A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting.

H eterodisulfide reductase (Hdr) was first discovered in CH 4 -producing species (methanogens) from the domain Archaea where one or the other of two classes (HdrABC or HdrDE) is essential for all methanogenic pathways (1). However, the genomes of diverse species in the domains Bacteria and Archaea are annotated with genes encoding HdrABC homologs, suggesting that these genes play roles in a greater diversity of energy-conserving metabolisms, which include the oxidation of methanol and inorganic sulfur compounds (2)(3)(4), the reduction of sulfate and ferric iron (4)(5)(6), syntrophic utilization of fatty acids (7), and the anaerobic oxidation of CH 4 (8)(9)(10). Indeed, the HdrABC class belongs to the core repertoire of the ancient prokaryotic common ancestor consistent with diverse physiological functions of extant species (11).
Although of ancient origin and widespread, the biochemical and physiological understanding of the HdrABC class is restricted to one homolog essential for the pathway of CO 2 reduction to CH 4 . The final step in all methanogenic pathways (equation 1) is the reductive demethylation of methyl coenzyme M (CH 3 -SCoM) for which coenzyme B (HSCoB) supplies reductant. The heterodisulfide product is reduced by Hdr (equation 2), releasing HSCoB and HSCoM for methylation.
CoMS-SCoB ϩ 2e Ϫ ϩ 2H ϩ → HSCoM ϩ HSCoB (2) The cytoplasmic HdrABC in H 2 -oxidizing CO 2 -reducing species is complexed with the MvhAGD hydrogenase. Electron pairs donated from the hydrogenase are bifurcated by HdrABC, reducing CoMS-SCoB and ferredoxin (Fdx), and Fdx donates electrons for the first step in the reduction of CO 2 to produce CH 3 -SCoM. The bifurcation is a flavin-based coupling of the endergonic reduction of Fdx with the exergonic reduction of CoMS-SCoB. Flavin-based electron bifurcation is considered to be an ancient energyconserving mechanism (12)(13)(14). Importantly, a comprehensive mechanistic understanding of the HdrABC class has been impeded by the unavailability of a recombinantly produced enzyme or an activity assay utilizing a physiological electron donor. Although the obligate two-electron carrier coenzyme F 420 (F 420 ) has diverse roles in species from the domains Bacteria and Archaea, roles in electron bifurcation and the donor to HdrABC homologs are still unknown (15). More-diverse roles are postulated for electron bifurcation catalyzed by HdrABC homologs in phylogenetically and physiologically diverse species in the domains Bacteria and Archaea (12,13,16). Bioinformatic analyses of methanogens and nonmethanogens from the domains Bacteria and Archaea predict an HdrABC homolog wherein the HdrA subunit is fused with MvhD (see Fig. S1 in the supplemental material). Genes encoding the homolog, designated HdrA2B2C2, are present in the methanogenic archaeon Methanosarcina acetivorans with metabolic capabilities distinct from those of obligate CO 2 -reducing methanogens (17,18). The fused HdrA2 subunit and low sequence identity of all subunits with subunits of the HdrABC homolog from obligate CO 2 -reducing methanogens (29 to 37%) predict differences in structure and function (19).
Here we report the individual heterologous production and characterization of HdrA2, HdrB2, and HdrB2C2 from M. acetivorans that form an HdrA2B2C2 complex with Fdx 2Ϫ -and F 420 H 2 -dependent heterodisulfide reductase activity. The experimental approach has advanced an understanding of intersubunit electron transfer for the HdrABC class and uncovered a previously unknown coenzyme F 420 H 2 -dependent electron bifurcation that predicts roles for HdrA2B2C2 homologs in pathways of anaerobic CH 4 oxidation in M. acetivorans and other species (20)(21)(22). The uncommon properties of HdrA2B2C2 identify it as representative of an HdrABC subclass with homologs in diverse species from the domains Bacteria and Archaea.

RESULTS
Bioinformatic analyses. A search of the nonredundant databases (https://www.ncbi. nlm.nih.gov/pubmed) with HdrA2 as the query retrieved 150 sequences, of which 77 were HdrA2 homologs containing HdrA and MvhD domains with greater than 46% identity and 96% coverage. Figure S1 in the supplemental material shows an alignment of representative sequences. A total of 48 HdrA2 homologs were from acetotrophic and methylotrophic methanogens. None of the homologs were from obligate CO 2 -reducing methanogens, indicating a role for HdrA2 specific to acetotrophic and methylotrophic methanogens. Of the 77 HdrA2 homologs, 29 were from physiologically and phylogenetically diverse nonmethanogenic species in the domains Bacteria and Archaea. Of the 150 sequences retrieved, 73 were homologs of the canonical HdrA from both methanogenic and nonmethanogenic species. These results indicate that HdrA2 homologs play roles in metabolically diverse species, warranting investigation of an HdrA2 representative chosen from M. acetivorans that grows by converting acetate to CH 4 (17).
