Advertisement

Evolution of complex I–like respiratory complexes

Open AccessPublished:May 02, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100740
      The modern-day respiratory complex I shares a common ancestor with the membrane-bound hydrogenase (MBH) and membrane-bound sulfane sulfur reductase (MBS). MBH and MBS use protons and sulfur as their respective electron sinks, which helped to conserve energy during early life in the Proterozoic era when the Earth's atmosphere was low in oxygen. MBH and MBS likely evolved from an integration of an ancestral, membrane-embedded, multiple resistance and pH antiporter and a soluble redox-active module encompassing a [NiFe] hydrogenase. In this review, we discuss how the structures of MBH, MBS, multiple resistance and pH, photosynthetic NADH dehydrogenase-like complex type-1, and complex I, which have been determined recently, thanks to the advent of high-resolution cryo-EM, have significantly improved our understanding of the catalytic reaction mechanisms and the evolutionary relationships of the respiratory complexes.

      Keywords

      Abbreviations:

      Fd (ferredoxin), MBH (membrane-bound hydrogenase), MBS (membrane-bound sulfane sulfur reductase), Mrp (multiple resistance and Ph), NDH (NADH dehydrogenase-like complex type-1), PQ (plastoquinone)
      Respiratory complexes couple the energy from redox reactions to the generation of electrochemical gradients and play a central role in cellular energy generation. Respiratory complex I is the first and most complicated enzyme in the electron transport chain of modern aerobic organisms. It catalyzes electron transfer from NADH to quinone and concomitantly drives protons across the membrane that are subsequently used to conserve energy by ATP synthesis (
      • Sazanov L.A.
      A giant molecular proton pump: Structure and mechanism of respiratory complex I.
      ,
      • Hirst J.
      Mitochondrial complex I.
      ,
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ,
      • Letts J.A.
      • Sazanov L.A.
      Clarifying the supercomplex: The higher-order organization of the mitochondrial electron transport chain.
      ). Complex I contains a 14-subunit core for redox catalysis and proton translocation. That core is highly conserved from bacteria to higher eukaryotes, although mammalian complex I contains an extra 31 supernumerary subunits (
      • Sazanov L.A.
      A giant molecular proton pump: Structure and mechanism of respiratory complex I.
      ,
      • Hirst J.
      Mitochondrial complex I.
      ,
      • Agip A.A.
      • Blaza J.N.
      • Fedor J.G.
      • Hirst J.
      Mammalian respiratory complex I through the lens of cryo-EM.
      ,
      • Berrisford J.M.
      • Baradaran R.
      • Sazanov L.A.
      Structure of bacterial respiratory complex I.
      ,
      • Parey K.
      • Wirth C.
      • Vonck J.
      • Zickermann V.
      Respiratory complex I - structure, mechanism and evolution.
      ,
      • Galemou Yoga E.
      • Angerer H.
      • Parey K.
      • Zickermann V.
      Respiratory complex I - mechanistic insights and advances in structure determination.
      ). Mechanistic insights into the function and intricacies of complex I have been gained through structural research, beginning with crystallographic studies of bacteria and yeast complex I, and recently from cryo-EM studies of the mammalian version and supercomplexes containing complex I as a component (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Fiedorczuk K.
      • Letts J.A.
      • Degliesposti G.
      • Kaszuba K.
      • Skehel M.
      • Sazanov L.A.
      Atomic structure of the entire mammalian mitochondrial complex I.
      ,
      • Guo R.
      • Zong S.
      • Wu M.
      • Gu J.
      • Yang M.
      Architecture of human mitochondrial respiratory megacomplex I2III2IV2.
      ,
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ,
      • Vinothkumar K.R.
      • Zhu J.
      • Hirst J.
      Architecture of mammalian respiratory complex I.
      ,
      • Letts J.A.
      • Fiedorczuk K.
      • Sazanov L.A.
      The architecture of respiratory supercomplexes.
      ,
      • Agip A.A.
      • Blaza J.N.
      • Bridges H.R.
      • Viscomi C.
      • Rawson S.
      • Muench S.P.
      • Hirst J.
      Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.
      ,
      • Letts J.A.
      • Fiedorczuk K.
      • Degliesposti G.
      • Skehel M.
      • Sazanov L.A.
      Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk.
      ,
      • Cabrera-Orefice A.
      • Yoga E.G.
      • Wirth C.
      • Siegmund K.
      • Zwicker K.
      • Guerrero-Castillo S.
      • Zickermann V.
      • Hunte C.
      • Brandt U.
      Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps.
      ).
      In oxygenic photosynthetic microorganisms, there are two types of electron transport. One is a linear electron flow, in which electrons extracted from water by photosystem II are passed through cytochrome b6f and plastoquinone (PQ), reducing NADP+ to NADPH in photosystem I and thereby establishing a proton gradient for ATP synthesis. The second is a cyclic flow in which electrons are cycled between cytochrome b6f and photosystem I via PQ. These organisms also contain an NADH dehydrogenase-like complex type-1 (NDH) that functions in cyclic electron flow by transferring electrons from ferredoxin (Fd) to PQ in order to pump protons across a thylakoid membrane, enabling ATP synthesis without concomitant production of NADPH (
      • Shikanai T.
      Chloroplast NDH: A different enzyme with a structure similar to that of respiratory NADH dehydrogenase.
      ,
      • Peltier G.
      • Aro E.M.
      • Shikanai T.
      NDH-1 and NDH-2 plastoquinone reductases in oxygenic photosynthesis.
      ). NDH is closely related to complex I; it contains 11 of its 14 core subunits as well as several oxygenic- and photosynthesis-specific subunits. Three cryo-EM structures of the 18-subunit NDH complex isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus have been determined recently from samples prepared at various pH values, revealing the overall organization and the Fd and PQ binding modes (
      • Laughlin T.G.
      • Bayne A.N.
      • Trempe J.F.
      • Savage D.F.
      • Davies K.M.
      Structure of the complex I-like molecule NDH of oxygenic photosynthesis.
      ,
      • Schuller J.M.
      • Birrell J.A.
      • Tanaka H.
      • Konuma T.
      • Wulfhorst H.
      • Cox N.
      • Schuller S.K.
      • Thiemann J.
      • Lubitz W.
      • Setif P.
      • Ikegami T.
      • Engel B.D.
      • Kurisu G.
      • Nowaczyk M.M.
      Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer.
      ,
      • Pan X.
      • Cao D.
      • Xie F.
      • Xu F.
      • Su X.
      • Mi H.
      • Zhang X.
      • Li M.
      Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.
      ).
      Energy conservation by complex I–related respiratory systems in some anaerobic microbes within the Archaea shows great variation and complexity; some even utilize Na+ rather than H+ as the coupling ion to drive ATP synthesis (
      • Sapra R.
      • Bagramyan K.
      • Adams M.W.
      A simple energy-conserving system: Proton reduction coupled to proton translocation.
      ,
      • Mayer F.
      • Muller V.
      Adaptations of anaerobic archaea to life under extreme energy limitation.
      ,
      • Kim Y.J.
      • Lee H.S.
      • Kim E.S.
      • Bae S.S.
      • Lim J.K.
      • Matsumi R.
      • Lebedinsky A.V.
      • Sokolova T.G.
      • Kozhevnikova D.A.
      • Cha S.S.
      • Kim S.J.
      • Kwon K.K.
      • Imanaka T.
      • Atomi H.
      • Bonch-Osmolovskaya E.A.
      • et al.
      Formate-driven growth coupled with H(2) production.
      ,
      • Lim J.K.
      • Mayer F.
      • Kang S.G.
      • Muller V.
      Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.
      ). Pyrococcus furiosus is a hyperthermophilic, anaerobic archaeon that resides in hot marine sediments and grows optimally at 100 °C, representing an ancestral life form with great potential in biotechnological applications (
