3.D.7 The H2:Heterodisulfide Oxidoreductase (HHO) Family

A single multicomponent system, the membranous H2:heterodisulfide oxidoreductase system of the methanogenic archaeon, Methanosarcina mazei Gö1 catalyzes (1) the H2-dependent reduction of 2-hydroxyphenazine by 2-hydroxyphenazine-dependent hydrogenase and (2) the dihydro-2-hydroxyphenazine-dependent reduction of the heterodisulfide of coenzyme M-SH and coenzyme B-SH (CoM-S-S-CoB) by heterodisulfide reductase. Washed inverted vesicles of M. mazei couple both processes with the transfer of H+ across the cytoplasmic membrane. The H+/2e- ratio measured was 0.9 for each reaction, and the electrochemical proton gradient generated could drive ATP synthesis via the H+-translocating ATP synthase.

M. mazei can grow on (1) H2 + CO2, (2) methanol, (3) methylamines and (4) acetate. In the pathways of methanogenesis, all substrates are converted to methyl-S-CoM (2-methylthioethane-sulfonate) either by reduction of CO2 or by demethylation of methanol, methylamine or acetate. Methane is formed from methyl-S-CoM by a two electron reduction reaction catalyzed by methyl-S-CoM reductase which uses HS-CoB (7-mercaptoheptanoylthreonine phosphate) as the electron donor. This reaction produces the heterodisulfide, CoM-S-S-CoB, which is the terminal electron acceptor of the membraneous electron transport system of M. mazei. If H2 is present, a membranous F420 [(N-L-lactyl-γ-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5&146;-phosphate]-nonreducing hydrogenase channels electrons via b-type cytochromes to the heterodisulfide reductase which reduces the terminal electron acceptor. This electron transport system, referred to as H2:heterodisulfide oxidoreductase, is coupled to proton translocation across the cytoplasmic membrane. When cells are grown on methanol, part of the methyl groups are oxidized to CO2, and reducing equivalents are transferred to coenzyme F420. In Methanosarcina strains, reduced F420 (F420H2) is reoxidized in a reaction catalyzed by the membrane-bound F420H2 dehydrogenase which is part of the F420H2:heterodisulfide oxidoreductase. Electrons are channeled to the heterodisulfide reductase, resulting in both the reduction of CoM-S-S-CoB and the formation of an electrochemical proton gradient. The resulting ΔμH+ (transmembrane electrochemical gradient of H+) is the driving force for ATP synthesis from ADP plus Pi as catalyzed by an A1A0-type ATP synthase (TC #3.2). The system is probably subject to respiratory control.

A proposed energy-coupling mechanism is presented in Figure 3 of Ide et al. (1999). Five gene products are involved where electrons are passed from H2 to the heterodimeric hydrogenase, VhoGA, then to a cytochrome b1, VhoC, then to methanophenazine (which may serve as the transmembrane proton carrier), and finally to the heterodisulfide oxidoreductase, HdrDE, which reduces CoB-S-S-CoM.

Homologues of VhoAG included numerous hydrogenases of bacteria and archaea; VhoC is distantly related to a quinone-reactive Ni2+/Fe2+-hydrogenase cytochrome b in Wolinella succinogenes; HtrD is homologous to numerous bacterial and archaeal enzymes including fumarate reductase and glycerol-3-P dehydrogenase, and HtrE, a second b-type cytochrome with five putative transmembrane spanners, has distant homologues in bacteria, other archaea and eukaryotes. In spite of the apparent mosaic origin of the components of the system, the complete system may be specific to methanogenic archaea. The H2:heterodisulfide oxidoreductase may be related to F420H2 and CO:heterodisulfide oxidoreductases which are also believed to generate a proton motive force by redox potential-driven H+ translocation.

The two reactions of the H2:heterodisulfide oxidoreductase which are coupled to H+ translocation (extrusion) are:

(1) H2 + 2-hydroxyphenazine + H+ (in) → dihydro-2-hydroxyphenazine + H+ (out)

(2) dihydro-2-hydroxyphenazine + CoM-S-S-CoB + H+ (in) →
2-hydroxyphenazine + HS-CoM + HS-CoB + H+ (out).


This family belongs to the Iron-Sulfur Protein (ISP) Superfamily.



Bertran, E., W.D. Leavitt, A. Pellerin, G.M. Zane, J.D. Wall, I. Halevy, B.A. Wing, and D.T. Johnston. (2018). Deconstructing the Dissimilatory Sulfate Reduction Pathway: Isotope Fractionation of a Mutant Unable of Growth on Sulfate. Front Microbiol 9: 3110.

Brodersen, J., S. Baümer, H.J. Abken, G. Gottschalk, and U. Deppenmeier. (1999). Inhibition of membrane-bound electron transport of the methanogenic archaeon Methanosarcina mazei Gö1 by diphenyleneiodonium. Eur. J. Biochem. 259: 218-224.