Recombinant Fdx. The conversion of acetate to CH 4 begins with activation to acetyl coenzyme A (acetyl-CoA) that is cleaved at the C-C and C-S bonds producing methyl and carbonyl groups catalyzed by CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) (23,24). The methyl group is transferred to HSCoM, and the carbonyl group is oxidized to CO 2 with reduction of Fdx. Thus, Fdx is a candidate electron donor/acceptor of HdrA2B2C2. Of multiple Fdx-encoding genes, the Fdx encoded by MA0431 is produced in acetate-grown M. acetivorans and is the electron acceptor for CODH/ACS (25). The Fdx produced in Escherichia coli strain BL21(DE3) ΔiscR (Fig. 1A) showed a prominent absorption band centered at~390 nm with an A 390 /A 280 ratio of 0.69, indicating a nearly full complement of the predicted two [Fe 4 S 4 ] clusters (25). The recombinant Fdx was competent in accepting electrons from CODH/ACS at a rate of 55.3 nmol/min/mg. Recombinant HdrA2, HdrB2, HdrC2, and HdrB2C2. HdrA2, HdrB2, HdrC2, and HdrB2C2 were produced in E. coli strain BL21(DE3) ΔiscR. All except HdrC2 were present in the cytoplasm and purified to homogeneity (Fig. S2). HdrC2 was present in inclusion bodies and not purified. When coproduced with HdrB2, HdrC2 was present in the cytoplasm and copurified with HdrB2 to homogeneity (Fig. S2). The results indicate that either HdrC2 was misfolded or the presence of HdrB2 is required for HdrC2 to fold properly. Regardless, the results indicate that HdrB2 and HdrC2 form an HdrB2C2 complex.
HdrA2 migrated in SDS-polyacrylamide gels with an apparent molecular mass of 87 kDa (Fig. S2) consistent with the molecular mass of 86.9 kDa calculated for the HdrA-MvhD fusion. The UV-visible (UV-Vis) spectrum of the protein as purified (aspurified protein) shows prominent absorption bands centered at~320 and~420 nm attributed to Fe-S clusters (Fig. 1B). Reconstitution of the as-purified protein with flavin adenine dinucleotide (FAD) resulted in the enhancement of an absorption band centered at~560 nm attributed to flavin. Indeed, upon reconstitution, the flavin content increased from 0.32 Ϯ 0.06 (n ϭ 3) to 1.2 Ϯ 0.1 (n ϭ 3) mol per mol of HdrA2. The FAD-reconstituted preparation had 20.6 Ϯ 2.4 atoms of nonheme iron and 18.6 Ϯ 1.3 atoms of acid-labile sulfur per molecule of HdrA2. The 420 was found to be 82.3 mMϪ1 cm Ϫ1 . These results are consistent with each molecule of HdrA2 containing one FAD molecule and four Fe 4 S 4 clusters in the HdrA domain, and one Fe 2 S 2 cluster in the MvhD domain, predicted by motifs in the deduced sequence (Fig. S3).
HdrB2 migrated in SDS-polyacrylamide gels with an apparent molecular mass of 33 kDa in agreement with a calculated molecular mass of 32.8 kDa (Fig. S2). The UV-Vis spectrum of as-purified preparations showed absorption bands centered at~320,~420, and~580 nm (Fig. 1C). The protein contained 5.0 Ϯ 0.1 (n ϭ 3) nonheme iron atoms and 5.1 Ϯ 0.2 (n ϭ 3) acid-labile sulfur atoms per molecule of HdrB2. The 420 was found to be 15.9 mM Ϫ1 cm Ϫ1 . These results are consistent with the presence of an Fe 4 S 4 cluster ligated by one of two cysteine-rich motifs (CX 31-39 CCX 35-36 CX 2 C) (Fig. S3) that are conserved in HdrB of obligate CO 2 -reducing methanogens and proposed to ligate a novel active site [Fe 4 S 4 ] 3ϩ cluster (26). The addition of CoMS-SCoB to dithionite-reduced HdrB2 increased the absorbance at~320 and~420 nm, indicating that the protein was competent to reduce the heterodisulfide to HSCoM and HSCoB (Fig. 1C). In an apparent anomaly, no significant forward activity (reduction of CoMS-SCoB with reduced methyl viologen [MV]) was detected, either with HdrB2 alone or in combination with HdrA2 and Fdx. However, reverse activity with HdrB2 alone was measurable, and there was no significant change in the V max when HdrA2 was present (Table 1). To our knowledge, this is the first heterologously produced and catalytically active HdrB from any source.