      • Nisbet E.G.
      • Sleep N.H.
      The habitat and nature of early life.
      ,
      • Kengen S.W.M.
      'Pyrococcus furiosus, 30 years on'.
      ). In P. furiosus, two Na+-dependent energy-conserving complexes have been characterized: a membrane-bound [NiFe] hydrogenase (MBH) complex and a membrane-bound sulfane sulfur reductase complex (MBS) (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ,
      • Sapra R.
      • Bagramyan K.
      • Adams M.W.
      A simple energy-conserving system: Proton reduction coupled to proton translocation.
      ,
      • Wu C.H.
      • Schut G.J.
      • Poole 2nd, F.L.
      • Haja D.K.
      • Adams M.W.W.
      Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.
      ,
      • McTernan P.M.
      • Chandrayan S.K.
      • Wu C.H.
      • Vaccaro B.J.
      • Lancaster W.A.
      • Yang Q.
      • Fu D.
      • Hura G.L.
      • Tainer J.A.
      • Adams M.W.
      Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.
      ). MBH functions when the organism grows without elemental sulfur (S°). It falls into group 4 [NiFe] hydrogenases; this is the only group that contains nonhydrogenases, including several other Na+-dependent energy-coupling complexes in related Thermococcus species such as carbon monoxide dehydrogenase and formate hydrogen lyase (
      • Sapra R.
      • Bagramyan K.
      • Adams M.W.
      A simple energy-conserving system: Proton reduction coupled to proton translocation.
      ,
      • Lim J.K.
      • Mayer F.
      • Kang S.G.
      • Muller V.
      Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.
      ,
      • Schut G.J.
      • Lipscomb G.L.
      • Nguyen D.M.
      • Kelly R.M.
      • Adams M.W.
      Heterologous production of an energy-conserving carbon monoxide dehydrogenase complex in the hyperthermophile Pyrococcus furiosus.
      ). Those complexes transfer electrons from various donors (Fd for MBH; carbon monoxide for carbon monoxide dehydrogenase; and formate for formate hydrogen lyase) to protons and evolve H2 gas. The electron transfer is coupled to the translocation of Na+ across the membrane for subsequent ATP synthesis through the Na+-dependent ATP synthase (
      • Mayer F.
      • Muller V.
      Adaptations of anaerobic archaea to life under extreme energy limitation.
      ,
      • Pisa K.Y.
      • Huber H.
      • Thomm M.
      • Muller V.
      A sodium ion-dependent A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus.
      ).
      However, when P. furiosus grows on S°, MBH is downregulated, whereas MBS is upregulated and is essential (
      • Wu C.H.
      • Schut G.J.
      • Poole 2nd, F.L.
      • Haja D.K.
      • Adams M.W.W.
      Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.
      ,
      • Schut G.J.
      • Bridger S.L.
      • Adams M.W.
      Insights into the metabolism of elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: Characterization of a coenzyme A- dependent NAD(P)H sulfur oxidoreductase.
      ,
      • Bridger S.L.
      • Clarkson S.M.
      • Stirrett K.
      • DeBarry M.B.
      • Lipscomb G.L.
      • Schut G.J.
      • Westpheling J.
      • Scott R.A.
      • Adams M.W.
      Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.
      ). MBS catalyzes the transfer of electrons from Fd to polysulfide and generates a Na+ gradient, conserving more energy (42 kJ/mol/2e) than MBH (12 kJ/mol/2e), with a maximal growth yield coefficient almost twice that obtained without S° (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ,
      • Schicho R.N.
      • Ma K.
      • Adams M.W.
      • Kelly R.M.
      Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus.
      ). MBS lacks a molybdopterin cofactor, and it therefore reduces sulfur by a mechanism distinct from the molybdopterin-containing sulfur reductases found in mesophilic bacteria (
      • Jormakka M.
      • Yokoyama K.
      • Yano T.
      • Tamakoshi M.
      • Akimoto S.
      • Shimamura T.
      • Curmi P.
      • Iwata S.
      Molecular mechanism of energy conservation in polysulfide respiration.
      ,
      • Enemark J.H.
      • Cosper M.M.
      Molybdenum enzymes and sulfur metabolism.
      ). The recent cryo-EM structure of MBS reveals its architecture and provides insights into its catalytic mechanism; together with the recent MBH structure, it sheds lights into their close evolutional relationship with complex I (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ).
      One of the most convincing evolutionary relations is the similarity between the [NiFe] hydrogenase catalytic subunits from group 4 and the Q module of complex I, as shown using phylogenetic analysis (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ,
      • Schut G.J.
      • Boyd E.S.
      • Peters J.W.
      • Adams M.W.
      The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications.
      ). These homologous modules are illustrated in Figure 1. A rather simple evolutionary pathway has been proposed in which complex I evolved from ancestral [NiFe] hydrogenases and multiple resistance and pH (Mrp) complexes (
      • Efremov R.G.
      • Sazanov L.A.
      The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.
      ), driven by changes in geochemistry and bioenergetics (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ). In addition to complex I, MBH, and MBS, several other related complexes have been identified based on genome sequence (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ). However, our understanding of the evolution of respiratory complexes has been complicated by the available structures of complex I, MBH, and MBS, which reveal different arrangements of the homologous modules between these complexes.
      Figure thumbnail gr1
      Figure 1Modular architecture of complex I–like respiratory complexes. AE, the structures and proposed working models of complex I–like family members: (A) complex I (Thermus thermophiles; Protein Data Bank [PDB] ID: 4HEA); (B) NDH (PDB ID: 6HUM) with OPS subunits highlighted in transparent green surface view; (C) MBH (PDB ID: 6CFW); (D) MBS (PDB ID: 6U8Y); and (E) Mrp (PDB ID: 6Z16). The outlined region is the membrane-anchored peripheral module conserved among these complexes. The colored and shaded boxes in the left panel mark the modules corresponding to the schematic representation in the right panel. MBH, membrane-bound hydrogenase; Mrp, multiple resistance and pH; NDH, NADH dehydrogenase-like complex type-1; OPS, oxygenic photosynthesis-specific.
      NDH, MBH, and MBS belong to the complex I–like superfamily of membrane-bound respiratory complexes (
      • Brandt U.
      Adaptations of an ancient modular machine.
      ). All the members of this family are believed to have evolved from a common Mrp Na+/H+ antiporter complex (
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ,
      • Efremov R.G.
      • Sazanov L.A.
      The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.
      ,
      • Hedderich R.
      Energy-converting [NiFe] hydrogenases from archaea and extremophiles: Ancestors of complex I.
      ), the structure of which was recently reported (
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ,
      • Li B.
      • Zhang K.
      • Nie Y.
      • Wang X.
      • Zhao Y.
      • Zhang X.C.
      • Wu X.L.
      Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.
      ).
      In spite of the great progress described previously, detailed molecular mechanisms for this complex I–like superfamily are still lacking, such as the ion translocating path, utilization of different substrates, coupling of redox reactions to proton translocation, and evolutionary relationship among these complexes. This review focuses on the differences and connections among complex I–like respiratory complexes. We describe how mechanistically relevant modules are differently organized, as well as the unique adaptions of the individual respiratory machineries for various energy needs.

