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.

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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.

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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.

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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 emerged through [NiFe]-hydrogenase enzymes in methanogens duplication of the gene encoding an ancestral suggests that they arose through a series of gene enzyme that also partnered with a flavin cofactor duplications. Because methanogen [NiFe]-hy- (Fig. 3). drogenase enzymes in group 3 (Fig. 1B) and Irrespective of coupling, the observation that group 4 (Fig. 1B and Fig. 2A) branch lower (basal) the basal branching methanogen enzymes in sub- in these lineages, we speculate that the triple groups 3 and 4 are involved in hydrogen oxida- branch point in the unrooted phylogenetic re- tion suggests that the ancestral [NiFe]-hydroge- construction (asterisk, Fig. 1) is the ancestral en- nase enzyme also was involved in hydrogen zyme complex and may have been associated oxidation, a hypothesis that is bolstered by the with a methanogen. group 1 and most of the identifıed group 2 en- This scenario requires two gene duplications zymes also being involved in hydrogen oxidation to generate these three phylogenetically related (Fig. 1). The group 1 enzymes, which indirectly

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couple with oxidants such as oxygen, nitrate, and NADH, and flavins in strict anaerobes, and they sulfate, likely evolved in response to selective may well have done so on early Earth. pressure to harvest energy associated with these oxidants after they became more available about [NiFe]-Hydrogenases as Progenitors 2.4 billion years ago. to Complex Respiratory Systems Group 2a enzymes are involved in harvesting hydrogen generated as a byproduct of nitrogen What were the oxidants that supported life on fıxation, which is not considered a property of the early Earth before oxygen became widely avail- Last Universal Common Ancestor and thus is not able? Oxidant-limited anaerobes that lack well- ancestral. Because methanogens harbor early defıned respiratory systems may provide a clue. branching hydrogenase subfamilies in two of These organisms can recycle reduced Fd by form- these three enzyme subgroups, we further suggest ing hydrogen. The anaerobic oxidation of glucose that this ancestral [NiFe]-hydrogenase enzyme is coupled with reduction of Fd and NADϩ to functioned as a hydrogen oxidizing enzyme that produce ATP. However, Fd has a redox potential ϩ coupled with a variety of other redox partners similar to or lower than that of the H /H2 couple Ј Ј such as flavin and Fd (Fig. 3). Such an enzyme (Eo – 420 mV, pH 7.0), while NADH (Eo – 320 may have functioned in the acetyl-CoA pathway mV) cannot be recycled through the reduction of in methanogens much like modern flavin-based protons to H2. EB [NiFe]-hydrogenase enzyme complexes that Several members of the euryarchaeal order also involve Fd. Thermococcales circumvented this problem by using Fd as the sole redox carrier during sugar metabolism. The electrons that Fd gains are then Hydrogen-Based Electron disposed of by producing hydrogen by means of a Bifurcation in Early Life group 4 ion-translocating, membrane-bound Generating cofactors with reduction potentials [NiFe]-hydrogenase (Mbh; Fig. 2B and Fig. 3). suitable for driving vital biosynthetic reactions is Our phylogenetic analyses indicate that these a paramount challenge in environments where group 4 [NiFe]-hydrogenases likely functioned energetic gradients are minimal. Methanogens early in evolution as oxidizing enzymes coupling and other anaerobic organisms, however, have hydrogen indirectly with carbon dioxide reduc- mechanisms to meet this challenge, enabling tion in methanogenesis. Later, this type of en- them to persist in such environments. zyme diversifıed in function, eventually coupling Ј For example, although reducing protons (Eo numerous types of metabolism involving Fd and Ј – 420 mV, pH 7.0) via NADH (Eo – 320 mV) is hydrogen (Fig. 2A and 2B). thermodynamically unfavorable, many anaer- Membrane-bound hydrogenases (Mbh), in- obes generate more hydrogen than expected cluding Eha/Ehb, form multisubunit complexes solely from Fd-dependent reactions. Thus, some with ion-translocating proteins, termed Mrp. To- hydrogen is being derived from NADH, even gether, these complexes couple the oxidation or though it is not thermodynamically favorable. reduction of Fd to the oxidation or production of The coupling of thermodynamically favorable hydrogen. The outcome of this reaction deter- reactions with thermodynamically unfavorable mines the direction of ion translocation across reactions is made possible through a process the cellular membrane (Fig. 2B and Fig. 3). Ions called electron bifurcation (EB). Several [FeFe]- enter the cell when Fd reduction is coupled to and [NiFe]-hydrogenase enzymes are part of hydrogen oxidation (Eha/Ehb), whereas ions flavin-containing EB complexes, most notably in leave the cell when Fd is oxidized and protons are microorganisms conducting fermentative, aceto- reduced. genic, and methanogenic metabolisms. For Some organisms harbor additional Mrp-Mbh instance, the [NiFe]-hydrogenase (group 3c)/het- complexes that contain carbon monoxide dehy- erodisulfıde reductase complex of hydrog- drogenase (CODH), formate dehydrogenase enotrophic methanogenesis—producing reduced (FDH), or NADPH modules that couple the oxi- Fd from hydrogen—is a critical energy-conserving dation of these substrates to hydrogen produc- step in methanogenesis. Thus, bifurcating mecha- tion while translocating ions outside the cell for nisms now play a central role in balancing the re- use in ATP synthesis (Fig. 2). Organisms that duced and oxidized forms of cofactors such as Fd, generate ATP through the coupling of Fd, carbon

