Review pubs.acs.org/CR
New Perspectives on Proton Pumping in Cellular Respiration Mårten Wikström,*,† Vivek Sharma,‡ Ville R. I. Kaila,§ Jonathan P. Hosler,∥ and Gerhard Hummer⊥ †
Institute of Biotechnology, University of Helsinki, Biocenter 3 (Viikinkaari 1), PB 65, Helsinki 00014, Finland Department of Physics, Tampere University of Technology, Korkeakoulunkatu 3, Tampere 33720, Finland § Department Chemie, Technische Universität München, Lichtenbergstraße 4, D-85748 Garching, Germany ∥ Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, United States ⊥ Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt am Main, Germany ‡
4.6. Proton Pumping in Heme-Copper Oxidases of Types B and C 5. Conclusions Associated Content Special Issue Paper Author Information Corresponding Author Notes Biographies Acknowledgments References
CONTENTS 1. Introduction 2. Proton-Motive Oxidoreduction Loop 2.1. Cytochrome bd Oxidase 2.2. Photosynthetic Reaction Centers and Photosystems I and II 2.3. Complex III (Cytochrome bc1 and b6 f) 3. Complex I 3.1. General 3.2. Electron Transfer 3.3. Binding and Dynamics of Ubiquinone 3.4. Membrane Domain 3.5. Coupling Principles and Pumping Mechanisms 3.5.1. Could the Bound Ubiquinone Work as a Piston? 3.5.2. Initiation of the Proton-Pumping Mechanism 3.5.3. Stoichiometry of Proton Pumping 4. Complex IV 4. 1. Major Structure and Function 4.2. Proton Channels 4.2.1. D-Channel and Subunit III 4.2.2. K-Channel 4.2.3. H-Channel 4.3. Catalytic Cycle: Mechanism and Thermodynamics 4.4. Oxygen Affinity 4.5. Proton Pumping Mechanism of the A-Type Oxidases 4.5.1. Dilemma Concerning the One-Electron Reduction of State OH 4.5.2. State PR and Proton Pumping During the “Oxidative Phase″ 4.5.3. Minimal Models of the Proton Pump
© 2015 American Chemical Society
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1. INTRODUCTION Cellular respiration has a long research history at the interface between biology, chemistry, and physics. Research in this field spans the entire range from fundamental mechanistic and structural considerations on the atomic level to cell biology, physiology, and biomedicine. Our current concepts of cell respiration were established by the now classical work of Otto Warburg,1 David Keilin,2 and Britton Chance.3,4 Albert Lehninger5 localized the process to the mitochondria of eukaryotic cells, and researchers at the Enzyme Institute in Madison, headed by David Green, first isolated and purified the complexes of the respiratory chain.6 Efraim Racker and coworkers7 identified and isolated the structures responsible for catalyzing oxidative ATP synthesis. Relatively soon it became clear that respiratory chain-linked ATP synthesis (i.e., oxidative phosphorylation) is catalyzed by very similar membrane-bound protein complexes in all three domains of life, archaea, bacteria, and eukaryota, suggesting common universal mechanistic principles. The mechanism of coupling of respiratory electron transfer (eT) to the synthesis of ATP from ADP and inorganic phosphate (Pi) was mysterious for several decades, and was dominated by proposals of high-energy forms of cytochromes and other respiratory redox carriers that were thought to be chemical reaction intermediates in the process,8 thus providing the required coupling between eT and phosphorylation.9−12 The proposal of the chemiosmotic hypothesis of oxidative and photosynthetic phosphorylation by Peter Mitchell in 196113 required no other intermediate than a transmembrane electrochemical potential difference of protons (a protonmotive force, pmf), and postulated that the transfer of reducing
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Figure 1. Respiratory chain and ATP synthase. The respiratory chain is depicted in a phospholipid membrane, as it is found in most mitochondria and in many bacteria. ATP synthase is shown to translocate 8 protons per 3 synthesized ATP molecules, as observed in animal mitochondria.