The HdrC2 of purified HdrB2C2 migrated in SDS-polyacrylamide gels with an apparent molecular mass of 18 kDa in agreement with a calculated molecular mass of 18.1 kDa (Fig. S2). The UV-Vis spectrum of as-purified HdrB2C2 showed absorption bands centered at~320,~420, and~580 nm (Fig. 1D). Preparations contained 14.6 Ϯ 1.0 (n ϭ 3) nonheme iron atoms and 11.9 Ϯ 0.8 (n ϭ 3) acid-labile sulfur atoms per molecule of HdrB2C2. The 420 was found to be 45.7 mM Ϫ1 cm Ϫ1 . These results are consistent with the presence of three Fe 4 S 4 clusters in the HdrB2C2 complex which is predicted from results obtained for HdrB2 in addition to the sequence of HdrC2 that contains two canonical Fe 4 S 4 -binding motifs (Fig. S3). Thus, the results indicate that as-purified HdrB2C2 had a full complement of iron-sulfur centers. The addition of CoMS-SCoB to dithionite-reduced HdrB2C2 increased the absorbance at~420 nm, indicating that the complex was active in reducing the heterodisulfide to HSCoM and HSCoB (Fig. 1D). No significant forward activity was detected unless HdrA2 was present, a result indicating that HdrB2C2 is incapable of accepting electrons from MV and that HdrA2 mediates electron transfer between MV and HdrB2C2. Kinetic analyses (Table 1) showed that V max nearly doubled in the presence of both HdrA2 and Fdx. These results, and the finding that HdrB2 in combination with HdrA2 was incapable of catalyzing MV-dependent activity, is consistent with a role for HdrC2 in mediating electron transfer from HdrA2 to HdrB2. Kinetic constants for the reverse activity of HdrB2C2 (Table 1) were not significantly different from those determined for HdrB2, which indicates that HdrC2 plays no role in catalysis by HdrB2.
Reconstitution of an active HdrA2B2C2 complex. Figure 2A shows representative time courses for reduction of CoMS-SCoB catalyzed by various combinations of HdrA2, HdrB2, and HdrB2C2 in the presence of CO, CODH/ACS, and Fdx as electron donors. Substantial initial rates were observed only with HdrB2C2, which indicates that Fdx mediates direct electron transfer from CODH/ACS to HdrB2C2. Rates with only HdrB2 were inconsequential, indicating a role for HdrC2 in the transfer of electrons from Fdx to HdrB2. Rates of Fdx-dependent reduction of HdrB2 or HdrB2C2 (Fig. S4) showed that HdrB2C2 was reduced at a rate fivefold greater than for HdrB2, providing further support for the role of HdrC2 in mediating electron transfer from Fdx to HdrB2. In contrast, the combination of HdrA2 and HdrB2 enhanced the initial rate of CoMS-SCoB to levels comparable with only HdrB2C2 (Fig. 2A). This result indicates that HdrA2 mediates electron transfer from Fdx to catalytic HdrB2 without HdrC2 participation. Reaction mixtures containing a combination of HdrA2 and HdrB2C2 catalyzed the reduction of CoMS-SCoB at initial rates approximately sixfold greater than the rate observed for the combination of HdrA2 and HdrB2 ( Fig. 2A). These results indicate that HdrC2 mediates electron transfer from HdrA2 to HdrB2 at a rate substantially greater Heterodisulfide Reductases of Bacteria and Archaea ® than the transfer of electrons from HdrA2 directly to HdrB2. Although hdrA2 (MA2868) is located distant from hdrB2 and hdrC2 (MA4237-MA4236), the results establish formation of an HdrA2B2C2 complex catalyzing the most efficient oxidation of Fdx and reduction of CoMS-SCoB. Nevertheless, gel filtration column chromatography of the reconstituted HdrA2B2C2 resulted in separation of HdrA2 from HdrB2C2, indicating a weakly bound catalytic complex.