      Distinct assembly of shared modules of respiratory complexes

      At first glance, all complex I–like respiratory complexes appear similar, with an L-shaped structure consisting of a peripheral arm and a membrane arm (Fig. 1). However, careful structural analyses have revealed that these complexes are assembled differently from a common set of highly conserved functional modules.
      Complex I has the longest peripheral arm among these complexes, containing an N module and a Q module (Fig. 1A). The N module is distal to the membrane arm and contains the NADH dehydrogenase activity that extracts low-potential high-energy electrons from NADH (
      • Sazanov L.A.
      • Hinchliffe P.
      Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.
      ). It is connected to the membrane proton pumps by the Q module, which is directly attached to the membrane arm via a membrane anchor (mammalian ND1; Thermus thermophilus Nqo8). The Q module contains four hydrophilic subunits that coordinate three [4Fe–4S] clusters that relay electrons from the N module to ubiquinone (
      • Sazanov L.A.
      • Hinchliffe P.
      Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.
      ). The membrane arm of complex I translocates protons with a stoichiometry of 4H+ to 2e, necessitating four proton-pumping units (
      • Galkin A.S.
      • Grivennikova V.G.
      • Vinogradov A.D.
      -->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.
      ,
      • Wikstrom M.
      Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone.
      ,
      • Galkin A.
      • Drose S.
      • Brandt U.
      The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes.
      ). It is generally agreed that three large membrane subunits—in mammals: ND2, ND4, and ND5; in T. thermophilus: Nqo12, 13, and 14; also called antiporter-like subunits—each form a proton translocation path called the P module in Figure 1A (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ).
      The identity of the fourth proton path is less certain. It may be composed of several smaller membrane subunits (mammalian, ND6, ND4L; T. thermophilus, Nqo10, 11) termed the Pc module here (for composite proton path) (
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Efremov R.G.
      • Sazanov L.A.
      Structure of the membrane domain of respiratory complex I.
      ,
      • Kaila V.R.
      • Wikstrom M.
      • Hummer G.
      Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I.
      ). Alternatively, it may be two half channels, with one half channel in the T. thermophilus Nqo8 (mammalian, ND1; the membrane anchor of Q module) and the other half channel in the Pc module (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ). The 11 core subunits of NDH (Fig. 1B) are arranged essentially the same as in complex I (
      • Laughlin T.G.
      • Bayne A.N.
      • Trempe J.F.
      • Savage D.F.
      • Davies K.M.
      Structure of the complex I-like molecule NDH of oxygenic photosynthesis.
      ,
      • Schuller J.M.
      • Birrell J.A.
      • Tanaka H.
      • Konuma T.
      • Wulfhorst H.
      • Cox N.
      • Schuller S.K.
      • Thiemann J.
      • Lubitz W.
      • Setif P.
      • Ikegami T.
      • Engel B.D.
      • Kurisu G.
      • Nowaczyk M.M.
      Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer.
      ,
      • Pan X.
      • Cao D.
      • Xie F.
      • Xu F.
      • Su X.
      • Mi H.
      • Zhang X.
      • Li M.
      Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.
      ). However, NDH has a shorter peripheral arm because of the lack of the three subunits found in complex I that oxidize NADH. Accordingly, NDH transfers electrons from Fd (rather than from NADH) to PQ.
      Both MBH and MBS establish a Na+ gradient across the membrane with redox energy (low-potential high-energy electrons) from Fd. However, MBH transfers electrons to protons rather than to quinone and thus evolves H2 gas, whereas MBS transfers electrons to reduce the S–S bond of sulfane sulfur (
      • Sapra R.
      • Bagramyan K.
      • Adams M.W.
      A simple energy-conserving system: Proton reduction coupled to proton translocation.
      ,
      • Wu C.H.
      • Schut G.J.
      • Poole 2nd, F.L.
      • Haja D.K.
      • Adams M.W.W.
      Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.
      ). Both MBH and MBS are L-shaped structures with a peripheral arm and a membrane arm (Fig. 1, C and D) (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). Interestingly, MBS has evolved specific structural elements that assemble a dimeric complex. These elements include multiple salt bridges and a tryptophan-rich C-terminal extension of one MbsD subunit that extends into a large hydrophobic cleft in the peripheral arm of the second subunit. Each monomeric complex is expected to be fully functional. The peripheral arms of MBH and MBS contain four hydrophilic subunits: MbhJKLN in MBH and MbsJKLN in MBS, and they are closely related to the Q module of complex I. These cytoplasmic subunits are attached to their respective membrane arms through a membrane-anchoring subunit, MbhM in MBH and MbsM in MBS, which are homologous to the membrane-anchoring subunit Nqo8 in complex I. The four hydrophilic subunits together with the membrane anchor form a membrane-anchored peripheral module, a prominent feature conserved among MBH, MBS, NDH, and complex I (Fig. 1).
      Despite the high degree of structural conservation, major architectural and mechanistic differences exist among the respiratory complexes. First, the membrane-anchored peripheral module is attached to the “left” side of the membrane module of complex I and NDH (Fig. 1, A and B) but is attached to the “right” side of the membrane module of MBH and MBS (Fig. 1, C and D). This architectural difference may suggest different proton translocation mechanisms, which are described later. Second, while neither complex I nor NDH translocate Na+, MBH and MBS each contains a Na+-translocating module that is homologous to the Na+-translocating module found in the Mrp antiporter (
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ,
      • Li B.
      • Zhang K.
      • Nie Y.
      • Wang X.
      • Zhao Y.
      • Zhang X.C.
      • Wu X.L.
      Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.
      ). In fact, Mrp has every ion-translocating unit found in MBS (Fig. 1E). However, the number of H+-translocating units varies in these complexes: MBH has two, MBS and Mrp each have three, and NDH and complex I each have four (Fig. 1, A, C and D). Nevertheless, in each complex, one H+-translocating unit is universally made of the composite Pc path.