Deppenmeier, U., M. Blaut, A. Mahlmann, and G. Gottschalk. (1990). Reduced coenzyme F 420H2-dependent heterosulfide oxidoreductase: a proton-translocating redox system in methanogenic bacteria. Proc. Natl. Acad. Sci USA 87: 9449-9453.

Deppenmeier, U., V. Müller, and G. Gottschalk. (1996). Pathways of energy conservation in methanogenic archaea. Arch. Microbiol. 165: 149-163.

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Ruppert, C., S. Wimmers, T. Lemker, and V. Müller. (1998). The A1A0-ATPase from Methanosarcina mazei: cloning of the 5’ end of the aha operon encoding the membrane domain and expression of the proteolipid in a membrane-bound form in Escherichia coli. J. Bacteriol. 180: 3448-3452.

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TC#NameOrganismal TypeExample

H2:heterodisulfide oxidoreductase, HHO. Two protons are consumed in the cytoplasm while two protons are released in the periplasm, contributing to the pmf (Simon et al., 2008; Welte and Deppenmeier 2013).  The HdrD/E-type heterodisulfide reductase probably can function independently of the other subunits.

Methanogenic archaea

HHO of Methanosarcina mazei Göl
VhoA: (CAA58114)
VhoG: (CAA58113)
VhoC: (CAA58115)
HdrD: (gbY09870)
HdrE: (gbY09870)


Putative heterodisulfide oxidoreductase, HdrDE (Pires et al. 2003). HdrD, 423 aas and 1 TMS; HdrE, 394 aas and 6 TMSs.

HdrDE of Desulfovibrio desulfuricans


Putative heterodisulfide reductase, HdrDE (Pires et al. 2003). HdrD, 663 aas and 6 TMSs; HdrE, 392 aas and 6 TMSs.

HdrDE of Chlorobium phaeobacteroides


Cytoplasmic adenylylsulfate reductase, ApsAB, with its associated electron transfer protein complex, QmoABC which may take electrons from a periplasmic electron donor or from the membrane quinone pool (Zane et al. 2010; Bertran et al. 2018).

ApsAB/QmoABC of Desulfovibrio vulgaris


CoB--CoM heterodisulfide reductase with 4 different proteins subunits plus two probable auxillary proteins:  OLS30722 - OLS30727.

CoB--CoM heterodisulfide reductase of Candidatus Thorarchaeota archaeon AB_25


Putative cytoplasmic adenylylsulfate reductase, ApsAB, with its associated electron transfer protein complex, QmoABC which may take electrons from a periplasmic electron donor or from the membrane quinone pool and transfer them to ApsAB (Zane et al. 2010; Bertran et al. 2018).

ApsAB/QmoABC of Desulfococcus multivorans


TC#NameOrganismal TypeExample

Membrane-bound H2 oxidizing hydrogenase, HydAB (Guiral et al., 2005). HydC has not been identified, but adjacent to hydB is an open reading frame (ORF; AAC07057; 226aas) showing similarity to cytochromes that could be HydC.


HydAB of Aquifex aeolicus
HydA (large subunit; 564 aas) (AAC07046)
HydB (small subunit; 349 aas) (AAC07047)


Membrane-bound Ni/Fe-hydrogenase complex, HydABC (Simon et al., 2008). HydC (P31875) resembles 5.A.3.5.1 (e-6), 5.A.3.2.1 (e-5), and 3.D.7.1.1 (e-4). Two protons are consumed in the cytoplasm while two protons are released in the periplasm, contributing to the pmf (Simon et al., 2008).


HydABC of Wolinella succinogenes
HydA (P31884)
HydB (P31883)
HydC (P31875)


Hydrogenase HyaABCDEF, important for H2 oxidation during fermentation (Zbell and Maier 2009).


HyaABCDEF of Salmonella enterica suspecies enterica serovar Typhimurium
HyaA (367 aas, 4 TMSs)
HyaB (600 aas, 0 TMSs)
HyaC (347 aas, 4 TMSs)
HyaD (198 aas, 0 TMSs)
HyaE (134 aas, 0 TMSs)
HyaF (353 aas, 0 TMSs)


F420-non reducing hydrogenase subunits A, D and G of 472, 141, and 308 aas, respectively, with 0, 0 and 3-5 TMSs, respectively. It is a [NiFe] hydrogenase  and part of a complex that provides reducing equivalents for heterodisulfide reductase (Stojanowic et al. 2003). Also called HdrABC. This system is also found in Archeoglobus profundus DSM5631 (acc#s P84625, P84624, P84627) and Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Imachi et al. 2020).

MvhADG of Methanothermobacter marburgensis


Hydrogenase-2, HybABCDEFG including the subunits of the hydrogenase as well as three maturation proteins (HybD, HybF and HybG).

HybABCDEFG of E. coli