The mitochondrial respiratory chain.

We present a brief review of the mitochondrial respiratory chain with emphasis on complexes I, III and IV, which contribute to the generation of protonmotive force across the inner mitochondrial membrane, and drive the synthesis of ATP by the process called oxidative phosphorylation. The basic structural and functional details of these complexes are discussed. In addition, we briefly review the information on the so-called supercomplexes, aggregates of complexes I-IV, and summarize basic physiological aspects of cell respiration.

On the role of ubiquinone in the proton translocation mechanism of respiratory complex I.

Complex I converts oxidoreduction energy into a proton electrochemical gradient across the inner mitochondrial or bacterial cell membrane. This gradient is the primary source of energy for aerobic synthesis of ATP. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) by ubiquinone (Q) yields NAD+ and ubiquinol (QH2 ), which is tightly coupled to translocation of four protons from the negatively to the positively charged side of the membrane. Electrons from NADH oxidation reach the iron-sulfur centre N2 positioned near the bottom of a tunnel that extends circa 30 Å from the membrane domain into the hydrophilic domain of the complex. The tunnel is occupied by ubiquinone, which can take a distal position near the N2 centre or proximal positions closer to the membrane. Here, we review important structural, kinetic and thermodynamic properties of ubiquinone that define its role in complex I function. We suggest that this function exceeds that of a mere substrate or electron acceptor and propose that ubiquinone may be the redox element of complex I coupling electron transfer to proton translocation.

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.

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba3 from Thermus thermophilus.

Cytochrome ba3 from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba3. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 µs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.

Architecture of bacterial respiratory chains.

Bacteria power their energy metabolism using membrane-bound respiratory enzymes that capture chemical energy and transduce it by pumping protons or Na+ ions across their cell membranes. Recent breakthroughs in molecular bioenergetics have elucidated the architecture and function of many bacterial respiratory enzymes, although key mechanistic principles remain debated. In this Review, we present an overview of the structure, function and bioenergetic principles of modular bacterial respiratory chains and discuss their differences from the eukaryotic counterparts. We also discuss bacterial supercomplexes, which provide central energy transduction systems in several bacteria, including important pathogens, and which could open up possible avenues for treatment of disease.

Thermodynamic efficiency, reversibility, and degree of coupling in energy conservation by the mitochondrial respiratory chain.

The protonmotive mitochondrial respiratory chain, comprising complexes I, III and IV, transduces free energy of the electron transfer reactions to an electrochemical proton gradient across the inner mitochondrial membrane. This gradient is used to drive synthesis of ATP and ion and metabolite transport. The efficiency of energy conversion is of interest from a physiological point of view, since the energy transduction mechanisms differ fundamentally between the three complexes. Here, we have chosen actively phosphorylating mitochondria as the focus of analysis. For all three complexes we find that the thermodynamic efficiency is about 80-90% and that the degree of coupling between the redox and proton translocation reactions is very high during active ATP synthesis. However, when net ATP synthesis stops at a high ATP/ADP.Pi ratio, and mitochondria reach "State 4" with an elevated proton gradient, the degree of coupling drops substantially. The mechanistic cause and the physiological implications of this effect are discussed.

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice.

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1lp.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cybp.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1lp.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc1 complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.

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.

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.

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.

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.

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.

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.

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.

Nitric oxide is a potent inhibitor of the cbb(3)-type heme-copper oxidases.

C-type heme-copper oxidases terminate the respiratory chain in many pathogenic bacteria, and will encounter elevated concentrations of NO produced by the immune defense of the host. Thus, a decreased sensitivity to NO in C-type oxidases would increase the survival of these pathogens. Here we have compared the inhibitory effect of NO in C-type oxidases to that in the mitochondrial A-type. We show that O2-reduction in both the Rhodobacter sphaeroides and Vibrio cholerae C-type oxidases is strongly and reversibly inhibited by submicromolar NO, with an inhibition pattern similar to the A-type. Thus, NO tolerance in pathogens with a C-type terminal oxidase has to rely mainly on other mechanisms.

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.

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.