
Methane-producing archaea (methanogens) are terminal organisms of anaerobic microbial food chains decomposing complex organic matter in Earth's anaerobic biosphere which includes the lower intestinal tract of humans, the hind gut of termites, the rumen of animals, natural wetlands and rice paddies. As such, methanogens are an essential link in the global carbon cycle (Figure 1). In step 1, CO 2 is incorporated into biomass by photosynthetic plants and microbes. In oxygenated environments, O 2 -respiring microbes oxidize the biomass producing CO 2 that re-enters the carbon cycle (step 2). A significant fraction of the biomass enters anaerobic biospheres where it is converted to CO 2 and CH 4 by microbial food chains comprised of at least four metabolic groups (steps 3-6). The fermentative group digests the complex biomass producing acetate, H 2 , and CO 2 along with other volatile fatty acids (step 3) that are oxidized to acetate plus either formate or H 2 (step 4) by syntrophic acetogens. The CO 2 -reducing methanogen group reduces CO 2 to CH 4 with electrons derived from oxidation of H 2 or formate (step 5). This group forms symbioses with the acetogens that supply H 2 or formate the methanogens metabolize to concentrations thermodynamically favorable for the acetogens in a process termed interspecies electron transfer (ISET) (Sieber et al., 2009). The acetate-utilizing (acetoclastic) methanogen group converts the methyl group to CH 4 and the carbonyl group to CO 2 (step 6). A portion of the CH 4 is oxidized to CO 2 (step 7) by the anaerobic oxidation of methane (AOM) proposed to involve the reversal of methanogenic pathways. The CO 2 and remaining CH 4 escapes into oxygenated zones where O 2 -respiring methanotrophic microbes oxidize CH 4 to CO 2 (step 8), closing the carbon cycle. As a greenhouse gas, methane is nearly 20-fold more potent than CO 2 ; thus, the aerobic and anaerobic oxidation of CH 4 plays an important role in controlling Earth's climate (Valentine, 2002;Rhee et al., 2009).
Electron transport is much less understood than the comprehensive biochemical understanding of carbon transformations in methanogenic and reverse methanogenic pathways. Herein is reviewed the current understanding of electron transport with a focus on the role of flavin-based electron bifurcation (FBEB) and confurcation (FBEC).

OBLIGATE CO 2 REDUCING METHANOGENS
As the name implies, this group only produces CH 4 by reducing CO 2 , primarily with electrons from oxidation of H 2 or formate. The pathway (Figure 2) is the subject of reviews (Liu and Whitman, 2008;Thauer et al., 2008;Ferry, 2010). The first step is reduction of CO 2 to formyl-methanofuran (CHO-MF) catalyzed by formylmethanofuran dehydrogenase (Fwd or Fmd) (Wagner et al., 2016). The reaction is endergonic and dependent on reduced ferredoxin (Fdx 2− ). The formyl group of CHO-MF is transferred to tetrahydromethanopterin (H 4 MPT) and reduced to yield CH 3 − H 4 MPT. Most methanogens contain H 4 MPT, whereas Methanosarcina species contain the FIGURE 1 | The global carbon cycle. Solid lines indicate aerobic (red) and anaerobic (blue) steps in the cycle and dotted lines indicate transfer of material between aerobic and anaerobic environments. (1) Photosynthesis, (2) aerobic decomposition, (3) fermentation, (4) syntrophic acetogenesis, (5) CO 2 reduction to methane with electrons derived from formate or H 2 , (6) acetotrophic methanogenesis, (7) anerobic oxidation of methane, and (8) aerobic oxidation of methane.
