Proton Translocation Research Articles

Deactivation of mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I): Extrinsically affecting factors

Mitochondrial NADH:ubiquinone oxidoreductase (proton translocating respiratory complex I) serves several essential functions in cell metabolism: it maintains the intramitochondrial NADH/NAD+ ratio, contributes to generation of the proton-motive force, and participates in physiological and/or pathophysiological production of so-called reactive oxygen species. A characteristic feature of complex I is a slow, compared with its catalytic turnover, transformation to its inactive (deactivated) state, a phenomenon operationally called A/D transition. Here we report data on several extrinsic factors affecting deactivation as observed in coupled or uncoupled bovine heart submitochondrial particles. The time course of the strongly temperature-dependent deactivation deviates from first-order kinetics, and this deviation is abolished in the presence of an SH-group-specific reagent. The residual fraction of activity attained upon extensive deactivation shows the same kinetics of NADH oxidation as the fully active enzyme does. The rate of complex I deactivation is only slightly pH dependent within the range of 7.0–8.5 and significantly increases at higher pH. ATP∙(Mg) decreases the rate of complex I deactivation in coupled SMP, and this effect is abolished if the proton-motive force generating ATPase activity of Fo∙F1 is precluded. Taken together, the data show that an equilibrium between the A and D forms of complex I exists. Possible mechanistic aspects of the deactivation process are discussed.

Proton motive function of the terminal antiporter-like subunit in respiratory complex I

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.

Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I

NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky "block" that is ∼13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.

Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.

Introduction of protonic potential of Brønsted−Lowry acids and bases to the quantification of the energy of proton translocation and elucidation of oxidative phosphorylation

ATP synthesis in mitochondria is driven by the pH gradient between lipid membranes, and a proton-motive force (PMF) forms along the respiratory electron-transport chain. The reasons for these phenomena remain unclear. In an unbuffered quinone solution, acids induce the formation of new cathodic peaks, whose peak potential is dependent on their pKa. Thus, the protons of acids have a potential defined as “protonic potential.” Herein, the energy of proton translocation was easily quantified by protonic potential. We observed that the redox potential of chemical reactions can be transformed into the protonic potential of Brønsted−Lowry acids and bases in an unbuffered solution. We postulated that in the mitochondrial membrane, the redox potential of the respiratory chain can be transformed to protonic potential and that the PMF originates from the protonic potential difference between compounds. Ultimately, the protonic potential energy derived from redox reactions was stored in the ATP.

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.

Molecular Dynamics Simulation of T10609C and C10676G Mutations of Mitochondrial ND4L Gene Associated With Proton Translocation in Type 2 Diabetes Mellitus and Cataract Patients.

The mutation rate of mitochondrial DNA (mtDNA) is 17 times higher than nuclear DNA, and these mutations can cause mitochondrial disease in 1 of 10.000 people. The T10609C mutation was identified in type 2 diabetes mellitus (T2DM) patients and the C10676G mutation in cataract patients, with both mutations occurring in the ND4L gene of mtDNA that encodes ND4L protein. ND4L protein, a subunit of complex I in the respiratory complex, has been shown to play a role in the proton translocation process. The purpose of this study was to investigate the effect of both mutations on the proton translocation mechanism. Mutation mapping showed changes in amino acids M47T (T10609C) and C69W (C10676G). The 100 ns molecular dynamics (MD) simulations performed on native and mutants of ND4L-ND6 subunits. It is revealed that the native model had a similar proton translocation pathway to that of complex I from other organisms. Interestingly, the mutant M47T and C69W showed the interruption of the translocation pathway by a hydrogen bond formation between Glu34 and Tyr157. It is observed that the mutations were restricting the passage of water molecules through the transmembrane region. These results could help to develop the computational assay for the validation of a specific genetic biomarker for T2DM and cataracts.

Proton-coupled electron transfer across benzimidazole bridges in bioinspired proton wires.

Designing molecular platforms for controlling proton and electron movement in artificial photosynthetic systems is crucial to efficient catalysis and solar energy conversion. The transfer of both protons and electrons during a reaction is known as proton-coupled electron transfer (PCET) and is used by nature in myriad ways to provide low overpotential pathways for redox reactions and redox leveling, as well as to generate bioenergetic proton currents. Herein, we describe theoretical and electrochemical studies of a series of bioinspired benzimidazole-phenol (BIP) derivatives and a series of dibenzimidazole-phenol (BI2P) analogs with each series bearing the same set of terminal proton-accepting (TPA) groups. The set of TPAs spans more than 6 pKa units. These compounds have been designed to explore the role of the bridging benzimidazole(s) in a one-electron oxidation process coupled to intramolecular proton translocation across either two (the BIP series) or three (the BI2P series) acid/base sites. These molecular constructs feature an electrochemically active phenol connected to the TPA group through a benzimidazole-based bridge, which together with the phenol and TPA group form a covalent framework supporting a Grotthuss-type hydrogen-bonded network. Infrared spectroelectrochemistry demonstrates that upon oxidation of the phenol, protons translocate across this well-defined hydrogen-bonded network to a TPA group. The experimental data show the benzimidazole bridges are non-innocent participants in the PCET process in that the addition of each benzimidazole unit lowers the redox potential of the phenoxyl radical/phenol couple by 60 mV, regardless of the nature of the TPA group. Using a series of hypothetical thermodynamic steps, density functional theory calculations correctly predicted the dependence of the redox potential of the phenoxyl radical/phenol couple on the nature of the final protonated species and provided insight into the thermodynamic role of dibenzimidazole units in the PCET process. This information is crucial for developing molecular “dry proton wires” with these moieties, which can transfer protons via a Grotthuss-type mechanism over long distances without the intervention of water molecules.

