FEATURE ARTICLE Hydrogen Metabolism and the Evolution of Biological Respiration Two separate families of enzymes that oxidize hydrogen and also produce it arose through convergent evolution Eric S. Boyd, Gerrit J. Schut, Michael W.W. Adams, and John W. Peters Electron transport phosphorylation (ETP), one particularly prevalent in Archaea and Bacteria, of three known biochemical mechanisms for where it plays a central role in aerobic and anaer- conserving energy, couples oxidation-reduction obic physiologies and autotrophic and hetero- reactions to the translocation of ions across cell trophic metabolisms. This wide distribution of membranes (Table 1). Although we tend to asso- hydrogen metabolism among diverse organisms ciate ETP with high potential acceptors such as is consistent with its ancient origin. oxygen, it also comes into play using low poten- Two families of hydrogenase enzymes, one tial acceptors. In such cases, energy can accumu- containing iron ([FeFe]-hydrogenase) and the late through sequential translocation of ions other nickel and iron ([NiFe]-hydrogenase) met- across cellular membranes through low-energy- alloclusters, function in the metabolism of hydro- yielding reactions. gen. Members of both these enzyme families can Like ETP, substrate-level phosphorylation oxidize hydrogen as well as produce it. Despite (SLP) occurs widely in both oxic and anoxic set- their structural similarities, however, these two tings, where it plays an important role in meta- types of hydrogenases are not related but appear bolic pathways such as glycolysis and the tricar- to be the result of convergent evolution. boxylic acid cycle. SLP, which can combine with electron bifurcation (EB) in anaerobes such as hydrogen-oxidizing methanogenic archaea and SUMMARY acetogenic bacteria, is crucial for conserving en- ➤ The ability of Archaea and Bacteria to metabolize hydrogen plays a central ergy in anoxic environments where electron ac- role in aerobic and anaerobic physiologies, and in autotrophic and hetero- ceptors, that is, oxidants, are limited. Presum- trophic metabolisms. ably, early anaerobic organisms linked the ➤ Two unrelated families of hydrogenase enzyme, one containing iron (FeFe) oxidation of hydrogen with the reduction of car- and the other nickel and iron ([NiFe]-hydrogenase) metalloclusters, arose bon dioxide, much as we observe among modern through convergent evolution to catalyze the reversible oxidation of methanogens and acetogens. Understanding hydrogen. how their hydrogenase enzymes evolved to har- ➤ Ancient duplications of genes encoding hydrogen-oxidizing [NiFe]-hydro- ness energy thus may provide insights into some genase likely occurred in methanogenic Archaea, suggesting this enzyme of the earliest forms of life on Earth. class first evolved to oxidize hydrogen. ➤ Much like modern methanogenic archaea and acetogenic bacteria, early Origins of Hydrogen Metabolism anaerobic organisms linked hydrogen oxidation with carbon dioxide reduc- tion. Water interacting with the peridotitic minerals ➤ In strict anaerobes, electron bifurcating mechanisms play a central role in olivine and pyroxene that contain high levels of balancing the reduced and oxidized forms of cofactors such as ferredoxin ferrous iron can form hydrogen, a process known (Fd), NADH, and flavins. as serpentinization (Fig. 1). This process was ➤ Membrane-bound hydrogenases form multisubunit complexes with ion- likely widespread on the early Earth and may translocating proteins, coupling the oxidation or reduction of Fd to have sustained early forms of hydrogen-depen- hydrogen metabolism and the translocation of ions across cellular mem- dent life. The ability to metabolize hydrogen is branes. These complexes are the progenitors of modern-day respiratory distributed across the three domains of life and is complexes. Microbe—Volume 9, Number 9, 2014 • 361 FEATURE ARTICLE FIGURE 1 ؊ (Left) Abiotic H2, CO, and HCOO produced through the interaction of ultramafic minerals and water create gradients in available energy, which are likely governed by increasing oxidant limitation with depth into ultramafic (e.g., peridotite) units. Hypothetical depiction of the distribution of specific groups of organisms and associated [NiFe]-hydrogenases (designated by “G”) that function to couple the oxidation or reduction of substrates produced through water-rock reactions to energy conserving reactions is indicated. (Right) Phylogenetic reconstruction of representatives of [NiFe]-hydrogenase and related paralogs (NuoD, MbxL) with group designations indicated. The inferred position of the progenitor of [NiFe]-hydrogenase is indicated by an asterisk, with arrows indicating the directionality of putative diversification from this ancestor through evolutionary