functionally equivalent tetrahydrosarcinapterin (H 4 SPT). The electron donor is reduced coenzyme F 420 (F 420 H 2 ) generated from H 2 or formate by F 420 -dependent hydrogenases (Fru, Frc, Frh) or formate dehydrogenase (Fdh) (Thauer et al., 2010). Coenzyme F 420 is an obligate two-electron carrier donating or accepting a hydride. The methyl group of CH 3 − H 4 MPT is transferred to coenzyme M (HS-CoM) catalyzed by methyltransferase (Mtr) to generate CH 3 − SCoM. This exergonic reaction is linked to translocation of Na + outside the membrane generating a gradient (high outside). Without cytochromes in obligate CO 2 reducing methanogens, this is the only mechanism generating an ion gradient that drives ATP synthesis (Thauer et al., 2008). Methyl-SCoM methylreductase (Mcr) catalyzes the reductive demethylation of CH 3 − SCoM to CH 4 involving coenzyme B (HS-CoB) accompanied by formation of CoMS-SCoB. Heterodisulfide reductase (HdrABC) reduces the disulfide bond with electrons supplied from the oxidation of 2H 2 or 2HCO 2 H (E • ∼ −420 mV) catalyzed by F 420independent hydrogenase or Fdh. The exergonic reduction of CoMS-SCoB (E • = -140 mV) drives the endergonic reduction of CO 2 (E • = -500 mV) in the first step (Figure 2) via FBEB by HdrABC (Buckel and Thauer, 2013).
Two variations are proposed for FBEB of obligate CO 2 -reducing methanogens (Figure 2). FBEB in strictly hydrogenotrophic Methanothermobacter marburgensis involves the soluble MvhAGD:HdrABC for which the crystal structure of the heterododecameric complex from Methanothermococcus thermolithotrophicus supports a proposed mechanism (Figure 3; Thauer et al., 2008;Kaster et al., 2011;Buckel and Thauer, 2013;Wagner et al., 2017  characterized FBEB enzymes for which a hydride is donated to FAD Peters and Lubner, 2017). Any of three conformational changes are proposed to overcome the >30 Å distance observed in the crystal structure that otherwise would prohibit electron transfer between the [2Fe-2S] cluster of MvhD and FAD of HdrA. At this juncture the electrons from reduced FAD (FADH − ) bifurcate into a high-potential and a low-potential electron. The high-potential electron from FADH − is transported via [4Fe-4S] clusters of HdrA and HdrC to the active-site non-cubane [4Fe-4S] clusters of HdrB where CoMS-SCoB is reduced. The low-potential electron of the resulting semiquinone radical (FADH) is transported via [4Fe-4S] clusters (HA3, HA5, and HA6) of HdrA to Fdx. It is proposed that a conformational change overcomes the 21.5 Å distance in the crystal structure that would otherwise prohibit electron transfer between HA3 and HA5 (Figure 3). The structure reveals residues adjacent to the isoalloxazine ring of FAD proposed to achieve the low-potential neutral FADH radical and a postulated anionic semiquinone (FAD − ) intermediate during reduction of FAD. The process occurs twice yielding HSCoM, HSCoB, and Fdx 2− that donates electrons to Fwd reducing CO 2 to CHO-MF (Figure 2). Generation of Fdx 2− by the membrane-bound energy-converting hydrogenase Eha (Figure 4) of obligate CO 2 reducing methanogens serves an anaplerotic role and validates the essentiality of FBEB (Lie et al., 2012).
In contrast to strictly hydrogenotrophic M. marburgensis, Methanococcus maripaludis utilizes either H 2 or formate as electron donors for reduction of CO 2 to CH 4 requiring FBEB mechanisms for each substrate (Figure 2). A protein complex isolated from formate-grown cells contains HdrABC, F 420 -nonreducing selenocysteine-containing hydrogenase (Vhu), Fdh, and the tungsten-containing Fwd (Costa et al., 2010). This result lead to the conclusion that Fdh oxidizes formate . The solid arrows indicate the electron-transfer pathway composed of iron-sulfur clusters at distances less than 13.5 Å. The dashed arrows correspond to a hypothetical electron-transfer pathway with distances longer than 15 Å between the redox centers. Reproduced by permission (Wagner et al., 2017).