Figure 2. Redox loops. (A) Classical redox loop. A hydrogen donor is oxidized by an electron accepting center (red circle) on the P-side of the membrane, and the electrons are transferred back across the membrane by electron carriers, to reach a hydrogen carrier (blue circle) on the N-side. (B) Redox loop modified by a “proton well”, which is a strict proton conductance pathway in the protein structure that may cross a considerable fraction of the membrane dielectric. (C) An approximation of the profiles of proton-motive force (pmf), electrical membrane potential (ΔΨ), and pH across the membrane at the level of the proton well in the protein.
equivalents (hydrogen and electrons) in the respiratory chain was tightly coupled to net proton translocation across the coupling membrane (refs 14−16, see below). The same concept was proposed for primary energy transduction in
photosynthesis, so that the pmf appeared to be a truly universal intermediate in all primary biological energy transduction. The subsequent debate, in the beginning of which Mitchell had only few supporters, was often fierce (the “ox phos wars”), 2197
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2. PROTON-MOTIVE OXIDOREDUCTION LOOP The key point of Mitchell’s chemiosmotic theory is that the oxidoreduction reactions of the respiratory chain generate an electrochemical proton gradient (pmf) across the inner mitochondrial (or bacterial) membrane, which is then utilized to drive the synthesis of ATP from ADP and inorganic phosphate (Pi). The chemiosmotic theory included an explicit description of how respiratory chain activity may generate pmf. On the basis of ideas proposed earlier by the Swedish plant physiologist Henrik Lundegårdh, the oxidoreduction reactions were postulated to be organized as oxidoreduction (or redox) loops with respect to the membrane, such that electrically neutral hydrogen is transported from the negatively charged Nside to the positively charged P-side, whereas transmembrane eT occurs in the opposite direction (Figure 2A). Such vectorial organization of the redox chemistry (“vectorial chemistry”) results in overall translocation of protons, but from a mechanistic viewpoint it is important to note that the proton itself never crosses the dielectric barrier of the membrane. Instead, the proton crosses the barrier electroneutrally in one direction together with the electron, whereas the translocation of charge is due to eT in the opposite direction, catalyzed by electron carriers positioned across the membrane. It is interesting to note today that the Mitchell−Lundegårdh redox loop principle turned out to be correct for a large number of proton-motive systems of primary energy transduction in biology. Thus, both Photosystems I and II in plants and photosynthetic bacteria generate pmf by this principle,44,45 and the same is true for bacterial nitrite and nitrate reduction,46 for the alternative bacterial ubiquinol oxidase cytochrome bd,47 and also for cytochrome bc1, i.e., Complex III, of the respiratory chain,48−50 as for the analogous cytochrome b6 f complex in photosynthesis.51 For mechanistic reasons we make a strict distinction here between such redox loops and true proton pumps. Here, we focus our endeavor on the true proton-pumping respiratory complexes I and IV, in which protons do cross the membrane dielectric against a pmf. Yet, a few brief comments on systems that catalyze different versions of the redox loop may be warranted.
intriguing details of which have been documented by science historians.17,18 Later on the chemiosmotic hypothesis received increasing experimental support and reached the status of a theory. It became accepted by an increasing number of researchers,19 and the notions of high-energy forms of respiratory redox carriers as intermediates in oxidative phosphorylation were refuted experimentally for the cases of both cytochrome b20,21 and cytochrome c oxidase (CcO).22,23 In the latter case presumed high-energy forms of heme a3 turned out to be two hitherto unknown reaction intermediates in the catalysis of O2 reduction to water, the so-called P (PM) and F states (ref 22, section 4.3). These intermediates accumulated in mitochondria treated with ATP because the electron and proton transfer reactions of energy conservation by CcO were driven backward at the high applied pmf and a high (oxidizing) redox potential at cytochrome c. In that sense these intermediates were indeed of high energy. Mitchell was a strong proponent of a so-called direct coupling principle, both between respiration and generation of proton-motive force,13−16 and between the latter and the synthesis of ATP by the F1Fo ATP synthase.24 This principle was based on the concept of “vectorial chemistry”, implying that the respiratory and photosynthetic electron and hydrogen transfer reactions themselves, as well as those of phosphorylation of ADP to ATP, would be directed in space relative to the membrane. We will discuss this concept here as far as respiration is concerned. For oxidative ATP synthesis, as catalyzed by the F1Fo complex, the work by Boyer,25 Junge,26 Walker,27 Yoshida,28 and others has ruled out such “directed” synthesis of ATP, and has shown beyond doubt that the proton-motive force drives ATP synthesis in a more indirect fashion, consisting