The redox potential for the CO/CO 2 couple (Ϫ558 mV) predicts two-electron reduction of Fdx (Ϫ520 mV) by CODH/ACS (27). Indeed, results shown in Fig. 2B confirm reduction to Fdx 2Ϫ in the presence of CODH/ACS and 1.0 atm of CO. However, Fdx 2Ϫ was only partially oxidized by HdrA2B2C2 in the presence of CoMS-SCoB. A total of 10.1 Ϯ 1.1 (n ϭ 3) nmol of free thiols were produced from CoMS-SCoB in the reaction mixture, which contained 9.4 nmol Fdx 2Ϫ . The results indicate that the physiologically relevant reduction of CoMS-SCoB catalyzed by CODH/ACS and HdrA2B2C2 proceeds according to equations 3 and 4 that are summed in equation 5.
Interaction of F 420 with HdrA2 and HdrA2B2C2. F 420 is an electron carrier with a multitude of functions in methanogenic and nonmethanogenic species in the domains Bacteria and Archaea that also contain genes encoding HdrA2B2C2 (15). F 420 is an obligatory two-electron carrier that requires Fdx:F 420 oxidoreductases to contain a flavin that accepts an electron from Fdx and generates the hydride for transfer to F 420 . Thus, the flavin-containing HdrA2 is a candidate for interacting with F 420 . Indeed, HdrA2 catalyzed the Fdx-dependent reduction of F 420 with a K m for F 420 of 6.4 M (Fig. S5), identifying a novel function for heterodisulfide reductases. NAD did not substitute for F 420 , although an HdrA homolog from the acetogen Moorella thermoacetica reduces the artificial electron acceptor benzyl viologen with NADH (28).
The ability of HdrA2 to interact with F 420 prompted asking whether HdrA2 together with HdrB2C2 is able to bifurcate electron pairs from F 420 H 2 for reduction of Fdx and CoMS-SCoB. Figure 3A and B are representative of results showing a dependence on Fdx for free thiol (HSCoM and HSCoB) formation and F 420 H 2 oxidation in the presence of CoMS-SCoB. A replot (Fig. 3C) of the replicated data shows one free thiol produced from CoMS-SCoB for each F 420 H 2 oxidized, a result consistent with simultaneous reduction of Fdx. These results show that HdrA2B2C2 catalyzes a thermodynamically favorable (ΔG°= ϭϪ38.6 kJ), flavin-based, coupling of the endergonic one-electron reduction of Fdx (E 0 = of ca. Ϫ520 mV) with F 420 H 2 (E 0 = of Ϫ380 mV) to the exergonic reduction of CoMS-SCoB (E 0 = of Ϫ140 mV) (Table S2) as previously predicted (29). Considering the inability of HdrA2B2C2 to utilize Fdx 1Ϫ as an electron donor (Fig. 2B), the results indicate the bifurcation proceeds according to equation 6. Figure 3D shows the dependence of F 420 H 2 oxidation on two limiting concentrations of CoMS-SCoB and a fixed nonlimiting concentration of Fdx that is stoichiometrically consistent with equation 6.
2F 420 H 2 ϩ 2Fdx ϩ CoMS-SCoB → 2Fdx 1Ϫ ϩ HSCoM ϩ HSCoB ϩ 2F 420 (6) Fdx:CoMS-SCoB oxidoreductase activity in cytoplasmic and membrane fractions of acetate-grown M. acetivorans. During growth of M. acetivorans with acetate, the reduced Fdx generated by oxidation of the carbonyl group donates its electrons to a membrane-bound electron transfer chain culminating with membrane-bound HdrDE that reduces CoMS-SCoB to the corresponding sulfhydryl forms of the cofactors (equation 2). The membrane-bound electron transport is coupled to H ϩ and Na ϩ translocation, forming gradients that drive ATP synthesis (30). Acetate-grown cells also upregulate genes encoding a homolog of HdrABC from CO 2 -reducing methanogens (HdrA1B1C1), HdrA2 and HdrB2C2 (18, 19, 31). Thus, it is hypothesized that either HdrA1B1C1 or HdrA2B2C2 catalyze cytoplasmic Fdx:CoMS-SCoB oxidoreductase activity in acetate-grown cells (19,25,31) for which biochemical evidence of cytoplasmic oxidoreductase activity is shown in Table 2. Of the total heterodisulfide reductase activity present in the extracts, a corresponding amount, namely, 56 and 29%, was recovered in the cytoplasmic and membrane fractions, respectively. The cytoplasmic fraction contained approximately 50% of the total oxidoreductase activity in extracts. Less than 10% of the total activity was recovered in the membrane fraction with the location of the remaining 40% unexplained. Nonetheless, the results support a cytoplasmic Fdx:CoMS-SCoB oxidoreductase system that accounts for approximately half of the total activity in acetate-grown cells.  a One unit is defined as micromoles of sulfhydryl produced per minute. b One unit is defined as micromoles of reduced methyl viologen oxidized per minute. c A sample from the same extract was loaded onto the sucrose gradient for preparation of cytoplasmic and membrane fractions.