FIGURE 4 | Schematic representation of the structure and function of the energy-converting [NiFe]-hydrogenases EchA-F, EhaA-T, EhbA-Q, and MbhA-N found in methanogenic archaea. The energy-converting hydrogenase EchA-F is composed only of the six conserved core subunits, which are shown in shades of gray. The energy-converting hydrogenases EhaA-T, EhbA-Q, and MbhA-N also contain several hydrophobic and hydrophilic subunits of unknown function. These subunits are symbolized by areas with dashed boundaries. Abbreviations: Fd ox , oxidized ferredoxin (Fdx); Fd red 2− , two-electron-reduced ferredoxin (Fdx 2− ). Adapted from (Thauer et al., 2010).
with direct transfer of electrons to HdrABC without first producing H 2 by a F 420 -dependent formic hydrogenlyase system and then oxidation of the H 2 by the F 420 -independent MvhAGD hydrogenase as in FBEB by M. marburgensis. Direct transfer without participation of H 2 as an intermediate is supported by robust growth with formate, although not H 2 , for a mutant deleted of genes encoding subunits of the selenocysteine-containing (vhu) and cysteine-containing (vhc) F 420 -independent hydrogenases associated with HdrABC. However, the mutant retained the vhuD and vhcD genes homologous to mvhD of M. marburgensis obscuring potential roles for VhuD and VhcD. When grown under conditions where both Fdh and Vhu are expressed, the enzymes compete for binding to VhuD, and are fully functional and bound to VhuD (Costa et al., 2013). Further, Fdh co-purifies with VhuD in the absence of other hydrogenase subunits. It was concluded that VhuD, also containing a [2Fe-2S] cluster, functions analogous to MvhD by mediating direct electron transfer from Vhu or Fdh to HdrABC (Figure 2; Costa et al., 2013). The mechanism for transfer of electrons from HdrABC to Fwd is unknown although likely mediated by Fdx as for M. marburgensis (Costa et al., 2010). Not reported is biochemical validation of electron bifurcation by the proposed complex as was shown for the MvhAGD:HdrABC and HdrA2B2C2 complexes of M. marburgensis and Methanosarcina acetivorans (Kaster et al., 2011;Yan et al., 2017). Nonetheless, an in silico genomescale metabolic reconstruction of M. maripaludis indicates the organism is unable to grow without the energy-conserving complex (Richards et al., 2016). In addition to supplying Fdx 2− for the first step in the CO 2reduction pathway of methanogenesis, it is proposed that energyconserving FBEB is instrumental for growth of Methanocella conradii when concentrations of H 2 are exceptionally low (Liu et al., 2014). A transcription unit comprised of genes encoding Fwd, HdrABC and MvhD is up regulated in M. conradii grown syntrophically in co-culture with H 2producing acetogens utilizing propionate and butyrate (Liu et al., 2014). Thus, it is proposed that an electron bifurcating MvhD/HdrABC/Fwd complex is essential for syntrophic growth with low concentrations of H 2 . As M. conradii encodes MvhGA remote from the up regulated transcription unit, the mechanism by which H 2 is oxidized and electrons transferred to HdrABC is unknown. Interestingly, obligate CO 2 -reducing methanogens of the order Methanomicrobiales are missing genes encoding MvhA and MvhG but encode MvhD and HdrABC (Browne et al., 2016). These methanogens could form an MvhD/HdrABC complex associated with energy-converting hydrogenases EchA-F, EhaA-T, or MbhA-N dependent on ion gradients to supply Fdx 2− for reduction of CO 2 to CHO-MF, although reduction of CoMS-SCoB would be energy consuming. Thus, it is proposed that these methanogens substitute MvhA and MvhG with FrhA and FrhG of the F 420 -reducing hydrogenase (FrhABG) contained in all methanogens without cytochromes (Thauer et al., 2010;Gilmore et al., 2017). In this way, FrhAG would be present in an FrhAG/MvhD/HdrABC complex with the potential for FBEB of H 2 that generates the Fdx 2− required for reduction of CO 2 to CHO-MF (Figure 5) with the added advantage of conserving energy. In this scenario, the energyconverting hydrogenases play a role in only providing Fdx 2− for biosynthesis (Major et al., 2010). However, the possibility of an FrhABG/MvhD/HdrABC complex (Figure 5) cannot be ruled out at this juncture. Inclusion of the F 420 -binding FrhB subunit invokes electron transport dependent on FrhABG producing F 420 H 2 for which the electron pair is bifurcated by MvhD/HdrABC reducing Fdx and CoMS-SCoB analogous to the HdrA2B2C2 of M. acetivorans (Vitt et al., 2014;Yan et al., 2017). However, it is unknown which FBEB pathway is physiologically relevant.