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AUTHOR PROFILE Boyd: from Baseball to the Rockies, and from Rocks to Hydrogenases and Energy Eric Boyd’s 7th-grade language arts teacher told him that he are trips to the Badlands, Rocky Mountain National Park, as could earn an “A” if he would stop worrying about his grade well as Yellowstone. Who would have known that several and also complete the coursework. “Ironically, it was not until decades later I would be studying hot-springs and the role of I was older before I realized what she was really trying to say minerals in sustaining life in these systems?” to me—focus on working hard and doing a good job,” he In his youth, Boyd spent much of his summers playing says. “I have taken this approach to much of my life.” pickup baseball. “I couldn’t get enough of it—and as a result Boyd, 35, is assistant professor of microbiology and immu- ended up wearing out my throwing arm and needing surgery nology at Montana State University in Bozeman, where he to repair it.” The orthopedist predicted Boyd would never studies microbial physiology and ecology as it relates to again throw a baseball or football. Ultimately, however, Boyd energy metabolism. “In particular, our work is focused on the played pitcher, third baseman, and quarterback on his high biochemical processes that are likely to have supported the school baseball and football teams. He earned a B.S. in biology earliest forms of life—that is, life that is supported not by in 2002 from Iowa State University and a Ph.D. in microbiology light-driven photosynthetic energy but rather chemical en- in 2007 from Montana State University (MSU). He was a NASA ergy supplied by water-rock interactions,” he says. “Much of Astrobiology Institute postdoctoral fellow and is currently a our work focuses on molecular hydrogen.” NASA Early Career fellow at MSU. Boyd was born in Des Moines, Iowa, and lived there as well He and his partner, Marisa, a self-employed gardener, live as in Wisconsin and South Dakota while growing up. “Wher- in Livingston, Mont., with their two dogs and a cat. “We used ever we lived, however, my parents made certain that we to have a flock of backyard chickens, but after nine years . . . were involved in sports and that we had access to the they joined a friend’s flock,” he says. In addition to hiking and outdoors,” he says. His father recently retired after 40 years camping, Boyd also enjoys “the history of places. It’s a lot of working at John Deere. His mother, a homemaker who cared fun reading about mining booms and busts, and then getting for Boyd, an older brother, and a younger sister until Boyd was in the car and driving to the old mines or towns, if they still in third grade, worked in the insurance industry and, later, as exist, and trying to imagine what it was like living during a librarian. His parents live in Des Moines. those times.’’ The family took many vacations in the West, including in the Rockies “where I developed a fascination with rocks, minerals, and the geological processes that are involved in Marlene Cimons their formation,’’ Boyd says. “Some of my earliest memories Marlene Cimons lives and writes in Bethesda, Md.