DISCUSSION
The results have produced several milestones that provide a greater comprehensive understanding of the HdrABC class. Fusion of the MvhD homolog to the HdrA domain of HdrA2 is an unusual feature that, together with unusual catalytic capabilities reported here, identifies HdrA2B2C2 as representative of a previously unrecognized HdrABC subclass distributed in diverse species from the domains Bacteria and Archaea.
Intersubunit electron transfer. The individual expression and characterization of recombinant HdrA2, HdrB2, and HdrB2C2 provided an experimental approach to identify roles for each subunit previously unknown for any HdrABC homolog (13). The results show that the flavin-containing HdrA2 interacts with F 420 and Fdx and that HdrA2B2C2 bifurcates electron pairs from F 420 H 2 directed to the reduction of Fdx and CoMS-SCoB. It was also shown that, although HdrC2 has no role in catalysis, this subunit mediates electron transfer from HdrA2 to the catalytic HdrB2. These results establish the path of electron transfer from Fdx to CoMS-SCoB (Fig. 4) that in all probability generally applies to the HdrABC class.
Role for the Fdx:CoMS-SCoB oxidoreductase activity of HdrAB2C2 in the pathway for conversion of acetate to CH 4 . Coenzyme F 420 does not participate in the pathway of acetate conversion to methane, ruling out a role for the F 420 H 2 bifurcating activity of HdrA2B2C2. However, a role is envisioned for the Fdx:CoMS-SCoB oxidoreductase activity of HdrAB2C2 as shown in Fig. 5. The canonical HdrABC class is a cytoplasmic enzyme that contains flavin, whereas the HdrDE class is membrane bound, flavin free, and contains heme (32,33). The cytoplasmic HdrABC class is considered specific to obligatory CO 2 -reducing methanogens, and the membrane-bound HdrDE class is considered specific to pathways converting acetate and methylotrophic substrates (methanol, methylamines, and methylsulfide) to CH 4 . However, homologs of the cytoplasmic HdrABC class are also encoded in the genomes of acetotrophic and methylotrophic species from the order Methanosarcinales consistent with an auxiliary function. Our results indicate HdrA2B2C2 is responsible for cytoplasmic Fdx:CoMS-SCoB oxidoreductase activity of M. acetivorans when metabolizing acetate to CH 4 . This conclusion is further supported by the reported upregulation of hdrA2, hdrB2, and hdrC2 in response to growth with acetate and genetic analyses indicating a role for HdrA2 while excluding a role for the canonical HdrA1B1C1 (19,31,34). Although HdrB2C2 was shown to catalyze the transfer of electrons from Fdx to CoMS-SCoB, the HdrA2B2C2 complex was shown to be more efficient, which suggests a greater role in cytoplasmic electron transport during conversion of acetate to CH 4 . Genes encoding HdrA2B2C2 of M. acetivorans constitute a clade with homologs of other acetotrophic methanogens, further supporting a role in the pathway of acetate conversion to methane (19). Yan et al.

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The hdrA2 gene in M. acetivorans is in an operon with a gene encoding a putative polyferredoxin that is upregulated during acetotrophic growth, which suggests a potential role in mediating electron transfer to HdrA2B2C2 (18,19,31). Attempts to produce the recombinant protein in E. coli strain BL21(DE3) ΔiscR resulted in preparations that contained only a partial complement of Fe-S clusters that resisted reconstitution. The preparations were unable to replace Fdx or stimulate reduction of CoMS-SCoB in complete reaction mixtures. Therefore, no definitive conclusions can be drawn regarding the role of the polyferredoxin except that it is not necessary for the transfer of electrons from CODH/ACS to HdrA2B2C2.