METHYLOTROPHIC METHANOGENS
Methanogens from the order Methanosarcinales grow and produce CH 4 with methyl-containing substrates (methanol, methylamines, and methyl sulfides) (Liu and Whitman, 2008). A few also grow by reducing CO 2 with H 2 . Unlike obligate CO 2 reducers, these methanogens contain cytochromes and generate a proton gradient dependent on electron transport involving hydrogenases (Thauer et al., 2008). Like obligate CO 2 reducers, Fdx 2− is required to supply electrons to Fwd/Fmd catalyzing reduction of CO 2 to CHO-MF; however, Fdx 2− is generated independent of FBEB by the membrane-bound energyconverting Ech hydrogenase driven by the proton gradient (Figure 4).
The methylotrophic pathway of the order Methanosarcinales involves transfer of substrate methyl groups to HSCoM forming a CH 3 -SCoM pool of which one-fourth of the methyl groups are oxidized to CO 2 via reversal of the CO 2 -reduction pathway to supply F 420 H 2 and Fdx 2− required for reductive demethylation of the remaining three-fourths CH 3 -SCoM to CH 4 (Figure 6). The F 420 H 2 is oxidized by a membrane-bound complex (Fpo) that donates electrons to a quinone-like electron carrier (methanophenazine, MP) coupled to generation of a proton gradient. It is proposed that Fdx 2− is re-oxidized by reducing F 420 although the mechanism is unknown. The production of CH 4 from CH 3 -SCoM is similar to obligate CO 2 reducing methanogens involving HSCoB and Mcr with the exception of the membrane-bound heterodisulfide reductase (HdrDE) that reduces CoMS-SCoB to the sulfhydryl forms of the cofactors. Electrons are supplied to HdrDE by MPH 2 with the scalar translocation of protons contributing to the proton gradient that drives ATP synthesis. However, the genomes of all sequenced Methanosarcinales also contain genes encoding the HdrABC homologs HdrA1B1C1 and HdrA2B2C2 (Buan and Metcalf, 2010). HdrA1B1C1 is elevated during methylotrophic growth of M. acetivorans for which the hdrA1B1C1 mutant strain is growth impaired. Thus, a role in methylotrophic growth is proposed wherein FBEB by HdrA1B1C1 reduces F 420 and CoMS-SCoB with electron pairs donated by two Fdx 2− generated in the oxidation of CHO-MF (Figure 6), thereby allowing energy conservation via Fpo (Buan and Metcalf, 2010).
Methanosphaera stadtmanae, isolated from the human gut, is also a methylotrophic methanogen reducing the methyl group of methanol to CH 4 although belonging to the order Methanobacteriales that do not contain cytochromes necessary for electron-transport coupled proton translocation that drives ATP synthesis. The genome also lacks a complete gene set necessary for reversal of the CO 2 reduction pathway and therefore requires the oxidation of H 2 to supply electrons for reductive demethylation of CH 3 -SCoM to CH 4 (Fricke et al., 2006). A scheme is proposed that includes FBEB of H 2 by an MvhADG:HdrABC complex to explain the finding that ATP synthesis is driven by an ion gradient (Figure 7; Sparling et al., 1993;Thauer et al., 2008). The Fdx 2− produced donates electrons to the membrane-bound energy-converting Ehb complex that generates a Na + gradient driving ATP synthesis and regenerates H 2 recycled for FBEB by the MvhADG:HdrABC complex. A similar FBEB/H 2 cycling scheme is proposed for a sixth class of methanogens, 'Candidatus Methanofastidiosa, ' based on metagenome-derived draft genomes that also lack cytochromes and genes encoding enzymes for reversal of the CO 2 reducing pathway (Nobu et al., 2016). However, this class is restricted to reducing methylthiols with H 2 . A seventh order, the Methanomassiliicoccales, also grows by reducing the methyl groups of methylotrophic substrates with H 2 . Analyses of several genomes show this class also lacks cytochromes and genes required for reversal of the CO 2 reducing pathway (Borrel et al., 2014;Kroninger et al., 2015;Lang et al., 2015). Unlike M. stadtmanae and the 'Candidatus Methanofastidiosa' class, genes encoding the membrane-bound energy-converting complexes are absent and genes encoding an Fpo-like complex and the HdrD subunit of HdrDE are present. Figure 8 shows the pathway proposed for Methanomassiliicoccus luminyensis. The Fpo complex oxidizes Fdx 2− generated via FBEB of H 2 by the MvhADG:HdrABC complex. HdrD accepts electrons from Fpo and reduces CoMS-SCoB coupled to generation of a H + gradient. Roles for involvement of the Ech1 and Ech2 hydrogenases are ruled out based on low abundance of transcripts and low membrane-bound hydrogenase activity (Kroninger et al., 2015). Thus, CoMS-SCoB is essential for both FBEB and the terminal electron acceptor which is distinct from that proposed for M. stadtmanae and the 'Candidatus Methanofastidiosa' class which involves H 2 cycling (Figure 7).