      Proton and sodium translocation

      Here, we present a detailed structural comparison to identify features important for proton and sodium translocation, in an attempt to understand how MBH, MBS, and Mrp have maintained the Na+/H+ antiporter activity and how NDH and complex I may have evolved to only pump protons by abolishing the Na+ translocation capability (
      • Sazanov L.A.
      A giant molecular proton pump: Structure and mechanism of respiratory complex I.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ,
      • Schut G.J.
      • Boyd E.S.
      • Peters J.W.
      • Adams M.W.
      The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications.
      ,
      • Efremov R.G.
      • Sazanov L.A.
      The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.
      ,
      • Hedderich R.
      Energy-converting [NiFe] hydrogenases from archaea and extremophiles: Ancestors of complex I.
      ).
      H+ translocation is the most conserved feature among these respiratory complexes. There are two types of proton paths in each complex. Type I is contained within antiporter-like subunits, whereas type II is the composite Pc path (Fig. 2). Type I proton paths are the most conserved and best understood (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ). MBH has one (MbhH) and MBS has two (MbsH and MbsH′), whereas NDH and complex I each have three (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Fiedorczuk K.
      • Letts J.A.
      • Degliesposti G.
      • Kaszuba K.
      • Skehel M.
      • Sazanov L.A.
      Atomic structure of the entire mammalian mitochondrial complex I.
      ,
      • Guo R.
      • Zong S.
      • Wu M.
      • Gu J.
      • Yang M.
      Architecture of human mitochondrial respiratory megacomplex I2III2IV2.
      ,
      • Vinothkumar K.R.
      • Zhu J.
      • Hirst J.
      Architecture of mammalian respiratory complex I.
      ,
      • Letts J.A.
      • Fiedorczuk K.
      • Sazanov L.A.
      The architecture of respiratory supercomplexes.
      ) (Fig. 2A). Key elements associated with proton translocation include two half channels in each transporter-like subunit, a transmembrane helix break in each half channel (in MbsH, TMH7 and 12; in MbsH′, TMH7 and 12), and a charged axis extending across membrane interior (in MbsH, E138, E219, K250, H330, and K393; in MbsH′, E128, K217, K248, H336, and K399) (Fig. 2B). These features are highly conserved among antiporter-like subunits of complex I–like machineries and suggest there is only one proton path within each antiporter-like subunit; this suggestion is supported by mutagenesis studies and molecular dynamics simulations (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Laughlin T.G.
      • Bayne A.N.
      • Trempe J.F.
      • Savage D.F.
      • Davies K.M.
      Structure of the complex I-like molecule NDH of oxygenic photosynthesis.
      ,
      • Schuller J.M.
      • Birrell J.A.
      • Tanaka H.
      • Konuma T.
      • Wulfhorst H.
      • Cox N.
      • Schuller S.K.
      • Thiemann J.
      • Lubitz W.
      • Setif P.
      • Ikegami T.
      • Engel B.D.
      • Kurisu G.
      • Nowaczyk M.M.
      Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer.
      ,
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ,
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ,
      • Di Luca A.
      • Gamiz-Hernandez A.P.
      • Kaila V.R.I.
      Symmetry-related proton transfer pathways in respiratory complex I.
      ,
      • Kaila V.R.I.
      Long-range proton-coupled electron transfer in biological energy conversion: Towards mechanistic understanding of respiratory complex I.
      ,
      • Euro L.
      • Belevich G.
      • Verkhovsky M.I.
      • Wikstrom M.
      • Verkhovskaya M.
      Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (complex I).
      ).
      Figure thumbnail gr2
      Figure 2Ion translocation mechanism in membrane arms of complex I–like complexes. A, comparison of membrane arms of complex I, Mrp, MBS, and MBH (the sources of the structures are the same as in ). Structural alignment was based on their respective antiporter-like subunit: Nqo14 of complex I, MrpD of Mrp, MbsH of MBS, and MbhH of MBH. The aligned structures are shown separately for clarity. Antiporter-like subunits are illustrated as cartoons; membrane anchor in the peripheral arm as semitransparent cartoons; and Na module and Pc module as green and pink surfaces, respectively. B, proposed ion translocation paths across each module (P module; Pc module; and Na module) are illustrated on the membrane arm of MBS. A prominent central hydrophilic axis (with the residues labeled) extending across the membrane arm is formed. For the Na+ translocation activity of MBS, the Na+ path and H+ path together may form a putative Na+:H+ antiporter module for the exchange of Na+ and H+. C, zoom view of the putative Na+ translocation path as marked by the dashed box in (B). The superscript letters on the amino acid labels refer to subunit identities. The Na+ path is illustrated as a solid gray arrow, and the H+ path is illustrated as a dashed gray arrow. This is based on the MBS structure (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ). An alternative Na+ path involving similar key residues (dashed blue arrow) was put forth based on the Mrp structure (
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ). MBH, membrane-bound hydrogenase; MBS, membrane-bound sulfane sulfur reductase; Mrp, multiple resistance and Ph.
      Notably, a lateral helix (helix HL) is universally conserved; it integrates all type I antiporter-like proton paths in each respiratory complex (Fig. 2A). Helix HL is the longest in complex I, as it connects together three antiporter-like subunits. Helix HL is shorter in MBH, MBS, and Mrp, just long enough to span one (in MBH) or two (in MBS and Mrp) type I proton paths. The sizes suggest an important role of the lateral helix in maintaining the assembly of varied numbers of antiporter-like subunits in complex I–like machineries (
      • Zhu S.
      • Vik S.B.
      Constraining the lateral helix of respiratory complex I by cross-linking does not impair enzyme activity or proton translocation.
      ,
      • Belevich G.
      • Knuuti J.
      • Verkhovsky M.I.
      • Wikstrom M.
      • Verkhovskaya M.
      Probing the mechanistic role of the long alpha-helix in subunit L of respiratory complex I from Escherichia coli by site-directed mutagenesis.
      ).
      The type II Pc proton paths are putative because their precise mechanism is not well understood (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Efremov R.G.
      • Sazanov L.A.
      Structure of the membrane domain of respiratory complex I.
      ). The Pc paths are half the size of type I paths and composed of multiple subunits. Pc paths resemble type I paths in terms of charged resides and a helical distortion. For example, the Pc path in MBS, composed of MbsD, MbsG, and TMH1-2 of MbsE, contains the conserved charged residues E58 in MbsD and E37 in MbsG, and a π-bulge distortion in TMH3 of MbsD (3D; Fig. 2B). These features are also present in complex I and MBH (
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ,
      • Agip A.A.
      • Blaza J.N.
      • Bridges H.R.
      • Viscomi C.
      • Rawson S.
      • Muench S.P.
      • Hirst J.
      Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.
      ,
      • Letts J.A.
      • Fiedorczuk K.
      • Degliesposti G.
      • Skehel M.
      • Sazanov L.A.
      Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ,
      • Efremov R.G.
      • Sazanov L.A.
      Structure of the membrane domain of respiratory complex I.
      ).
      Located adjacent to the type II Pc proton path in MBH, MBS, and Mrp is another multiple-subunit module with Na+ translocation activity (Fig. 2, A and B). This composite Na+ path is absent in complex I, which does not translocate Na+. The deletion of this module diminished Na+-dependent H2 production in MBH (
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). The MBH, MBS, and Mrp structures have several conserved features essential for Na+ translocation (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ), and we will use the MBS structure as an example. At the cytoplasmic side are four conserved charged residues (in MbsB, D30 and R31; in MbsC, D31 and R35) that may serve as the entry sites for Na+ (Fig. 2C). Replacement of the MbsB D30 and R31 equivalents in Mrp by alanine abolished or reduced its H+/Na+ antiporter activity (
      • Morino M.
      • Natsui S.
      • Ono T.
      • Swartz T.H.
      • Krulwich T.A.
      • Ito M.
      Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.
      ). There are two conserved negatively charged cavities below the Na+ entry site that form a complete Na+ translocating path (Fig. 2C). The first cavity reaches halfway through the membrane and is close to the absolutely conserved MbsB D36. Replacing a leucine for the equivalent aspartate residue in Mrp abolished antiporter activity (
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ).
      Adjacent to the first negatively charged cavity is the conserved P83 in MbsC that breaks TMH3 into two half helices; this is a prominent feature found in other H+/Na+ antiporters (
      • Coincon M.
      • Uzdavinys P.
      • Nji E.
      • Dotson D.L.
      • Winkelmann I.
      • Abdul-Hussein S.
      • Cameron A.D.
      • Beckstein O.
      • Drew D.
      Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters.
      ,
      • Lee C.
      • Kang H.J.
      • von Ballmoos C.
      • Newstead S.
      • Uzdavinys P.
      • Dotson D.L.
      • Iwata S.
      • Beckstein O.
      • Cameron A.D.
      • Drew D.
      A two-domain elevator mechanism for sodium/proton antiport.
      ,
      • Wohlert D.
      • Kuhlbrandt W.
      • Yildiz O.
      Structure and substrate ion binding in the sodium/proton antiporter PaNhaP.
      ,
      • Hunte C.
      • Screpanti E.
      • Venturi M.
      • Rimon A.
      • Padan E.
      • Michel H.
      Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.
      ). The second cavity is close to the outside of the membrane, involving D60 of MbsB (conserved) and K70 of MbsC and Y119 of MbsE (conserved only as polar residues). It is less negatively charged, perhaps to facilitate the release of Na+ outside the membrane. An alternative Na+ translocation path was suggested based on the cryo-EM structure of Anoxybacillus flavithermus Mrp (Fig. 2C) (
      • Steiner J.
      • Sazanov L.
      Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
      ). It has a similar Na+ entry site involving the conserved MbsB D30 and MbsA H139 and a similar first negatively charged cavity containing MbsB D36; but it has a different Na+ outlet, which coincides with the proton entry pathway in the proposed Pc unit of MBH and MBS (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). Yet another cryo-EM structure of the Dietzia Mrp suggested that the antiporter-like subunit MrpD may translocate Na+ (
      • Li B.
      • Zhang K.
      • Nie Y.
      • Wang X.
      • Zhao Y.
      • Zhang X.C.
      • Wu X.L.
      Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.
      ), which is different from both models described previously. Therefore, the current Na+ translocation paths in the MBH and MBS respiratory complexes are speculative and require further investigation.
      The four-proton translocation stoichiometry of complex I is generally accepted and enjoys strong experimental support, although its fourth type II proton path (the Pc path) is less defined (Fig. 1A). However, the proton translocation mechanisms of MBH and MBS are primarily based on structures and therefore are much less well defined (Figs. 1 and 2B). Here, we distinguish type II Pc path into two categories: first, the Pc path in complex I or NDH, which is highly constrained (sandwiched between the membrane-anchoring subunit to the left and the HL-integrated type I proton path to the right), and second, the Pc path in MBH, MBS, or Mrp, which is less constrained because the membrane-anchoring subunit is either absent (in Mrp) or moved away to the far right side (in MBH and MBS) (Figs. 1 and 2A). We hypothesize that tight constraint in complex I and NDH forces the Pc unit to actively translocate protons in unison—in the same direction—with the three type I proton paths. Without the constraint of the membrane-anchoring unit, the type II Pc unit in Mrp, MBH, or MBS pairs with the Na+-translocating unit to function as a passive Na+/H+ exchanger (Fig. 1, C and D) (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). Our hypothesis provides a rationale for the opposing locations (“left” versus “right”) of the membrane-anchored peripheral redox modules among the MBH, MBS, NDH, and complex I respiratory complexes. The scheme of separating type II Pc units into active proton pumps and passive Na+/H+ exchangers is consistent with the energy conservation efficiency of these complexes (calculated under standard conditions): 81 kJ/mol/2e for complex I with four proton pumps, 41 kJ/mol/2e for MBS with two proton pumps, and 12 kJ/mol/2e for MBH with only a single proton pump (
      • Schut G.J.
      • Zadvornyy O.
      • Wu C.H.
      • Peters J.W.
      • Boyd E.S.
      • Adams M.W.
      The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
      ).

