Electric fields control water-gated proton transfer in cytochrome c oxidase.

Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome c oxidase (CcO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O2 reduction into proton pumping. Here we show that CcO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways. By combining large-scale quantum chemical density functional theory (DFT) calculations with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations and atomistic molecular dynamics (MD) explorations, we find that reduction of the electron donor, heme a, leads to dissociation of an arginine (Arg438)-heme a3 D-propionate ion-pair. This ion-pair dissociation creates a strong electric field of up to 1 V Å-1 along a water-mediated proton array leading to a transient proton loading site (PLS) near the active site. Protonation of the PLS triggers the reduction of the active site, which in turn aligns the electric field vectors along a second, "chemical," proton pathway. We find a linear energy relationship of the proton transfer barrier with the electric field strength that explains the effectivity of the gating process. Our mechanism shows distinct similarities to principles also found in other energy-converting enzymes, suggesting that orientated electric fields generally control enzyme catalysis.

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing.

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.

Redox-coupled quinone dynamics in the respiratory complex I.

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

Oxygen Activation and Energy Conservation by Cytochrome c Oxidase.

This review focuses on the type A cytochrome c oxidases (C cO), which are found in all mitochondria and also in several aerobic bacteria. C cO catalyzes the respiratory reduction of dioxygen (O2) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound C cO couples the O2 reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of C cO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase.

Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and uses the released free energy to pump protons against the transmembrane proton gradient. To better understand the proton-pumping mechanism of the wild-type (WT) CcO, much attention has been given to the mutation of amino acid residues along the proton translocating D-channel that impair, and sometimes decouple, proton pumping from the chemical catalysis. Although their influence has been clearly demonstrated experimentally, the underlying molecular mechanisms of these mutants remain unknown. In this work, we report multiscale reactive molecular dynamics simulations that characterize the free-energy profiles of explicit proton transport through several important D-channel mutants. Our results elucidate the mechanisms by which proton pumping is impaired, thus revealing key kinetic gating features in CcO. In the N139T and N139C mutants, proton back leakage through the D-channel is kinetically favored over proton pumping due to the loss of a kinetic gate in the N139 region. In the N139L mutant, the bulky L139 side chain inhibits timely reprotonation of E286 through the D-channel, which impairs both proton pumping and the chemical reaction. In the S200V/S201V double mutant, the proton affinity of E286 is increased, which slows down both proton pumping and the chemical catalysis. This work thus not only provides insight into the decoupling mechanisms of CcO mutants, but also explains how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO.

Multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free-energy profiles and calculated rates) the internal proton transport events that enable proton pumping during first steps of oxidation of the fully reduced enzyme. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored whereas the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates agree with experimental measurements. The backflow of the pumped proton from the PLS to E286 and from E286 to the inside of the membrane is prevented by large free-energy barriers for the backflow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways and, importantly, vice versa.

Proton pumping by cytochrome c oxidase - A 40 year anniversary.

Cytochrome c oxidase is a remarkable energy transducer that seems to work almost purely by Coulombic principles without the need for significant protein conformational changes. In recent years it has become possible to follow key partial reactions of the catalytic cycle in real time, both with respect to electron and proton movements. These experiments have largely set the stage for the proton pump mechanism. The structures of the catalytic binuclear heme­copper site that is common to the huge family of heme­copper oxidases, are today well understood throughout the catalytic cycle of oxygen reduction to water based on both spectroscopic studies and quantum chemical calculations. Here, we briefly review this progress, and add some recent details into how the proton pump mechanism is protected from failure by leakage.

Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase.

Molecular oxygen acts as the terminal electron sink in the respiratory chains of aerobic organisms. Cytochrome c oxidase in the inner membrane of mitochondria and the plasma membrane of bacteria catalyzes the reduction of oxygen to water, and couples the free energy of the reaction to proton pumping across the membrane. The proton-pumping activity contributes to the proton electrochemical gradient, which drives the synthesis of ATP. Based on kinetic experiments on the O-O bond splitting transition of the catalytic cycle (A → P(R)), it has been proposed that the electron transfer to the binuclear iron-copper center of O2 reduction initiates the proton pump mechanism. This key electron transfer event is coupled to an internal proton transfer from a conserved glutamic acid to the proton-loading site of the pump. However, the proton may instead be transferred to the binuclear center to complete the oxygen reduction chemistry, which would constitute a short-circuit. Based on atomistic molecular dynamics simulations of cytochrome c oxidase in an explicit membrane-solvent environment, complemented by related free-energy calculations, we propose that this short-circuit is effectively prevented by a redox-state-dependent organization of water molecules within the protein structure that gates the proton transfer pathway.

Redox-induced activation of the proton pump in the respiratory complex I.

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

Time-resolved generation of membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH states.