of a unique rotatory mechanical motion, fuelled by proton translocation that sets forth a series of changes in the conformation of the catalytic domains of the soluble F1 portion of the complex. The composition of the respiratory chain varies among species, particularly among prokaryotes where variation may occur even within a single species under different conditions of growth. In mitochondria of eukaryotic cells the variation is much less, and the chain is usually composed of Complexes I, III, and IV (Figure 1). We do not consider Complex II (succinate-ubiquinone oxidoreductase29) as a member of the respiratory chain, because it has no proton-motive activity. It is rather a component of Krebs’ tricarboxylate cycle, and bound to the inner mitochondrial membrane only due to the membranous location of its electron acceptor, ubiquinone. As will become apparent, the mechanistic principle of energy transduction and proton-motive function in respiratory Complex III differs fundamentally from those that govern the function of Complexes I and IV, which are genuine proton pumps, the topic of this review. Several reviews have been published recently on the proton-motive function of the respiratory chain complexes I and IV (e.g., refs 30−43). Here, we will refer extensively to these papers and instead focus on some of the key remaining problems in understanding their function, which includes cases where current interpretations of experimental data appear conflicting to the extent of hampering progress. We aim at a novel synthesis of available information, which we hope will lead to the emergence of a deeper mechanistic understanding and to stimulate further research.
2.1. Cytochrome bd Oxidase
The first example of a redox loop is the bacterial alternative quinol oxidase cytochrome bd (for a review, see ref 47), which is structurally completely unrelated to the family of hemecopper oxidases discussed in detail below (section 4). Although there is no 3D structure yet for this type of terminal oxygen reductase, the proton-motive arrangement in the membrane is believed to be as shown in Figure 2B. The sites of quinol oxidation, as well as of O2 binding and reduction, are all located near the P-side of the membrane. The electrons from quinol oxidation near heme b558 are transferred parallel to the membrane to the heme b595/heme d pair where O2 is bound and reduced to water. However, the protons released on quinol oxidation take a different route and are released to the P-side (the bacterial periplasm), while the protons required to form water from reduced dioxygen are taken from the N-side. This arrangement obviously requires a proton-transferring pathway (a “proton well”) in the protein structure that extends from the aqueous N-side almost entirely across the membrane (Figure 2B). Due to this, the electrical potential difference is created entirely, or nearly so, by transmembrane proton transfer across the “well”, involving amino acid residues, two of which have 2198
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one of the two redox couples involving ubiquinone, viz., Q/Q•− or Q•−/QH2, and the b-type cytochromes equilibrate with the other, then oxidation of cytochrome c will either create ubisemiquinone (Q•−) to reduce cytochrome b, or it will consume the ubisemiquinone and thus favor heme b reduction by raising the QH2/Q•− ratio. The former version has been subsequently adopted as the more likely one, because the usually quite unstable semiquinone renders the Q/Q•− and Q•−/QH2 couples low and high potential, respectively. Such a bifurcation of eT predicts that a redox titration of cytochrome b with the succinate/fumarate couple in the presence of antimycin should yield an apparent Nernstian nvalue of 2 (for a two-electron donor/acceptor), because one of the two electrons from succinate (ubiquinol) would always be lost via the high-potential branch to dioxygen, and only the other would reduce cytochrome b. This prediction was indeed fulfilled,53 and provided strong support to the idea of bifurcated ubiquinol oxidation. This idea was subsequently incorporated as an essential element of Mitchell’s proton-motive Q-cycle mechanism,57,58 which is presented schematically in Figure 4. It is evident that this mechanism is basically a redox loop, albeit somewhat complicated in that the electrons fed into cytochrome b are looped back to reduce a second molecule of ubiquinone. Two turnovers are required for complete oxidation of ubiquinol by cytochrome c. However, the cytochrome bc1 complex is dimeric, and Mulkidjanian59 proposed an “activated” Q cycle in which the primed physiologically active state already contains two electrons, one in heme bH and another as semiquinone near heme bH, each in separate monomers. It is noteworthy that the proton translocation catalyzed by the Q cycle is thermodynamically equivalent to translocation of 1 H+/ e− as expected from a classical redox loop. The fact that 2 H+/ e− are nevertheless released on the P-side of the membrane has created several misunderstandings in the literature, but one of these two protons is trivially released from QH2 on its oxidation near the P-side of the membrane. Moreover, the release of this proton into the well-buffered aqueous medium on the P-side has little effect on the ΔpH term of the pmf.