time. [FeFe]-hydrogenases are found in a limited [FeFe]-hydrogenase evolved after the divergence number of strict anaerobic bacteria and a few of Archaea and Bacteria from the Last Universal unicellular eukaryotes but not in archaea. In con- Common Ancestor, while [NiFe]-hydrogenase trast, [NiFe]-hydrogenase are widely distributed likely arose prior to the divergence of Archaea in both the archaeal and bacterial domains but and Bacteria, and may have played a role in pri- not in eukaryotes. This pattern suggests that mordial metabolism. TABLE 1. Modes of energy conservation in modern biology. Mode of Energy Conservation Description Electron Transport Phosphorylation (ETP) Coupling of oxidation-reduction reactions to the translocation of ions across cellular or organelle membranes. The retranslocation of ions to the cytoplasm is used to drive the synthesis of ATP. This process is common in Bacteria and Archaea and in the mitochondria of Eukarya and is more efficient than fermentative SLP. Substrate Level Phosphorylation (SLP) The transfer of a phosphoryl group from a donor compound to ADP to form ATP. This process is common in glycolytic and the tricarboxylic acid cycle and functions in all metabolisms. Electron Bifurcation (EB) The coupling of endergonic and exergonic reactions such that thermodynamically unfavorable reactions can be driven by thermodynamically favorable reactions. This process involves flavins and is common in anaerobic Bacteria and Archaea. 362 • Microbe—Volume 9, Number 9, 2014 FEATURE ARTICLE FIGURE 2 (A) Phylogenetic reconstruction of a concatenation of small and large subunits of group 4 [NiFe]-hydrogenase and related homologs (NuoBD, MbxJL). The distribution of lineages among Bacteria and Archaea, and among between archaeal phyla is indicated in parentheses. Abbreviations: Nuo, NADH dehydrogenase; Mbx: membrane bound ؉ ؉ oxidoreductase; Fpo: F420H2 dehydrogenase; Ech: energy conserving hydrogenase; Mrp: Na /H antiporter domain module; Mbh: membrane bound hydrogenase; CODH: carbon monoxide dehydrogenase module; FDH: formate dehydrogenase module; Eha/Ehb: membrane associated energy converting hydrogenase. The phylogeny is rooted with representatives of groups 1, 2 and 3 [NiFe]-hydrogenase. (B) Coupling of hydrogen and ferredoxin redox cycling with ion translocation in Mbh/Ech and Eha/Ehb. The [NiFe]-hydrogenases are prominent function in a complex with a flavin-containing among hydrogenotrophic methanogens, which heterodisulfıde reductase, coupling hydrogen ox- typically contain three phylogenetically and idation to exergonic reduction of the disulfıde physiologically distinct hydrogen-oxidizing bond (S-S) linking coenzyme M (CoM) and co- [NiFe]-hydrogenase enzymes (Fig. 1B and Fig. enzyme B (CoB) (Fig. 3). The energy released 2A). Among the [NiFe]-hydrogenases encoded during this reaction drives the thermodynami- by hydrogenotrophic methanogens are two rep- cally unfavorable reduction of ferredoxin (Fd), in resentatives of the group 3 subclass of this en- a process termed electron bifurcation. Methano- zyme, both of which are involved in reducing gens also contain group 4 membrane-bound flavin cofactors. Group 3a enzymes [8-hydroxy- [NiFe]-hydrogenase enzymes (Eha/Ehb) that 5-deazaflavin (F420) reducing [NiFe]-hydroge- couple oxidation of hydrogen with reduction of nases] couple the oxidation of hydrogen with re- Fd, which is coupled with sodium ion transloca- duction of F420, which serves as an electron tion into the cell (Fig. 2B and Fig. 3). Reduced Fd carrier during methanogenesis (Fig. 3). then drives the initial reduction of carbon dioxide Group 3c [NiFe]-hydrogenase enzymes (Mvh) during methanogenesis. Microbe—Volume 9, Number 9, 2014 • 363 FEATURE ARTICLE FIGURE 3 An evolutionary scheme based on phylogenetic reconstruction (Fig. 1B) explaining the series of duplication and gene recruitment events that led to the evolution of modern hydrogenotrophic methanogen [NiFe]-hydrogenase and simple (Mbh/Ech) and complex respiratory systems (Nuo). The directionality of redox coupled ion translocation in Eha/Ehb, Mbh/Ech, and Nuo is indicated by a positive or negative delta (⌬) and the change in membrane potential associated with a given reaction is indicated by a positive or negative psi (⌿). Abbreviations: G3, Group ,G3a, Group 3a; G3c, Group 3c; G4, Group 4; Nuo, NADH dehydrogenase; Mrp, Na؉/H؉ antiporter domain; Flav ;3 flavoprotein; CoM-S-S-CoB, Coenzyme M (CoM)-Coenzyme B (CoB) heterodisulfide; HS-CoM, reduced CoM; HS-CoB, reduced CoB; Quin, quinone. Likely Origins of the Hydrogenases enzymes (Fig. 3). Because both group 3 enzymes The occurrence of multiple phylogenetically re- are coupled to a bound flavin as a redox partner, lated and hydrogen-oxidizing group 3a/3c and 4 the group 3 enzymes possibly