      Comparison of the redox reaction site

      As described previously, the peripheral arm and its membrane anchor constitute the membrane-anchored peripheral module that is conserved among complex I, NDH, MBS, and MBH (Fig. 1) (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). The proximal half (for MBS, MbsL and MbsJ; MBH, MbhL and MbhJ; and T. thermophilus complex I, Nqo4 and Nqo6) directly contacts the membrane anchor (for MBS, MbsM; MBH, MbhM; and T. thermophilus complex I, Nqo8), and there is a large chamber at the interface that is of similar size in all these complexes (Fig. 3A). However, the entrance to the chamber is more open in MBS and complex I than in MBH (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ). This is consistent with the need to allow access to hydrophobic polysulfide and ubiquinone in MBS and complex I, respectively.
      Figure thumbnail gr3
      Figure 3Redox active sites in complex I–like respiratory complexes. A, the peripheral module is conserved among MBH, MBS, and complex I. A chamber of similar size is formed between the peripheral arm and its membrane anchor (MBH, MbhM; MBS, MbsM; complex I, Nqo8) in all three complexes and is shown in salmon surface. Structural alignment is based on the MBH MbhL, MBS MbsL, and complex I Nqo4; the aligned structures are shown separately for clarity. The sources of structures are the same as in . BF, comparison of the mode of coordination of proximal [4Fe–4S] cluster in complex I–like complexes. The complexes are aligned by MbhL in MBH (Protein Data Bank [PDB] ID: 6CFW), MbsL in MBS (PDB ID: 6U8Y), Nqo4 in complex I (PDB ID: 4HEA), the large subunit of [NiFe] hydrogenase (PDB ID: 2FRV), and NdhH in NDH (PDB ID: 6HUM). The aligned structures are shown separately for clarity. MBH, membrane-bound hydrogenase; MBS, membrane-bound sulfane sulfur reductase; NDH, NADH dehydrogenase-like complex type-1.
      The conserved peripheral arm coordinates a chain of three [4Fe–4S] clusters in all these complexes where the distances between cluster edges are less than 12 Å, enabling efficient electron tunneling (
      • Page C.C.
      • Moser C.C.
      • Chen X.
      • Dutton P.L.
      Natural engineering principles of electron tunnelling in biological oxidation-reduction.
      ). The distal and medial [4Fe–4S] clusters are each coordinated by four cysteines: MbsN C61, C64, C67, and C106 for the distal and C71, C96, C99, and C102 for the medial cluster in MBS. This is similar among MBS, MBH, NDH, and complex I (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ).
      The proximal [4Fe–4S] clusters are the top of the substrate-binding chambers, and they are differently coordinated, consistent with their distinct redox substrates, that is, ubiquinone for complex I, protons for MBH, and polysulfide for MBS (Fig. 3, BF). In MBH, four cysteines in MbhJ coordinate the proximal [4Fe–4S] cluster, holding it within the electron-tunneling distance of the [NiFe] cluster, which is site of H2 catalysis and also coordinated by four cysteines in MbhL. This mode of coordination resembles that of soluble dimeric [NiFe] hydrogenase and enables electron flow from the proximal [4Fe–4S] cluster to the [NiFe] cluster for proton reduction (Fig. 3, B and C) (
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). In complex I and NDH, the proximal [4Fe–4S] clusters (also called N2) are also coordinated by four cysteines. However, two of the four cysteines are consecutive in the primary sequence: Nqo6 C45 and C46 in complex I and NdhK C52 and C53 in NDH. They form a unique coordination geometry that allows for direct protonation and electron transfer to ubiquinone or PQ, respectively, although the exact mechanism is still unknown (Fig. 3, D and E) (
      • Sazanov L.A.
      • Hinchliffe P.
      Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.
      ). In MBS, however, the proximal [4Fe–4S] is coordinated by three, not four, cysteines (Fig. 3F). Therefore, one of the four Fe ions is unliganded and has been proposed to function as the catalytic center for reducing inorganic polysulfide (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ). This sulfur respiration mechanism is novel, distinct from the known sulfur reduction mechanism in mesophilic bacteria involving a molybdopterin (
      • Jormakka M.
      • Yokoyama K.
      • Yano T.
      • Tamakoshi M.
      • Akimoto S.
      • Shimamura T.
      • Curmi P.
      • Iwata S.
      Molecular mechanism of energy conservation in polysulfide respiration.
      ,
      • Enemark J.H.
      • Cosper M.M.
      Molybdenum enzymes and sulfur metabolism.
      ).
      Compared with MBH, complex I and NDH have lost all four cysteines coordinating the [NiFe] cluster and do not have hydrogenase activity (Fig. 3, BE). In MBS, however, there is a vestige of the [NiFe] binding site: of the four cysteines coordinating the [NiFe] in MBH, two remain in MBS (C85 and C385), although they can no longer coordinate a [NiFe] cluster (Fig. 3F). Surprisingly, they are not involved in catalysis even though they are conserved (
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ). Therefore, the structures suggest a gradual evolution of the [NiFe] site from a soluble [NiFe] hydrogenase, to MBH, then to MBS, and finally to NDH and complex I (
      • Brandt U.
      Adaptations of an ancient modular machine.
      ,
      • Kashani-Poor N.
      • Zwicker K.
      • Kerscher S.
      • Brandt U.
      A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I.
      ). While the active site among these complexes and the associated substrates are vastly different, the structure around the three [4Fe–4S] clusters is highly conserved, including the three-loop structure as depicted in Figures 3 and 4. It is likely that this chain of three clusters is more important for energy transduction than the substrate active site, and it may explain the evolutionary success of these respiratory complexes.
      Figure thumbnail gr4
      Figure 4Conserved structural elements for energy conversion in complex I–like respiratory complexes. A, the three interfacial loops (in the shaded area) are in a comparable configuration in respiratory complex MBH (in red), MBS (in magenta), and complex I (in gray). The three loops are looptwo-TMs (loop 1), loopβ1–β2 (loop 2), and loopTM5–6 (loop 3). They are adjacent to the proximal [4Fe–4S] cluster, at the interface between the peripheral arm and its membrane anchor in each complex. The structural conservation is revealed by aligning MBH MbhL, MBS MbsL, and complex I Nqo4, as in , CF. Notably, the C-terminal transmembranes immediately following loop 1 (highlighted in semitransparent surface view) are distinctively configured to coordinate the different numbers of the ion translocation units. B, the three-loop systems (marked by the shaded area) in complex I (top), MBH (middle), and MBS (bottom) are differently linked to membrane arm (semitransparent surface) through the variable C-terminal transmembranes following loop 1. However, a central charged axis from the proximal [4Fe–4S] through the membrane arms is maintained in each complex. Red spheres represent negatively charged residues and blue spheres positively charged residues. MBH, membrane-bound hydrogenase; MBS, membrane-bound sulfane sulfur reductase.