ACETOTROPHIC METHANOGENS
Methanosarcina and Methanosaeta are the only described genera of acetotrophic methanogens that are the subject of recent reviews (Ferry, 2013;Schlegel and Muller, 2013;Welte and Deppenmeier, 2014;Ferry, 2015). Most biochemical investigations have involved Methanosarcina species. M. acetivorans is a model for species that do not metabolize H 2 which constitute the majority of Methanosarcina species (Figure 9A). Acetate is converted to acetyl-CoA at the expense of one ATP followed by cleavage of the C-C and C-S bonds yielding a methyl group that is transferred to H 4 SPT and a carbonyl group that is oxidized to CO 2 with transfer of electrons to Fdx. The methyl group of CH 3 -H 4 SPT is transferred to HS-CoM followed by reductive demethylation of CH 3 S-CoM to methane involving reactions common to all methanogenic pathways. The Mtr complex catalyzes the exergonic methyl transfer coupled to generation of a Na + gradient. The reduced Fdx 2− is electron donor to the Na + -pumping Rnf complex that donates electrons to cytochrome c that is the electron donor to MP (Wang et al., 2011). As in the methylotrophic pathway, HdrDE oxidizes MPH 2 and reduces CoMS-SCoB with scalar translocation of H + that generates a gradient. The multisubunit Na + /H + antiporter Mrp FIGURE 8 | Proposed role for FBEB by heterodisulfide reductase in energy-conservation by Methanomassiliicoccus luminyensis. Ech1 and Ech2 are covered with crosses to indicate non-involvement in generating the proton gradient driving ATP synthesis (Kroninger et al., 2015). The question mark indicates a reaction not experimentally verified, although includes 2H + pumped by the Fpo complex and another 2H + translocated scalar via reduction and re-oxidation of methanophenazine. Fd ox , oxidized ferredoxin (Fdx); Fd red , two-electron-reduced ferredoxin (Fdx 2− ). Reproduced by permission (Kroninger et al., 2015).
adjusts the Na + /H + ratio optimal for the ATP synthase which is dependent on both Na + and H + gradients (Schlegel et al., 2012a;Jasso-Chavez et al., 2013. When switched from growth with methanol to growth with acetate, M. acetivorans up regulates an electron bifurcating heterodisulfide reductase (HdrA2B2C2) that oxidizes F 420 H 2 (E m = −380 mV) and reduces Fdx (E m = −520 mV) driven by reduction of CoMS-SCoB (E m = −140 mV) (Yan et al., 2017). A role has been proposed for HdrA2B2C2 dependent on reduction of NAD-like coenzyme F 420 (F 420 ) by the Rnf complex analogous to Fdx-dependent reduction of NAD + by homologous Rnf complexes from the domain Bacteria (Figure 9B; Buckel and Thauer, 2018a,b). In this way, Fdx reduced by HdrA2B2C2 is re-oxidized by Rnf thereby supplementing the translocation of Na + . The Na + gradient formed by Rnf and Mtr could be exchanged with H + by Mrp to adjust the Na + /H + ratio optimal for ATP synthesis. The process generates more ATP than electron transport involving MP and HdrDE (Figure 9). However, it is reported that HdrDE is essential for acetotrophic growth suggesting the possibility of both electron transport pathways oxidizing Fdx 2− and reducing CoMS-SCoB (Buan and Metcalf, 2010). Having alternate electron transport pathways with different thermodynamic efficiencies could provide the cell with options for responding to fluctuations in available free energy proportional to levels of acetate in the environment. Indeed, the conversion of acetate to CH 4 and CO 2 provides only a marginal amount of energy available for growth ( G • = −36 kJ/CH 4 ) that requires cells to maximize the thermodynamic efficiency.