Two electrogenic phases with characteristic times of ~14µs and ~290µs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba3 oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between CuA and heme b. The slow phase includes electron redistribution from both CuA and heme b to heme a3, and electrogenic proton transfer coupled to reduction of heme a3. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a3 is reduced, but there is no proton pumping and no reduction of CuB. Single-electron reduction of the oxidized "unrelaxed" state (OH→EH transition) is accompanied by electrogenic reduction of the heme b/heme a3 pair by CuA in a "fast" phase (~22µs) and transfer of protons in "middle" and "slow" electrogenic phases (~0.185ms and ~0.78ms) coupled to electron redistribution from the heme b/heme a3 pair to the CuB site. The "middle" and "slow" electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach CuB the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~0.1 H+/e-, probably due to the formed membrane potential in the experiment.

The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase.

The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active-site tyrosine (Tyr244) and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Molecular simulation and modeling of complex I.

Molecular modeling and molecular dynamics simulations play an important role in the functional characterization of complex I. With its large size and complicated function, linking quinone reduction to proton pumping across a membrane, complex I poses unique modeling challenges. Nonetheless, simulations have already helped in the identification of possible proton transfer pathways. Simulations have also shed light on the coupling between electron and proton transfer, thus pointing the way in the search for the mechanistic principles underlying the proton pump. In addition to reviewing what has already been achieved in complex I modeling, we aim here to identify pressing issues and to provide guidance for future research to harness the power of modeling in the functional characterization of complex I. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I.

Complex I serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. It catalyzes the reduction of quinones by electron transfer from NADH, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. The membrane domain of the enzyme extends ∼180 Šfrom the site of quinone reduction to the most distant proton pathway. To elucidate possible mechanisms of the long-range proton-coupled electron transfer process, we perform large-scale atomistic molecular dynamics simulations of the membrane domain of complex I from Escherichia coli. We observe spontaneous hydration of a putative proton entry channel at the NuoN/K interface, which is sensitive to the protonation state of buried glutamic acid residues. In hybrid quantum mechanics/classical mechanics simulations, we find that the observed water wires support rapid proton transfer from the protein surface to the center of the membrane domain. To explore the functional relevance of the pseudosymmetric inverted-repeat structures of the antiporter-like subunits NuoL/M/N, we constructed a symmetry-related structure of a possible alternate-access state. In molecular dynamics simulations, we find the resulting structural changes to be metastable and reversible at the protein backbone level. However, the increased hydration induced by the conformational change persists, with water molecules establishing enhanced lateral connectivity and pathways for proton transfer between conserved ionizable residues along the center of the membrane domain. Overall, the observed water-gated transitions establish conduits for the unidirectional proton translocation processes, and provide a possible coupling mechanism for the energy transduction in complex I.

Role of subunit III and its lipids in the molecular mechanism of cytochrome c oxidase.

The terminal respiratory enzyme cytochrome c oxidase (CcO) reduces molecular oxygen to water, and pumps protons across the inner mitochondrial membrane, or the plasma membrane of bacteria. A two-subunit CcO harbors all the elements necessary for oxygen reduction and proton pumping. However, it rapidly undergoes turnover-induced irreversible damage, which is effectively prevented by the presence of subunit III and its tightly bound lipids. We have performed classical atomistic molecular dynamics (MD) simulations on a three-subunit CcO, which show the formation of water wires between the polar head groups of lipid molecules bound to subunit III and the proton uptake site Asp91 (Bos taurus enzyme numbering). Continuum electrostatic calculations suggest that these lipids directly influence the proton affinity of Asp91 by 1-2pK units. We surmise that lipids bound to subunit III influence the rate of proton uptake through the D-pathway, and therefore play a key role in preventing turnover-induced inactivation. Atomistic MD simulations show that subunit III is rapidly hydrated in the absence of internally bound lipids, which is likely to affect the rate of O2 diffusion into the active-site. The role of subunit III with its indigenous lipids in the molecular mechanism of CcO is discussed.

Computational study of the activated O(H) state in the catalytic mechanism of cytochrome c oxidase.

Complex IV in the respiratory chain of mitochondria and bacteria catalyzes reduction of molecular oxygen to water, and conserves much of the liberated free energy as an electrochemical proton gradient, which is used for the synthesis of ATP. Photochemical electron injection experiments have shown that reduction of the ferric/cupric state of the enzyme's binuclear heme a3/CuB center is coupled to proton pumping across the membrane, but only if oxidation of the reduced enzyme by O2 immediately precedes electron injection. In contrast, reduction of the binuclear center in the "as-isolated" ferric/cupric enzyme is sluggish and without linkage to proton translocation. During turnover, the binuclear center apparently shuttles via a metastable but activated ferric/cupric state (O(H)), which may decay into a more stable catalytically incompetent form (O) in the absence of electron donors. The structural basis for the difference between these two states has remained elusive, and is addressed here using computational methodology. The results support the notion that CuB[II] is either three-coordinated in the O(H) state or shares an OH(-) ligand with heme a3 in a strained µ-hydroxo structure. Relaxation to state O is initiated by hydration of the binuclear site. The redox potential of CuB is expected, and found by density functional theory calculations, to be substantially higher in the O(H) state than in state O. Our calculations also suggest that the neutral radical form of the cross-linked tyrosine in the binuclear site may be more significant in the catalytic cycle than suspected so far.