been tentatively identified as carboxylates, and perhaps water molecules.47 Overall, this arrangement of quinol oxidation by molecular oxygen leads to net translocation of one proton per electron transferred, as expected. The particular arrangement in cytochrome bd emphasizes one extreme of the redox loop concept, where the translocation of charge across the membrane is due entirely (or almost entirely) to proton transfer to the site of proton consumption by the redox chemistry (here the heme b595/d site), whereas eT contributes little to generation of pmf. Assuming that the “proton channel” has a high and specific conductance for protons, this type of arrangement means that the local profile of the transmembrane pmf at the level of the cytochrome bd complex shows a steep gradient near the P-side interface (Figure 2C). 2.2. Photosynthetic Reaction Centers and Photosystems I and II
In contrast to the arrangement of cytochrome bd, the redox components of Photosystem II of plants, algae, and cyanobacteria,44 and the photosynthetic bacterial reaction centers,52 are arranged across the membrane in such a way as to maximize the component of transmembrane eT. Reduction of quinone on the N-side of the membrane and water splitting on the P-side (in oxygenic photosynthesis) create the pH gradient, which is the major component of the pmf in chloroplasts. Also Photosystem I (plastocyanin: ferredoxin oxidoreductase) catalyzes transmembrane eT, and consumes protons on the N-side by reduction of NADP+ (Figure 3).
Figure 3. Proton-motive force generation in photosynthesis. Photosystem II (PS II), the cytochrome b6 f complex, and Photosystem I (PS I) are shown schematically in the thylakoid membrane of chloroplasts. Q, plastoquinone; PC, plastocyanin. The classical redox loop arrangement is emphasized (see Figure 2A).
3. COMPLEX I 3.1. General
NADH:ubiquinone oxidoreductase, or Complex I, functions as the entry point for electrons into the respiratory chains of eukaryotes and many bacteria.30−34 Complex I couples the free energy released on reduction of quinone to quinol (Q + 2 e− + 2 H+ → QH2; Em,7 ∼90 mV for ubiquinone in membranes) by oxidation of nicotinamide adenine dinucleotide (NADH → NAD+ + 2 e− + H+; Em,7 = −320 mV) to translocation of protons across the inner mitochondrial membrane (or the cytoplasmic membrane of bacteria), by which pmf is generated across the membrane. Under standard conditions and zero pmf the driving force is thus ∼410 mV, or ∼820 mV for the twoelectron process of NADH oxidation by Q. Complex I is a fully reversible redox-coupled proton pump, which implies that in a sealed membrane system it readily catalyzes the reverse reaction, viz. pmf-driven reduction of NAD+ by QH2.30−34,60 Complex I is the largest enzyme of the respiratory chain with 13−14 core subunits that are conserved in bacteria and eukaryotes, and up to 34 additional subunits in higher organisms.61−64 Seven of the core subunits form the membrane domain of this L-shaped protein complex, and 6−7 core subunits form an extramembranous domain that contains all
2.3. Complex III (Cytochrome bc1 and b6 f)
Complex III (the cytochrome bc1 complex) of the respiratory chain has a close analogue in photosynthesis, the cytochrome b6 f complex. A unique feature of Complex III function is the bifurcation of the two-electron oxidation of ubiquinol such that one of the two electrons is carried via the Rieske Fe/S center and heme c1 to cytochrome c and cytochrome c oxidase, whereas the other is transferred to the diheme b-type cytochrome. Such a bifurcation of eT was first suggested in 197253 to explain the paradoxical phenomenon of “oxidantinduced reduction” of cytochrome b, where oxidation of cytochrome c, either by O2 via cytochrome c oxidase, or directly by an artificial oxidant, leads to reduction of the b-type hemes in the complex. This phenomenon had baffled researchers since its discovery by Britton Chance in the 1950s, and the notion prevailed that it was due to an energylinked increase in the standard redox potential of cytochrome b,54,55 or to a conformational change that altered electronic accessibility.56 However, if cytochrome c equilibrates with only 2199
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Figure 4. Q cycle. The Q cycle mechanism is described as the sum of two turnovers (A and B), each starting with oxidation of hydroquinone (QH2) near the P-side of the membrane. The low- and high-potential hemes b are called heme bL and heme bH, respectively. Q•−, semiquinone; 2Fe-2S, the Rieske nonheme iron center primarily accepting an electron from QH2 and delivering it to heme c1.