A genome-wide analysis of Methanosaeta thermophila revealed genes encoding enzymes catalyzing carbon transformation reactions in the pathway of acetate to CH 4 similar to Methanosarcina species (Smith and Ingram-Smith, 2007). However, genes encoding the Rnf complex are absent in the genome of Methanosaeta suggesting an unknown alternative electron transport pathway and mechanism for energy conservation.

REVERSE METHANOGENESIS
It is postulated that AOM is accomplished by a reversal of methanogenic pathways based on environmental metagenomic and metatranscriptomic analyses of sediments (Hallam et al., 2004;McGlynn, 2017;Timmers et al., 2017). Although discovered nearly four decades ago, the unavailability of pure cultures prevented biochemical investigations of AOM. However, M. acetivorans is capable of "trace methane oxidation" (TMO) defined as reverse methanogenesis during net CH 4 production from growth substrates (Moran et al., 2005(Moran et al., , 2007Timmers et al., 2017). More recently, methanotrophic growth dependent on reduction of Fe(III) was documented for M. acetivorans (Soo et al., 2016). Figure 10 illustrates the reverse methanogenesis pathway proposed for M. acetivorans based on a biochemical understanding of Fe(III)-dependent mechanisms driving endergonic reactions and energy conservation essential for methanotrophic growth (Yan et al., 2018). It is remarkably similar to the pathway proposed for anaerobic methanotrophic archaea (ANME) based on metagenomic and transcriptomic analyses of uncultured Methanosarcinales sp. ANME-2a (Wang et al., 2014).
The M. acetivorans pathway is a reversal of established acetateutilizing and CO 2 -reducing methanogenic pathways (Li et al., 2005(Li et al., , 2007Lessner et al., 2006;Ferry, 2008;Wang et al., 2011;Schlegel et al., 2012b;Welte and Deppenmeier, 2014). Methane is oxidized by Mcr (Rxn. 1) with the methyl group of CH 3 -SCoM transferred to H 4 SPT by Mtr (Rxn. 2) representing the reversal of reactions common to all methanogenic pathways. The HdrDE oxidizes HSCoM and HSCoB coupled to reduction of Fe(III) that regenerates CoMS-SCoB (Rxn. 4). Removal of HSCoM, HSCoB, and CH 3 -SCoM products by HdrDE and Mtr drives the endergonic oxidation of CH 4 by Mcr. The endergonic methyl transfer producing CH 3 -H 4 SPT is driven with the Na + gradient generated by the Rnf/cytochrome c complex catalyzing the highly exergonic oxidation of Fdx 2− and reduction of Fe(III) (Rxn. 3). Fdx 2− is also utilized in reduction of CO 2 that supplies the carbonyl group for condensation with the methyl group of CH 3 -H 4 SPT producing acetate (Rxns. 5 and 6). Fdx 2− and F 420 H 2 are generated in reversal of the CO 2 reduction pathway (Rxns. 7 and 8). F 420 H 2 is oxidized by the Fpo complex (Rxn. 9) with transfer of electrons to MP and Fe(III) coupled to generation of a H + gradient. The H + gradient, together with the Na + gradient, drives ATP synthesis assisted by the Mrp antiporter that optimizes the H + /Na + ratio optimal for the ATP synthase dependent on both H + and Na + (Rxns. 14 and 15) (Schlegel et al., 2012a;Jasso-Chavez et al., 2013). The reverse methanogenesis pathway is remarkably similar to that proposed for uncultivated Methanosarcinales sp. ANME-2a present in marine sediments that perform AOM It is also proposed that Fdx 2− is generated by HdrA2B2C2 previously shown to oxidize F 420 H 2 and reduce Fdx coupled to reduction of CoMS-SCoB via energy-conserving FBEB (Rxns. 10-12) (Yan et al., 2017). The HSCoM and HSCoB produced are oxidized by HdrDE coupled to the reduction of Fe(III) regenerating CoMS-SCoB (Rxn. 13). This proposed role would be essential in the environment where low availability of Fe(III) limits the generation of Na + and H + gradients by the Rnf and Fpo complexes. In this scenario, the Fdx 2− produced by HdrA2B2C2 is used to reduce CO 2 for the synthesis of acetate and ATP by substrate level phosphorylation (Rxns. 5 and 6). Notably, the Methanosarcinales sp. ANME-2a metagenome encodes HdrA2, HdrB2, and HdrC2 homologs with 59, 72, and 59% identities (Supplementary Figure S1) consistent with a role in reverse methanogenesis by ANME.