A few Methanosarcina species contain a hydrogenase (Ech) that oxidizes the Fdx with production of H 2 and translocation of H ϩ (24). A second hydrogenase (Vho) oxidizes H 2 and reduces methanophenazine (MP), a quinone-like electron carrier that donates electrons to HdrDE and translocates an additional 4H ϩ . Thus, together with the   (25). The membrane-bound transfer of electrons from Fdx to CoMS-SCoB supports Na ϩ translocation by the Rnf complex and H ϩ translocation by HdrDE (35). Thus, a possible three coupling sites generate Na ϩ and H ϩ gradients that together drive ATP synthesis in M. acetivorans (30). The free energy available from conversion of acetate to CH 4 and CO 2 under standard conditions of equimolar reactants and products (ΔG°= ϭ Ϫ36 kJ) provides only a marginal amount of energy for growth considering the ATP requirement for activating acetate to acetyl-CoA in the first step in the pathway (ΔG°= ϭ ϩ31.8 kJ) (36). Thus, growth with acetate is at the extreme thermodynamic limit, requiring extraordinary mechanisms for maximizing the thermodynamic efficiency. Our finding of a cytoplasmic Fdx:CoMS-SCoB oxidoreductase system in acetate-grown M. acetivorans, and reconstitution of an active HdrA2B2C2, supports the divergent electron transport pathways from Fdx to CoMS-SCoB shown in Fig. 5A. Methanosarcina mazei and Methanosarcina barkeri, species that produce and consume H 2 during growth on acetate, also encode HdrA2, HdrB2, and HdrC2 homologs with greater than 80% amino acid sequence identity, predicting these species also synthesize HdrA2B2C2 participating in a soluble electron transport pathway (Fig. 5B). Acetyl-CoA-dependent methanogenesis catalyzed by M. barkeri cell lysate is not dependent on the membrane fraction consistent with a cytoplasmic electron transport system (37). For Methanosarcina species with documented respiratory control (38), HdrA2B2C2 provides a mechanism for modulating cytoplasmic versus membranebound electron transport proportional to ATP demand. We posit that the HdrA2B2C2dependent electron transport allows cells to maximize thermodynamic efficiency by circumventing membrane-bound electron transport when nonstandard concentrations of acetate encountered in the environment constrain the free energy available for multiple ion translocation sites. With this mechanism, Methanosarcina species could maximize the thermodynamic efficiency and metabolize acetate more rapidly, thereby outcompeting others for acetate. Indeed, M. acetivorans must compete with the more thermodynamically favorable acetate utilization by sulfate-reducing species (CH 3 COO Ϫ ϩ SO 4 2Ϫ ¡ 2HCO 3 Ϫ ϩ HS Ϫ ; ΔG°= ϭ Ϫ71.7 kJ) in the marine environment where M. acetivorans was isolated (17,39).
A proposed role for the F 420 H 2 bifurcation activity of HdrA2B2C2 homologs in Fe(III)-dependent anaerobic CH 4 oxidation (ANME). Although the F 420 H 2 bifurcation activity of HdrA2B2C2 is unanticipated in the pathway of acetate conversion to methane, a role for this activity can be envisioned for homologs in pathways of Fe(III)dependent anaerobic methane oxidation as shown in Fig. 6. For more than a decade, it was assumed that ANME required a consortium of at least two metabolic groups. However, recent reports indicate uncultured Archaea species in ANME group 2 (ANME-2) environments oxidize CH 4 alone, albeit dependent on Fe(III) as a direct electron acceptor (40,41). Furthermore, genomic analyses of an ANME-2a environment implicate a role for HdrABC homologs and acetate as a product (9). Homologs of HdrA2 and HdrB2 are encoded in the genome of "Candidatus Methanoperedens nitroreducens" (see Fig. S1 in the supplemental material) consistent with roles in the NO 3 Ϫ -dependent ANME pathway of this and possibly other ANME species dependent on external electron acceptors (42). Notably, Fe(III)-dependent oxidation of CH 4 was recently reported for an enrichment culture containing archaea of the order Methanosarcinales related to "Candidatus Methanoperedens nitroreducens" (43). Isolated ANME species have not been reported; however, M. acetivorans is capable of Fe(III)-dependent conversion of CH 4 to acetate and is phylogenetically related to uncultured species identified in ANME-2 consortia (44). Our current understanding leads to a proposed ANME pathway for M. acetivorans involving HdrA2B2C2 (Fig. 6; Table S2) that may also be operable in ANME-2 environments.