monoxide, or formate oxidation while generating CODH, or NADPH modules that enable cou- hydrogen (group 4 enzymes) operate near the pling with formate, carbon monoxide, and thermodynamic limits of life (-8 to -20 kJ/mol). NADPH, respectively, for both Ech and Mrp- They are considered to be among the most prim- Mbh (Fig. 2A). The incorporation of FDH and itive respiratory complexes in extant life. CODH modules into these enzyme complexes A more detailed phylogenetic analysis of would allow cells to oxidize these substrates, gen- group 4 [NiFe]-enzymes (Fig. 2A) indicates a erating hydrogen and a Naϩ-based PMF capable complex history between the archaeal and bacte- of driving ATP synthesis. rial lineages. Following the early branching of The large and small subunits of group 4 Eha/Ehb, two main lineages emerged. One of [NiFe]-hydrogenases show substantial homology these lineages includes Mrp-Mbh from Crenar- with the subunits NuoD and NuoB of the NADH chaeaota in the order Desulfurococcales and other dehydrogenase (complex I), respectively, in Ar- enzymes that do not activate hydrogen, including chaea, Bacteria, and Eukarya. Phylogenetic anal- Fpo, Mrp-Mbx, and Nuo. The other lineage in- ysis indicates that Nuo proteins derive from Mbh cludes archaeal and bacterial enzymes that pro- or Ech (Fig. 1B and Fig. 2A). Additional subunits duce hydrogen, including an energy conserving in the multimeric [NiFe]-hydrogenases (group 4) hydrogenase (Ech) and an archaeal Mrp-Mbh. show sequence similarity to other subunits of The diversifıcation of these lineages was likely respiratory complex I (NuoK, L, C, H, and I). driven in part by recruitment of accessory FDH, In addition to NuoBD, the small and large

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subunits of [NiFe]-hydrogenase share homology SUGGESTED READING: with FpoBD, which catalyzes the reduction of Hedderich, R. 2004. Energy-converting [NiFe] hydroge- methanophenazine with F420H2 in methanogens, nases from Archaea and extremophiles: ancestors of and with Mrp-MbxJL, which may be involved in complex I. J. Bioenerg. Biomemb. 36:65–75. respiring elemental sulfur. While we continue to Martin, W., and M. J. Russell. 2007. On the origin of make progress understanding the evolution of biochemistry at an alkaline hydrothermal vent. Phil. microbial respiratory systems, much remains to Trans. R. Soc. B 362:1887–1925. be learned. Martin, W. F. 2012. Hydrogen, metals, bifurcating elec- Eric S. Boyd is Assistant Professor in the Department of trons, and proton gradients: the early evolution of Microbiology and Immunology, Montana State University, biological energy conservation. FEBS Lett. 586:485– Bozeman, and the Wisconsin Astrobiology Research 493. Consortium, University of Wisconsin, Madison; Gerrit Schut is a Sapra, R., K. Bagramyan, and M. W. W. Adams. 2003. A Research Scientist and Michael W. W. Adams is Professor in the simple energy-conserving system: proton reduction Department of Biochemistry & Molecular Biology, University of coupled to proton translocation. Proc. Natl. Acad. Sci. Georgia, Athens; and John W. Peters is Professor in the USA 100:7545–7550. Department of Chemistry and Biochemistry, Montana State Schut, G. J., E. S. Boyd, J. W. Peters, and M. W. W. University, Bozeman, and is director of the Biological Electron Adams. 2013. The modular respiratory complexes in- Transfer and Catalysis Energy Froutiers Research Center. volved in hydrogen and sulfur metabolism by hetero- trophic hyperthermophilic archaea and their evolu- tionary implications. FEMS Microbiol. Rev. 37:182– ACKNOWLEDGMENTS 203. This work was supported by NASA (NNX13AI11G and Thauer, R. K., A.-K. Kaster, H. Seedorf, W. Buckel, and NNA13AA94A), the Division of Chemical Sciences, R. Hedderich. 2008. Methanogenic archaea: ecologi- Geosciences and Biosciences, Offıce of Basic Energy cally relevant differences in energy conservation. Na- Sciences of the Department of Energy (DE-FG05- ture Rev. Microbiol. 6:579–591. 95ER20175), and the Biological Electron Transfer and Vignais, P. M., and B. Billoud. 2007. Occurrence, classi- Catalysis Energy Frontiers Research Center funded by fıcation, and biological function of hydrogenases: an the Department of Energy, Offıce of Science. overview. Chem. Rev. 107:4206–4272.

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