      Energy conversion by physical coupling of redox reaction with proton translocation

      The exact mechanism for coupling electron transfer to the spatially separated proton translocation remains unresolved (
      • Sazanov L.A.
      A giant molecular proton pump: Structure and mechanism of respiratory complex I.
      ,
      • Hirst J.
      Mitochondrial complex I.
      ,
      • Berrisford J.M.
      • Baradaran R.
      • Sazanov L.A.
      Structure of bacterial respiratory complex I.
      ,
      • Parey K.
      • Wirth C.
      • Vonck J.
      • Zickermann V.
      Respiratory complex I - structure, mechanism and evolution.
      ,
      • Galemou Yoga E.
      • Angerer H.
      • Parey K.
      • Zickermann V.
      Respiratory complex I - mechanistic insights and advances in structure determination.
      ,
      • Yu H.
      • Haja D.K.
      • Schut G.J.
      • Wu C.H.
      • Meng X.
      • Zhao G.
      • Li H.
      • Adams M.W.W.
      Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
      ,
      • Yu H.
      • Wu C.H.
      • Schut G.J.
      • Haja D.K.
      • Zhao G.
      • Peters J.W.
      • Adams M.W.W.
      • Li H.
      Structure of an ancient respiratory system.
      ). A dynamic region at the interface between the peripheral arm and membrane arm is proposed to be important for the energy transduction in complex I (
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Zhu J.
      • Vinothkumar K.R.
      • Hirst J.
      Structure of mammalian respiratory complex I.
      ,
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ). Specifically, quinone reduction triggers conformational changes in three protein loops around the substrate pocket, which propagate along a central charge axis in the membrane arm to drive proton translocation (
      • Galemou Yoga E.
      • Angerer H.
      • Parey K.
      • Zickermann V.
      Respiratory complex I - mechanistic insights and advances in structure determination.
      ,
      • Baradaran R.
      • Berrisford J.M.
      • Minhas G.S.
      • Sazanov L.A.
      Crystal structure of the entire respiratory complex I.
      ,
      • Zickermann V.
      • Wirth C.
      • Nasiri H.
      • Siegmund K.
      • Schwalbe H.
      • Hunte C.
      • Brandt U.
      Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
      ,
      • Parey K.
      • Brandt U.
      • Xie H.
      • Mills D.J.
      • Siegmund K.
      • Vonck J.
      • Kuhlbrandt W.
      • Zickermann V.
      Cryo-EM structure of respiratory complex I at work.
      ). This mechanism has gained strong support from recent biochemical and structural studies (
      • Agip A.A.
      • Blaza J.N.
      • Bridges H.R.
      • Viscomi C.
      • Rawson S.
      • Muench S.P.
      • Hirst J.
      Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.
      ,
      • Letts J.A.
      • Fiedorczuk K.
      • Degliesposti G.
      • Skehel M.
      • Sazanov L.A.
      Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk.
      ,
      • Cabrera-Orefice A.
      • Yoga E.G.
      • Wirth C.
      • Siegmund K.
      • Zwicker K.
      • Guerrero-Castillo S.
      • Zickermann V.
      • Hunte C.
      • Brandt U.
      Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps.
      ). Recent trapping of multiple states of complex I provide a glimpse of this complicated coupling mechanism: substrate reduction can initiate the reorganization of neighboring loops, which then induce conformational coupling (at the interface between the anchor for the peripheral arm and the proton translocation arm) and subsequent electrostatic coupling in a central hydrophilic axis across the membrane arm (
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ). A detailed structural comparison revealed that key elements implied in the coupling mechanism of complex I also exist in other members of complex I–like superfamily (Fig. 4).
      First, loops 1 to 3—corresponding to the Nqo7, Nqo4, and Nqo8 loops in T. thermophilus complex I or the ND3, 49 kDa, and ND1 loops in Bos taurus complex I—near the proximal [4Fe–4S] cluster are structurally conserved among complex I, MBH, and MBS, although there is little sequence-level conservation (Fig. 4A). These loops were proposed to sense quinone reduction and induce the initial conformational change in complex I (
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ). Second, despite the distinct location of the membrane-anchored peripheral module between MBS/MBH and complex I (Fig. 1), all the C-terminal regions of their loop 1 function to maintain the interface between the membrane-anchored peripheral arm and the ion-translocation module. These are helix HL and TMH2–3 of Nqo7 in complex I; helix HL and TMH1–2 of MbhI in MBH; and helix HL and TMH15–16 of MbsH' in MBS (Fig. 4B). This dynamic interface was proposed for conformational coupling in complex I (
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ). Third, a prominent, central, charged axis extending from the membrane anchor to the H+-translocating unit is maintained in MBH and MBS, as in complex I (Fig. 4B). This continuous hydrophilic axis within the membrane interior is key for electrostatic coupling in complex I (
      • Kampjut D.
      • Sazanov L.A.
      The coupling mechanism of mammalian respiratory complex I.
      ). Thus, in spite of their distinct overall architectures, the presence of these equivalent coupling elements implicates that the coupling mechanism is probably conserved among the respiratory complexes.

      Summary

      The complex I–like family of respiratory complexes now includes complex I, NDH, MBH, and MBS, which are evolutionarily related to the Mrp H+/Na+ antiporter. The most recent structures of these complexes have enabled us to understand their evolutionary trajectory at the atomic level. Structural comparison of these respiratory machineries highlights their modular evolution and reinforces the idea that the membrane-anchored peripheral module, the H+-translocation unit, and the Na+-translocation unit are independently evolving building blocks that are assembled differently in these respiratory complexes (Figs. 1 and 2) (
      • Efremov R.G.
      • Sazanov L.A.
      The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.
      ). Accordingly, the structures reveal how the respiratory machinery has evolved to acquire unique redox capabilities by utilizing different redox substrates as they became available on the evolutionary time scale (Fig. 3).
      Despite the different redox chemistries, the mechanism of energy conversion from redox potential to membrane proton gradient has been largely conserved across evolution (Fig. 4). The cryo-EM structures of the complex I–like superfamily provide us unprecedented knowledge into these complicated machineries. The determination of their ion translocation stoichiometry and molecular dynamics simulations based on these new structures will greatly help to understand their mechanisms. Solving the structures of substrate-bound or other coupling states will be required to answer important points such as the utilization of different substrates, ion translocation, and the coupling mechanism between them. Likewise, the differential arrangement of H+ and Na+ translocation modules in the currently available structures demonstrates that sequence homology is not sufficient to predict function, further demonstrating the need for the structures of additional complex I–related respiratory complexes.