The core of the proposed ANME pathway (reactions 1 to 5, 7, 8, 16, 18, and 19) is a reversal of the established acetate-utilizing CH 4 -producing pathway in M. acetivorans (45). Table S2 lists thermodynamic calculations for reactions shown in Fig. 6. Reactions 1 and 2 in the ANME pathway are thermodynamically unfavorable (ΔG°= ϭ ϩ121 kJ), requiring oxidation of HSCoM and HSCoB coupled to reduction of Fe(III) (reactions 1 to 6), yielding an overall ΔG°= of Ϫ176 kJ (Table S2). The oxidation is catalyzed by HdrDE with transfer of electrons to methanophenazine (MP) and cytochrome c where Fe(III) is the terminal electron acceptor, thereby pulling reactions 1 and 2. Scalar proton translocation is accomplished by a "Q loop" mechanism involving MP and driven by reactions 3 to 6 (ΔG°= ϭ Ϫ297 kJ). Acetate-grown M. acetivorans is rich in multiheme cytochrome c that shuttles electrons between the Rnf complex and MP (25,46). Reduction of Fe(III) at cytochrome c of M. acetivorans is consistent with the documented role of multiheme c-type cytochromes as electron shuttles to Fe(III) minerals outside microbial cells (47,48). Moreover, metagenomic analyses of ANME environments reveal homologs of genes encoding c-type cytochromes and Rnf complexes hypothesized to function in ANME pathways (9,41,49).
The product of reaction 2 (CH 3 -H 4 SPT) is metabolized by divergent pathways leading to either acetate or CO 2 . The acetogenic pathway begins with reaction 7 (ΔG°= ϭ Ϫ40.5 kJ) requiring reduced Fdx for reduction of CO 2 that provides the carbonyl group of acetyl-CoA. The requisite reduced Fdx is generated in the pathway oxidizing the methyl group of CH 3 -H 4 SPT to CO 2 (reactions 9 to 15). The oxidation pathway is identical to that which functions in the dismutation of methylotrophic substrates to CO 2 and CH 4 wherein reaction 9 involves multiple steps, including two oxidations dependent on F 420 as the electron acceptor (50). The F 420 H 2 is oxidized by HdrA2B2C2 with bifurcation of electrons directed to Fdx and CoMS-SCoB (reaction 11). The bifurcation (ΔG°= ϭ Ϫ38.6 kJ) is coupled to reactions 12 to 15 (ΔG°= ϭ Ϫ148.5 kJ), reducing Fe(III) with an overall ΔG°= of Ϫ187.1 kJ. Reactions 12 to 15 are catalyzed by the same proteins and electron carriers as those used in reactions 3 to 6 albeit with different stoichiometry. Scalar proton translocation generates a proton gradient (high outside) accomplished by a "Q loop" mechanism involving MP. The exergonic oxidation of Fdx and reduction of Fe(III) in reactions 16 and 17 (ΔG°= ϭ Ϫ249.3 kJ) drives a vectorial translocation of Na ϩ consistent with the previously reported reduction of cytochrome c and pumping of Na ϩ by Rnf (25,35). The thermodynamically unfa-  Table S2.
Heterodisulfide Reductases of Bacteria and Archaea ® vorable reaction 2 (ΔG°= ϭ ϩ29.2 kJ) is catalyzed by the membrane-bound methyltransferase (Mtr) and driven by the Na ϩ gradient. ATP synthesis is catalyzed by the ATP synthase (reaction 18) dependent on the Na ϩ and H ϩ gradients (30). ATP is also synthesized by substrate level phosphorylation (reaction 8) catalyzed by phosphotransacetylase and acetate kinase. As previously proposed, multisubunit Na ϩ /H ϩ antiporter (Mrp) functions to adjust the ratio of Na ϩ /H ϩ (reaction 19) optimal for the ATP synthase and methyltransferase (51,52). Finally, the stoichiometry shown in Fig. 6 assumes a low availability of Fe 3ϩ that limits the Fe(III)-dependent oxidation of Fdx (reactions 16 and 17), allowing for the Fdx-dependent synthesis of acetate (reactions 7 and 8). Reactions 16 and 17 are more thermodynamically favorable than reactions 7 and 8 (Table S2); therefore, complete oxidation of CH 4 to CO 2 would be expected when Fe 3ϩ is nonlimiting. Thus, the availability of Fe 3ϩ in the native environment is expected to modulate the stoichiometry of CH 4 oxidation to acetate and CO 2 . It is also possible that reactions 7 and 10, or reactions 7 and 11, are spatially coupled, circumventing the Fe(III)-dependent oxidation of reduced Fdx (Fdx R ) by reactions 16 and 17, resulting in the stoichiometry shown in Fig. 6 when Fe(III) is nonlimiting.
Additional electron bifurcating roles are possible for HdrA2B2C2 homologs in nonmethanogenic species with diverse metabolisms (Fig. S1) that may replace F 420 H 2 (E 0 = ϭ Ϫ380 mV) with the analogous two-electron carrier NADH (E 0 = ϭ Ϫ320 mV) and replace CoMS-SCoB with disulfides such as the DsrC protein postulated for the FlxlABCD-HdrABC bifurcating complex of Desulfovibrio vulgaris (12).