Figure 5. Complex I based on the structure from T. thermophilus,77 placed within a phospholipid membrane. Antiporter-like subunits L (yellow), M (brown), and N (green) are shown in the membrane arm. The L-subunit includes a long amphipathic α helix parallel to the membrane plus two transmembrane helices (yellow) that surround these subunits. In the middle of the membrane arm there is a remarkable row of polar residues (blue, red, and green). The polar arm shows the iron sulfur centers and the FMN where NADH is bound and oxidized. A ubiquinone molecule is modeled in a cavity (insert) where its headgroup encounters FeS center N2 via the conserved tyrosine 87, and the histidine 38−aspartate 139 pair.
the redox-active cofactors (Figure 5). Understanding the function of Complex I is not only of crucial importance for molecular bioenergetics, but is also of considerable biomedical relevance since several neurodegenerative and neuromuscular diseases are of mitochondrial origin, and the majority of these are linked to mutations in the seven genes of mitochondrial
DNA that encode the proteins of the membrane domain. Complex I is moreover believed to be the major producer of reactive oxygen species in cells, and it is therefore relevant for understanding processes such as aging and cancerogenesis.65−67 2200
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3.2. Electron Transfer
explanation is based on evolution, and points out that the hydrophilic subunits and Fe/S centers in Complex I are homologous to soluble Fe and NiFe hydrogenases from which they may have evolved.32 It may furthermore be advantageous to have the NADH-binding site at a significant distance from the unstirred layer of the membrane surface to maximize the rate of diffusion of the substrate (NADH) and product (NAD+) to and from the site.34
The eT between NADH and ubiquinone (Q) takes place in the soluble extramembrane domain of Complex I at the negatively charged side of the membrane (Figure 5). NADH is bound at a site near the flavin mononucleotide (FMN) cofactor. Electrons are transferred by quantum-mechanical tunneling from FMN onward via 7 of a total of 8−9 iron sulfur (Fe/S) centers, of which two are binuclear ([Fe2 S 2 ]Cys4 ) and 6−7 are tetranuclear ([Fe4S4]Cys4) (Figure 5).30,31,33,34,68−72 The Xray structure of the soluble domain of Complex I from Thermus thermophilus was resolved at 3.0 Å resolution in 200673 (PDB ID: 2FUG), followed by the whole structure of the complex from Yarrowia lipolytica at 6.3 Å resolution in 2010,74 and the membrane domain of Complex I from T. thermophilus and Escherichia coli at 4.5 Å75 (PDB ID: 3M9S) and 3.0 Å76 (PDB ID: 3RKO) resolutions. A recent breakthrough took place in 2013 when the complete structure of Complex I from T. thermophilus was resolved at 3.3 Å resolution77 (PDB ID: 4HEA). While one can expect some variability in, e.g., amino acid side chain positions at this resolution (as reflected by the high variability in B factors), we now have a detailed view of the architecture of the entire complex. It may be noted that the preparations of Complex I from T. thermophilus used for crystallization did not contain ubiquinone, but the crystals were subsequently soaked with Q and Q-site inhibitors,77 and structures of the bound states determined, although the released structure does not contain the bound ubiquinone. By contrast, preparations of Complex I from bovine heart78 and from E. coli34 contain about 1 Q/FMN. The Fe/S centers of Complex I have been studied by electron paramagnetic resonance (EPR) spectroscopy since the 1960s.79,80 Only six of the clusters are EPR-visible,80 probably due to electrostatic interactions between neighboring clusters.81,82 Ultrafast freeze−quench EPR experiments70 showed that the N2 cluster at the end of the chain (Figure 5) is reduced by NADH in ∼90 μs, which is very close to the rate expected from electron transfer theory.69−72 Computational studies using semiempirical, quantum chemical, and continuum electrostatics calculations of the eT wire in Complex I have been consistent with the experimentally obtained redox potentials and the tunneling mechanism72,83,84 despite some criticism of the freeze−quench results.31,82 One of the Fe/S clusters (N7) is located far away (∼21 Å) from the main eT chain (Figure 5). It does not seem