The causes of reduced proton-pumping efficiency in type B and C respiratory heme-copper oxidases, and in some mutated variants of type A.

The heme-copper oxidases may be divided into three categories, A, B, and C, which include cytochrome c and quinol-oxidising enzymes. All three types are known to be proton pumps and are found in prokaryotes, whereas eukaryotes only contain A-type cytochrome c oxidase in their inner mitochondrial membrane. However, the bacterial B- and C-type enzymes have often been reported to pump protons with an H(+)/e(-) ratio of only one half of the unit stoichiometry in the A-type enzyme. We will show here that these observations are likely to be the result of difficulties with the measuring technique together with a higher sensitivity of the B- and C-type enzymes to the protonmotive force that opposes pumping. We find that under optimal conditions the H(+)/e(-) ratio is close to unity in all the three heme-copper oxidase subfamilies. A higher tendency for proton leak in the B- and C-type enzymes may result from less efficient gating of a proton pump mechanism that we suggest evolved before the so-called D-channel of proton transfer. There is also a discrepancy between results using whole bacterial cells vs. phospholipid vesicles inlaid with oxidase with respect to the observed proton pumping after modification of the D-channel residue asparagine-139 (Rhodobacter sphaeroides numbering) to aspartate in A-type cytochrome c oxidase. This discrepancy might also be explained by a higher sensitivity of proton pumping to protonmotive force in the mutated variant. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Oxidoreduction properties of bound ubiquinone in Complex I from Escherichia coli.

The exploration of the redox chemistry of bound ubiquinone during catalysis is a prerequisite for the understanding of the mechanism by which Complex I (nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase) transduces redox energy into an electrochemical proton gradient. Studies of redox dependent changes in the spectrum of Complex I from Escherichia coli in the mid- and near-ultraviolet (UV) and visible areas were performed to identify the spectral contribution, and to determine the redox properties, of the tightly bound ubiquinone. A very low midpoint redox potential (<-300mV) was found for the bound ubiquinone, more than 400mV lower than when dissolved in a phospholipid membrane. This thermodynamic property of bound ubiquinone has important implications for the mechanism by which Complex I catalyzes proton translocation.

Stoichiometry of proton translocation by respiratory complex I and its mechanistic implications.

Complex I (NADH-ubiquinone oxidoreductase) in the respiratory chain of mitochondria and several bacteria functions as a redox-driven proton pump that contributes to the generation of the protonmotive force across the inner mitochondrial or bacterial membrane and thus to the aerobic synthesis of ATP. The stoichiometry of proton translocation is thought to be 4 H(+) per NADH oxidized (2 e(-)). Here we show that a H(+)/2 e(-) ratio of 3 appears more likely on the basis of the recently determined H(+)/ATP ratio of the mitochondrial F(1)F(o)-ATP synthase of animal mitochondria and of a set of carefully determined ATP/2 e(-) ratios for different segments of the mitochondrial respiratory chain. This lower H(+)/2 e(-) ratio of 3 is independently supported by thermodynamic analyses of experiments with both mitochondria and submitochondrial particles. A reduced H(+)/2 e(-) stoichiometry of 3 has important mechanistic implications for this proton pump. In a rough mechanistic model, we suggest a concerted proton translocation mechanism in the three homologous and tightly packed antiporter-like subunits L, M, and N of the proton-translocating membrane domain of complex I.

Mechanistic stoichiometry of proton translocation by cytochrome cbb3.

Cytochrome cbb(3) belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. The cbb(3)-type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. The mechanistic H(+)/e(-) stoichiometry of proton translocation in these cbb(3)-type cytochrome c oxidases has remained controversial. A stoichiometric efficiency of only one-half that of the mitochondrial aa(3)-type enzyme was recently proposed to be related to adaptation of the organism to microaerobic environments. Here, proton translocation by the Rhodobacter sphaeroides enzyme was studied using purified cytochrome cbb(3) reconstituted into liposomes. An H(+)/e(-) stoichiometry of proton translocation close to unity was observed using the oxygen pulse method, but solely in conditions in which the vast majority of the enzyme was fully reduced in the anaerobic state before the O(2) pulse. These data were compared with results using whole cells or spheroplasts, and the discrepancies in the literature data were discussed. Our results suggest that a proton-pumping efficiency of 1 H(+)/e(-) may be achieved using the single-proton uptake pathway identified in the structure of cytochrome cbb(3). The mechanism of proton pumping thus differs from that of the aa(3)-type oxidases of mitochondria and bacteria.

The fifth electron in the fully reduced caa(3) from Thermus thermophilus is competent in proton pumping.

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa(3) cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) pair is resolved as a single transition with τ~40 µs. In the reaction of the fully reduced cytochrome caa(3) with oxygen, the first electrogenic phase (τ~30 µs) is linked to OO bond cleavage and generation of the P(R) state. The next electrogenic component (τ~50 µs) is associated with the P(R)→F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ~0.25 ms, ~1.4 ms and ~4 ms, are linked to the conversion of the binuclear center through the F→O(H)→E(H) transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.