Conclusions. HdrA2B2C2 represents a subclass of the HdrABC class with homologs in diverse species of the domains Bacteria and Archaea. Results revealed a previously unknown electron bifurcation system and intersubunit electron transport pathway generally applicable to the HdrABC class. Properties of HdrA2B2C2 predict that homologs participate in anaerobic CH 4 oxidation pathways and that HdrA2B2C2 is essential for optimal growth of acetotrophic methanogens in native environments.

MATERIALS AND METHODS
Cell growth and materials. Acetate-grown Methanosarcina acetivorans was mass cultured and harvested as previously described (53). E. coli strain BL21(DE3) ΔiscR was a gift from J. Golbeck. HSCoB and CoMS-SCoB was a gift from T. Wood. All chromatography columns, resins, and prepacked columns were purchased from GE Healthcare. Purification of F 420 from methanol-grown M. acetivorans cells and preparation of F 420 H 2 was performed as described elsewhere (54). All other chemicals were purchased from Sigma-Aldrich or VWR International.
Cloning of M. acetivorans genes. Genes encoding Fdx (MA0431), HdrA2 (MA2868), and HdrB2 (MA4237) were amplified by PCR from genomic DNA using primers shown in Table S1 in the supplemental material and cloned into pET22b (Novagen) using In-Fusion (Clontech) for expression of the proteins with a C-terminal His 6 tag. Genes encoding HdrC2 (MA4236) and HdrB2 (MA4237) were amplified by PCR and cloned into the first and second site, respectively, of pETDuet (Novagen) for coexpression with a C-terminal His 6 tag on HdrB2. All constructs were validated by sequencing.
Expression and purification of recombinant proteins. The expression plasmids (Table S1) were transformed into E. coli strain BL21(DE3) ΔiscR and grown with 100 g/ml ampicillin. A single colony was used to inoculate 100 ml of LB medium buffered with 50 mM morpholinepropanesulfonic acid (MOPS) (pH 7.4) and supplemented with 100 g/ml of ampicillin that was incubated at 37°C for 12 h. The starter culture was subcultured (1:50 dilution) in 3 liters of the above medium supplemented with 1 mM ferric ammonium citrate that was contained in a spinner flask (Chemglass) modified to allow sparging with 100% Ar. Cultures were grown aerobically at 37°C to an optical density at 600 nm (OD 600 ) of 0.6 that was then supplemented with 1 mM cysteine and induced with 200 M isopropyl-␤-D-thiogalactopyranoside (IPTG). Anaerobic metabolism was facilitated by the addition of glucose (0.5%, wt/vol) and sodium fumarate (25 mM) followed by incubation at 21°C for 20 h with sparging. Cells were harvested by centrifugation in sealed tubes containing 95% N 2 -5% H 2 , resuspended (1:5, wt/vol) in 50 mM Tris (pH 8.0) containing 10% glycerol (buffer C), and stored at Ϫ80°C until use. The anaerobic purification of recombinant proteins is described in Text S1. Purified HdrA2 was reconstituted with FAD by anaerobic incubation at 4°C for 10 h in buffer containing 0.1 mM FAD and passed through a PD-10 column to remove excess flavin.
Purification of CODH/ACS. The five-subunit enzyme was purified from acetate-grown M. acetivorans as described previously with modifications noted in Text S1 (55). A typical preparation reduced the recombinant Fdx (33.3 M) at a rate of 55.3 nmol/min/mg CODH/ACS. Enzyme assays. All enzyme assays were performed at 21°C in stoppered cuvettes containing the indicated atmosphere. Heterodisulfide reductase forward and reverse activities were performed as described elsewhere with modifications noted in Text S1 (56). Fdx:CoMS-SCoB oxidoreductase activity was performed as described in Text S1.
Other analytical procedures. All protein concentrations, except for Fdx, were determined with either the Bradford assay kit (Bio-Rad Laboratories) or Pierce assay kit (Thermo Scientific) that gave similar results. The protein concentration for Fdx was determined using the 390 of 30 mM Ϫ1 cm Ϫ1 (57). Iron and acid-labile sulfur contents were determined as described elsewhere (58,59). The flavin content of HdrA2 was determined by UV-Vis and fluorescence spectrometry as described elsewhere (60). For the electron bifurcation experiments, F 420 H 2 oxidation was determined by monitoring the fluorescence intensity with excitation at 420 and emission at 480 nm.