      Major questions on this intriguing group of respiratory complexes remain

      • Are the charge core of the membrane domain and three-loop cluster the main elements responsible for energy transduction?
      • Is the change of the NQ-P module angle in complex I, which might be part of the energy transduction process (
        • Kampjut D.
        • Sazanov L.A.
        The coupling mechanism of mammalian respiratory complex I.
        ), a universal mechanism, and can it also be applied to MBH/MBS-type complexes?

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank members of the Li and Adams labs for their contributions to some of the work reviewed in this article. We thank David Nadziejka for technical editing of the article.

      Author contributions

      H. Y. prepared all figures. H. Y., G. J. S., D. K. H., M. W. W. A., and H. L. wrote and edited the article.

      Funding and additional information

      This work was funded by grants from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the US Department of Energy ( DE-SC0020085 to H. L. and DE-FG02-95ER20175 to M. W. W. A.).

      References

        • Sazanov L.A.
        A giant molecular proton pump: Structure and mechanism of respiratory complex I.
        Nat. Rev. Mol. Cell Biol. 2015; 16: 375-388
        • Hirst J.
        Mitochondrial complex I.
        Annu. Rev. Biochem. 2013; 82: 551-575
        • Schut G.J.
        • Zadvornyy O.
        • Wu C.H.
        • Peters J.W.
        • Boyd E.S.
        • Adams M.W.
        The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.
        Biochim. Biophys. Acta. 2016; 1857: 958-970
        • Letts J.A.
        • Sazanov L.A.
        Clarifying the supercomplex: The higher-order organization of the mitochondrial electron transport chain.
        Nat. Struct. Mol. Biol. 2017; 24: 800-808
        • Agip A.A.
        • Blaza J.N.
        • Fedor J.G.
        • Hirst J.
        Mammalian respiratory complex I through the lens of cryo-EM.
        Annu. Rev. Biophys. 2019; 48: 165-184
        • Berrisford J.M.
        • Baradaran R.
        • Sazanov L.A.
        Structure of bacterial respiratory complex I.
        Biochim. Biophys. Acta. 2016; 1857: 892-901
        • Parey K.
        • Wirth C.
        • Vonck J.
        • Zickermann V.
        Respiratory complex I - structure, mechanism and evolution.
        Curr. Opin. Struct. Biol. 2020; 63: 1-9
        • Galemou Yoga E.
        • Angerer H.
        • Parey K.
        • Zickermann V.
        Respiratory complex I - mechanistic insights and advances in structure determination.
        Biochim. Biophys. Acta Bioenerg. 2020; 1861: 148153
        • Baradaran R.
        • Berrisford J.M.
        • Minhas G.S.
        • Sazanov L.A.
        Crystal structure of the entire respiratory complex I.
        Nature. 2013; 494: 443-448
        • Zickermann V.
        • Wirth C.
        • Nasiri H.
        • Siegmund K.
        • Schwalbe H.
        • Hunte C.
        • Brandt U.
        Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.
        Science. 2015; 347: 44-49
        • Zhu J.
        • Vinothkumar K.R.
        • Hirst J.
        Structure of mammalian respiratory complex I.
        Nature. 2016; 536: 354-358
        • Fiedorczuk K.
        • Letts J.A.
        • Degliesposti G.
        • Kaszuba K.
        • Skehel M.
        • Sazanov L.A.
        Atomic structure of the entire mammalian mitochondrial complex I.
        Nature. 2016; 538: 406-410
        • Guo R.
        • Zong S.
        • Wu M.
        • Gu J.
        • Yang M.
        Architecture of human mitochondrial respiratory megacomplex I2III2IV2.
        Cell. 2017; 170: 1247-1257.e1212
        • Kampjut D.
        • Sazanov L.A.
        The coupling mechanism of mammalian respiratory complex I.
        Science. 2020; 370eabc4209
        • Vinothkumar K.R.
        • Zhu J.
        • Hirst J.
        Architecture of mammalian respiratory complex I.
        Nature. 2014; 515: 80-84
        • Letts J.A.
        • Fiedorczuk K.
        • Sazanov L.A.
        The architecture of respiratory supercomplexes.
        Nature. 2016; 537: 644-648
        • Agip A.A.
        • Blaza J.N.
        • Bridges H.R.
        • Viscomi C.
        • Rawson S.
        • Muench S.P.
        • Hirst J.
        Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.
        Nat. Struct. Mol. Biol. 2018; 25: 548-556
        • Letts J.A.
        • Fiedorczuk K.
        • Degliesposti G.
        • Skehel M.
        • Sazanov L.A.
        Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk.
        Mol. Cell. 2019; 75: 1131-1146.e1136
        • Cabrera-Orefice A.
        • Yoga E.G.
        • Wirth C.
        • Siegmund K.
        • Zwicker K.
        • Guerrero-Castillo S.
        • Zickermann V.
        • Hunte C.
        • Brandt U.
        Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps.
        Nat. Commun. 2018; 9: 4500
        • Shikanai T.
        Chloroplast NDH: A different enzyme with a structure similar to that of respiratory NADH dehydrogenase.
        Biochim. Biophys. Acta. 2016; 1857: 1015-1022
        • Peltier G.
        • Aro E.M.
        • Shikanai T.
        NDH-1 and NDH-2 plastoquinone reductases in oxygenic photosynthesis.
        Annu. Rev. Plant Biol. 2016; 67: 55-80
        • Laughlin T.G.
        • Bayne A.N.
        • Trempe J.F.
        • Savage D.F.
        • Davies K.M.
        Structure of the complex I-like molecule NDH of oxygenic photosynthesis.
        Nature. 2019; 566: 411-414
        • Schuller J.M.
        • Birrell J.A.
        • Tanaka H.
        • Konuma T.
        • Wulfhorst H.
        • Cox N.
        • Schuller S.K.
        • Thiemann J.
        • Lubitz W.
        • Setif P.
        • Ikegami T.
        • Engel B.D.
        • Kurisu G.
        • Nowaczyk M.M.
        Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer.
        Science. 2019; 363: 257-260
        • Pan X.
        • Cao D.
        • Xie F.
        • Xu F.
        • Su X.
        • Mi H.
        • Zhang X.
        • Li M.
        Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.
        Nat. Commun. 2020; 11: 610
        • Sapra R.
        • Bagramyan K.
        • Adams M.W.
        A simple energy-conserving system: Proton reduction coupled to proton translocation.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7545-7550
        • Mayer F.
        • Muller V.
        Adaptations of anaerobic archaea to life under extreme energy limitation.
        FEMS Microbiol. Rev. 2014; 38: 449-472
        • Kim Y.J.
        • Lee H.S.
        • Kim E.S.
        • Bae S.S.
        • Lim J.K.
        • Matsumi R.
        • Lebedinsky A.V.
        • Sokolova T.G.
        • Kozhevnikova D.A.
        • Cha S.S.
        • Kim S.J.
        • Kwon K.K.
        • Imanaka T.
        • Atomi H.
        • Bonch-Osmolovskaya E.A.
        • et al.
        Formate-driven growth coupled with H(2) production.
        Nature. 2010; 467: 352-355
        • Lim J.K.
        • Mayer F.
        • Kang S.G.
        • Muller V.
        Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.
        Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 11497-11502
        • Nisbet E.G.
        • Sleep N.H.
        The habitat and nature of early life.
        Nature. 2001; 409: 1083-1091
        • Kengen S.W.M.
        'Pyrococcus furiosus, 30 years on'.
        Microb. Biotechnol. 2017; 10: 1441-1444
        • Wu C.H.
        • Schut G.J.
        • Poole 2nd, F.L.
        • Haja D.K.
        • Adams M.W.W.
        Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.
        J. Biol. Chem. 2018; 293: 16687-16696