Unlike the HdrA1B1C1 of M. acetivorans and the HdrABC of obligate CO 2 -reducing methanogens, the C-terminal domain of HdrA2 extends with sequences homologous to MvhD (Yan et al., 2017). Although the function of this fused MvhD is unknown, HdrA2 homologs are ubiquitous in acetotrophic and methylotrophic species of the order Methanosarcinales suggesting important functions. Remarkably, HdrA2 and HdrBC homologs are present in non-methanogenic species of the domain Bacteria signaling diverse functions.
A metagenomics-based metabolic model of electron transport is proposed for the nitrate-dependent reverse methanogenesis by Methanoperedens-like ANME (Figure 11). Apart from the F 420 H 2 , HSCoM/HSCoB, and Fdx 2− generated by reverse methanogenesis, the model contrasts with the Fe(III)-dependent pathway of M. acetivorans (Arshad et al., 2015). Foremost, the genome encodes a Rieske-type protein, cytochromes c and b, and a nitrate reductase that reduces nitrate to nitrite with reduced menaquinone (MQH 2 ) generated by a F 420 H 2 dehydrogenase (Fqo) that combine to generate a proton gradient driving ATP synthesis. Reduction of MQ is also accomplished by oxidation of HSCoM/HSCoB with HdrDE. An energy-converting hydrogenase homolog (Ech) is proposed to oxidize Fdx 2− and contribute to the proton gradient although the fate of produced H 2 is unknown. Alternatively, Fdx 2− is the electron donor to a flavin-based electron confurcating complex comprised of an HdrABC homolog oxidizing HSCoM/HSCoB and donating electrons to the F 420 -dependent hydrogenase subunit (FrhB) proposed to oxidize Fdx 2− and reduce F 420 . Energy is conserved in the confurcation reaction rather than lost as heat should FrhB alone oxidize Fdx 2− and reduce F 420 ( E • = 120 mV). The genome of "Candidatus Methanoperedens nitroreducens" encodes a homolog of M. acetivorans HdrA2 which presents the possibility of an HdrA2B2C2 homolog catalyzing the confurcation reaction (Berger et al., 2017;Yan et al., 2017).

CONCLUSION
Methanogenic and reverse methanogenic pathways are proposed to involve FBEB or FBEC in electron transport that also serve as mechanisms of energy conservation. However, there is a significant lack of understanding requiring further investigation.
(1) Biochemical confirmation of FBEB is needed for the several proposed complexes other than that shown for the purified MvhADG:HdrABC of M. marburgensis and HdrA2B2C2 of M. acetivorans.
(3) Validation is needed for the proposed role of HdrA1B1C1 in the methylotrophic pathway of M. acetivorans and related methylotrophic methanogens; in particular, the proposal that HdrA1B1C1 of M. acetivorans oxidizes F 420 H 2 in analogy to that shown for HdrA2B2C2. Also worthy of investigation are the uncharacterized HdrA2B2C2 homologs in the order Methanosarcinales and the domain Bacteria.
(4) Investigations are in order to determine the mechanism by which H 2 is oxidized and electrons are delivered to the proposed MvhD/HdrABC and MvhD/HdrABC/Fwd complexes of methanogens in the orders Methanocellales and Methanomicrobiales. (5) The proposed roles for FBEB and FBEC in reverse methanogenesis pathways require validation via analyses of deletion mutants.