to participate in the eT process, but is essential for stabilizing the structure.85 Center N1a is also on a “side path” with regard to electron transfer, and its functional role is unclear. A pure electron tunneling process, as appears to be the case in Complex I, would neither involve protonation/deprotonation, nor conformational changes,70,71 which implies that the conservation of free energy would have to take place beyond the transfer of the electrons to center N2 at the end of the Fe/S chain. The very fast eT down the Fe/S chain, relative to the overall turnover rate of Complex I, also implies that no free energy is lost by reducing the relatively high-potential center N2 (Em ∼ −200 mV or higher) by NADH, as sometimes suggested, because the fast eT relative to the much slower electron throughput assures near-equilibrium between the NAD+/NADH pair and center N2. Instead, the high Em of center N2 ensures that it has maximal electron occupancy in its further reaction with bound Q (see below). One may therefore ask what the purpose may be of such a long chain of Fe/S centers if it only functions as a passive “electrical wire”. One
3.3. Binding and Dynamics of Ubiquinone
The position of the ubiquinone binding site77 observed using crystals soaked with the cofactor (Figure 5 inset) is consistent with previous mutagenesis data,86 labeling experiments,87,88 and the low-resolution structure from the Yarrowia lipolytica complex.74 The Q headgroup and part of the hydrophobic tail is embedded between the Nqo4 and Nqo6 subunits (49 kDa and PSST subunits in bovine Complex I; NuoCD and NuoB in E. coli), with the headgroup ∼14 Å from the Fe/S cluster N277 (PDB ID: 4HEA), very close to the distance determined on the basis of the magnetic interaction between N2 and bound ubisemiquinone.86 The Q headgroup is bridged between a tyrosine and a histidine/aspartate motif, as had been suggested by site-directed mutagenesis studies,87 and lies at the bottom of a narrow ∼35 Å tunnel that opens on the opposite side toward the phospholipid membrane between transmembrane helices of subunits NuoH and NuoA (ND1 and ND3 in bovine; Figure 5; see below). The narrow tunnel is suggested to be sealed from water,77 and accommodates nearly all 10 isoprene units of ubiquinone-10. On one side it has a dipolar array of charged residues, and on the other nonpolar residues,77 which may provide the “hydrophobic ramp” suggested earlier88,89 for Q to slide across. In 2003 Zickermann et al.88 had already made the remarkable discovery, using labeling by monoclonal antibodies and EM single particle imaging, that the Fe/S center N2, known already then to be close to the binding site of the headgroup of ubiquinone,80 lies at a considerable distance f rom the membrane, later confirmed to be ∼35 Å in Complex I from the yeast Yarrowia lipolytica.74 In good agreement with this, we find this distance to be ∼30 Å in our membrane model of the Complex I structure from T. thermophilus (4HEA, ref 77), and the shortest mean distance from the headgroup of bound Q, perpendicular to the membrane, to the projection of the membrane surface to be ∼20 Å. The Q-binding tunnel is lined by several charged residues of the membrane-embedded NuoH subunit (Figure 5). Mutations of several residues in the NuoH subunit cause severe inhibition of activity (summarized in ref 77), and many such mutations have been implicated in mitochondrial diseases (reviewed in refs 77, 90), suggesting that this subunit serves an important function in the junction between the hydrophobic and hydrophilic arms of Complex I. The bound ubiquinone lies well within eT distance from the Fe/S center N2, but too far from an acceptor ubiquinone in the phospholipid membrane to give rise to eT from one to the other at a rate sufficient for catalysis.69,71 Two major alternative mechanisms of ubiquinone dynamics have been considered in the literature. Although the mechanisms proposed by Brandt91 and Efremov and Sazanov92 are otherwise quite different (see below), both favor a single Q-binding site, viz., the one observed crystallographically, and that the Q molecule exits from the tunnel into the membrane after reduction, and that an oxidized Q from the membrane replaces it by entering the tunnel. A second possibility bears similarity with the QA/QB 2201