        • McTernan P.M.
        • Chandrayan S.K.
        • Wu C.H.
        • Vaccaro B.J.
        • Lancaster W.A.
        • Yang Q.
        • Fu D.
        • Hura G.L.
        • Tainer J.A.
        • Adams M.W.
        Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.
        J. Biol. Chem. 2014; 289: 19364-19372
        • Schut G.J.
        • Lipscomb G.L.
        • Nguyen D.M.
        • Kelly R.M.
        • Adams M.W.
        Heterologous production of an energy-conserving carbon monoxide dehydrogenase complex in the hyperthermophile Pyrococcus furiosus.
        Front. Microbiol. 2016; 7: 29
        • Pisa K.Y.
        • Huber H.
        • Thomm M.
        • Muller V.
        A sodium ion-dependent A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus.
        FEBS J. 2007; 274: 3928-3938
        • Schut G.J.
        • Bridger S.L.
        • Adams M.W.
        Insights into the metabolism of elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: Characterization of a coenzyme A- dependent NAD(P)H sulfur oxidoreductase.
        J. Bacteriol. 2007; 189: 4431-4441
        • Bridger S.L.
        • Clarkson S.M.
        • Stirrett K.
        • DeBarry M.B.
        • Lipscomb G.L.
        • Schut G.J.
        • Westpheling J.
        • Scott R.A.
        • Adams M.W.
        Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.
        J. Bacteriol. 2011; 193: 6498-6504
        • Schicho R.N.
        • Ma K.
        • Adams M.W.
        • Kelly R.M.
        Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus.
        J. Bacteriol. 1993; 175: 1823-1830
        • Jormakka M.
        • Yokoyama K.
        • Yano T.
        • Tamakoshi M.
        • Akimoto S.
        • Shimamura T.
        • Curmi P.
        • Iwata S.
        Molecular mechanism of energy conservation in polysulfide respiration.
        Nat. Struct. Mol. Biol. 2008; 15: 730-737
        • Enemark J.H.
        • Cosper M.M.
        Molybdenum enzymes and sulfur metabolism.
        Met. Ions Biol. Syst. 2002; 39: 621-654
        • Yu H.
        • Haja D.K.
        • Schut G.J.
        • Wu C.H.
        • Meng X.
        • Zhao G.
        • Li H.
        • Adams M.W.W.
        Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.
        Nat. Commun. 2020; 11: 5953
        • Yu H.
        • Wu C.H.
        • Schut G.J.
        • Haja D.K.
        • Zhao G.
        • Peters J.W.
        • Adams M.W.W.
        • Li H.
        Structure of an ancient respiratory system.
        Cell. 2018; 173: 1636-1649.e1616
        • Schut G.J.
        • Boyd E.S.
        • Peters J.W.
        • Adams M.W.
        The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications.
        FEMS Microbiol. Rev. 2013; 37: 182-203
        • Efremov R.G.
        • Sazanov L.A.
        The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.
        Biochim. Biophys. Acta. 2012; 1817: 1785-1795
        • Brandt U.
        Adaptations of an ancient modular machine.
        Science. 2019; 363: 230-231
        • Hedderich R.
        Energy-converting [NiFe] hydrogenases from archaea and extremophiles: Ancestors of complex I.
        J. Bioenerg. Biomembr. 2004; 36: 65-75
        • Steiner J.
        • Sazanov L.
        Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.
        Elife. 2020; 9e59407
        • Li B.
        • Zhang K.
        • Nie Y.
        • Wang X.
        • Zhao Y.
        • Zhang X.C.
        • Wu X.L.
        Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.
        Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 31166-31176
        • Sazanov L.A.
        • Hinchliffe P.
        Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.
        Science. 2006; 311: 1430-1436
        • Galkin A.S.
        • Grivennikova V.G.
        • Vinogradov A.D.
        -->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.
        FEBS Lett. 1999; 451: 157-161
        • Wikstrom M.
        Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone.
        FEBS Lett. 1984; 169: 300-304
        • Galkin A.
        • Drose S.
        • Brandt U.
        The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes.
        Biochim. Biophys. Acta. 2006; 1757: 1575-1581
        • Efremov R.G.
        • Sazanov L.A.
        Structure of the membrane domain of respiratory complex I.
        Nature. 2011; 476: 414-420
        • Kaila V.R.
        • Wikstrom M.
        • Hummer G.
        Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I.
        Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6988-6993
        • Di Luca A.
        • Gamiz-Hernandez A.P.
        • Kaila V.R.I.
        Symmetry-related proton transfer pathways in respiratory complex I.
        Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6314-E6321
        • Kaila V.R.I.
        Long-range proton-coupled electron transfer in biological energy conversion: Towards mechanistic understanding of respiratory complex I.
        J. R. Soc. Interface. 2018; 15: 20170916
        • Euro L.
        • Belevich G.
        • Verkhovsky M.I.
        • Wikstrom M.
        • Verkhovskaya M.
        Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (complex I).
        Biochim. Biophys. Acta. 2008; 1777: 1166-1172
        • Zhu S.
        • Vik S.B.
        Constraining the lateral helix of respiratory complex I by cross-linking does not impair enzyme activity or proton translocation.
        J. Biol. Chem. 2015; 290: 20761-20773
        • Belevich G.
        • Knuuti J.
        • Verkhovsky M.I.
        • Wikstrom M.
        • Verkhovskaya M.
        Probing the mechanistic role of the long alpha-helix in subunit L of respiratory complex I from Escherichia coli by site-directed mutagenesis.
        Mol. Microbiol. 2011; 82: 1086-1095
        • Morino M.
        • Natsui S.
        • Ono T.
        • Swartz T.H.
        • Krulwich T.A.
        • Ito M.
        Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.
        J. Biol. Chem. 2010; 285: 30942-30950
        • Coincon M.
        • Uzdavinys P.
        • Nji E.
        • Dotson D.L.
        • Winkelmann I.
        • Abdul-Hussein S.
        • Cameron A.D.
        • Beckstein O.
        • Drew D.
        Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters.
        Nat. Struct. Mol. Biol. 2016; 23: 248-255
        • Lee C.
        • Kang H.J.
        • von Ballmoos C.
        • Newstead S.
        • Uzdavinys P.
        • Dotson D.L.
        • Iwata S.
        • Beckstein O.
        • Cameron A.D.
        • Drew D.
        A two-domain elevator mechanism for sodium/proton antiport.
        Nature. 2013; 501: 573-577
        • Wohlert D.
        • Kuhlbrandt W.
        • Yildiz O.
        Structure and substrate ion binding in the sodium/proton antiporter PaNhaP.
        Elife. 2014; 3e03579
        • Hunte C.
        • Screpanti E.
        • Venturi M.
        • Rimon A.
        • Padan E.
        • Michel H.
        Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.
        Nature. 2005; 435: 1197-1202
        • Page C.C.
        • Moser C.C.
        • Chen X.
        • Dutton P.L.
        Natural engineering principles of electron tunnelling in biological oxidation-reduction.
        Nature. 1999; 402: 47-52
        • Kashani-Poor N.
        • Zwicker K.
        • Kerscher S.
        • Brandt U.
        A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I.
        J. Biol. Chem. 2001; 276: 24082-24087
        • Parey K.
        • Brandt U.
        • Xie H.
        • Mills D.J.
        • Siegmund K.
        • Vonck J.
        • Kuhlbrandt W.
        • Zickermann V.
        Cryo-EM structure of respiratory complex I at work.
        Elife. 2018; 7e39213