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suggested to form a “fourth” proton-pumping channel,77 although this was originally placed at the NuoN/K interface.76 Part of this proposed fourth channel (“E-channel”) is formed by a remarkable quartet of three conserved glutamate and one aspartate residues (E130, E163, E213 in NuoH and D72 in NuoA) placed centrally in the membrane domain (Figure 5). As discussed below, they form a link between a central chain of polar residues in the membrane arm and the Q-binding tunnel. The point in the Q-tunnel closest to this carboxylate-quartet lies >20 Å from the region that binds the headgroup of Q, and is at about halfway along the tunnel length. There is no direct experimental evidence to support the proposal that this structure functions as a fourth proton-pumping channel, although this is a possibility. Alternatively, the carboxylate quartet may be part of the energy-transmitting machinery that couples redox-dependent changes in the electrical charge of ubiquinone to the proton-translocating structures in the membrane arm (see below). Similar to carrier-type transporters, where separate conformational states make connections of the transported entity to each side of the membrane (see, e.g., refs 111, 112), the architecture of the TM helices suggest that Complex I could employ conformational state switching to catalyze proton pumping. The TM helices 4−6 and 9−11 of the L/M/N subunits seem to be in a similar conformation, whereas structural alignment suggests that TM helices 7−8 and 12−13 occupy different relative conformations. A possibility for a conformationally driven coupling mechanism would be to interchange the symmetry of the helices leading to opening and closing of proton uptake/release channels. NuoH/N and NuoL are structurally connected via a unique amphipathic HL-helix, which is orientated parallel to the membrane plane (Figure 5, refs 74, 76, 77). This helix was suggested to function as a piston, a crucial coupling-element, which mechanically transmits long-range energy propagation in Complex I. However, site-directed mutagenesis experiments show that the enzyme can function with unchanged activity despite insertion of relatively long sequences (6−9 residues) in the helix.113 This implies that mechanical stiffness is not a crucial property of the HL-helix, or for the proton translocation function, which might have been expected if the helix would serve a piston function. However, it was also observed that longer HL-insertion sequences leads to problems in the assembly of the enzyme, which is why Belevich et al.113 suggested that the helix might be of structural importance clamping the subunits together. Hence, the HL-helix may secure strong electrostatic interactions between their titratable residues (see below). The β-sheet structures of the antiporterlike domains,77 located at the negatively charged side of the membrane, have also been suggested to mediate energypropagation in Complex I, but so far there are no experimental studies supporting this view. Another remarkable feature of the membrane domain, already mentioned, is a chain of highly conserved and quite densely distributed charged and polar protonatable residues that extends in the center of the membrane arm all the way from the Q-binding tunnel to the distal subunit NuoL (refs 34, 77, 109, Figure 5). A chain of carboxylic acid residues in the membrane core of subunits NuoA, NuoH, and NuoK link this chain to the ubiquinone tunnel at a point about halfway along its length. Most of the residues forming this chain are essential for function, and their mutations lead to blockade of both Q reductase activity and proton translocation.77 Such an unusual
structure in bacterial photosynthetic reaction centers and in Photosystem II.52 Here, the reduced “crystallographic” ubiquinone donates electrons to another more loosely bound ubiquinone molecule closer to the membrane, which in turn exchanges with Q in the membrane.34,93,94 Although there is some experimental support for such a second Q-binding site in Complex I,95−99 Baradaran et al.77 excluded this alternative for the latest crystal structure of Complex I from T. thermophilus due to the apparent lack of space for a second ubiquinone molecule bound at a distance from the first Q short enough for effective eT between them (