Site-Directed Mutagenesis Reveals Crucial Residues in Escherichia coli Resistance-Nodulation-Division Efflux Pump OqxB.

Resistance-nodulation-division (RND) efflux pumps are important determinants of multidrug resistance in Gram-negative bacteria. As one of the typical members of the RND superfamily, Escherichia coli OqxAB multidrug efflux pump confers resistance to antimicrobial agents, such as olaquindox and fluoroquinolone. In the present study, site-directed mutagenesis and antimicrobial susceptibility measurement assay were applied to identify the crucial residues within OqxB, the transporter component of the OqxAB efflux pump system. It was found that alanine substitution of proton translocation pathway residues D410, D411, and R976 resulted in a complete loss of the transport function. Further studies revealed that the charge property of these residues is important for proper function of OqxB. Alanine replacement of residues involved in substrate-binding domains, including V141, F180, Y330, and F626, exhibited different responses toward different antimicrobial agents. Conservative replacement of Y330, F626, and F180 with amino acids having similar aromatic ring structure resulted in full or partial recovery of the efflux function. Molecular docking analysis demonstrated that olaquindox may form hydrogen bonds with F626, Y330, and V141, whereas only Y330 and F180 may interact with ciprofloxacin, implicating the different roles played by these residues when transporting different kinds of substrates. Graphical Abstract [Figure: see text].

Convergent patterns of evolution of mitochondrial oxidative phosphorylation (OXPHOS) genes in electric fishes.

The ability to generate and detect electric fields has evolved in several groups of fishes as a means of communication, navigation and, occasionally, predation. The energetic burden required can account for up to 20% of electric fishes' daily energy expenditure. Despite this, molecular adaptations that enable electric fishes to meet the metabolic demands of bioelectrogenesis remain unknown. Here, we investigate the molecular evolution of the mitochondrial oxidative phosphorylation (OXPHOS) complexes in the two most diverse clades of weakly electric fishes-South American Gymnotiformes and African Mormyroidea, using codon-based likelihood approaches. Our analyses reveal that although mitochondrial OXPHOS genes are generally subject to strong purifying selection, this constraint is significantly reduced in electric compared to non-electric fishes, particularly for complexes IV and V. Moreover, analyses of concatenated mitochondrial genes show strong evidence for positive selection in complex I genes on the two branches associated with the independent evolutionary origins of electrogenesis. These results suggest that adaptive evolution of proton translocation in the OXPHOS cellular machinery may be associated with the evolution of bioelectrogenesis. Overall, we find striking evidence for remarkably similar effects of electrogenesis on the molecular evolution of mitochondrial OXPHOS genes in two independently derived clades of electrogenic fishes. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

AbstractRespiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

A 3.3 Å-Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability.

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc 1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c 1 was found to greatly enhance the interaction between cyt. b and cyt. c 1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

New Structures Reveal Interaction Dynamics in Respiratory Supercomplexes

Mitochondrial energy conversion involves a chain of membrane-bound proteins that are wired to conduct an electron current, which drives transmembrane proton translocation. These enzymes associate to form supercomplexes, but the functional relevance of the higher-order structures is unknown. A recent study by Letts etal. presents structures of a supercomplex, which suggest how the interaction choreography may control overall functionality.

Explaining leak states in the proton pump of heme-copper oxidases observed in single-molecule experiments

Heme-copper oxidases couple the exergonic oxygen reduction with the endergonic proton translocation. Redox-linked structural changes have been localized in deeply buried regions of the protein, near the low-potential heme. How these movements can modulate distant gating events along the intramolecular proton path, where the entry (exit) of pumped proton occurs, is a major concern for the proton pump models. Generally, these models associate, more or less directly, all translocation events with redox transitions. Although they can account for many phenomenological aspects of the pump, evidences from single-molecules experiments about leak states of the pump represent a formidable challenge. Disconnecting the redox-linked pKa shifts of the proton loading site from the external barriers, we obtain a simple stochastic mechanism which behaves similarly to the real enzyme, able to reverse the flow of the proton transfer.

Deiodination in the presence of Dehalococcoides mccartyi strain CBDB1: comparison of the native enzyme and co-factor vitamin B12.

Triiodinated benzoic acid derivatives are widely used as contrast media for medical examinations and are found at high concentrations in urban aquatic environments. During bank filtration, deiodination of iodinated contrast media has been observed under anoxic/anaerobic conditions. While several bacterial strains capable of dechlorination and debromination have been isolated and characterized, deiodination has not yet been shown for an isolated strain. Here, we investigate dehalogenation of iodinated contrast media (ICM), triiodobenzoic acids (TIBA), and analogous chlorinated compounds by Dehalococcoides mccartyi strain CBDB1 and its corrinoid co-factor vitamin B12. No cell growth of CBDB1 was observed using iodinated compounds as electron acceptor. Only negligible deiodination occurred for ICM, whereas 2,3,5-TIBA was nearly completely deiodinated by CBDB1 without showing cell growth. Furthermore, TIBA inhibited growth with hexachlorobenzene which is usually a well-suited electron acceptor for strain CBDB1, indicating that TIBA is toxic for CBDB1. The involvement of CBDB1 enzymes in the deiodination of TIBA was verified by the absence of deiodination activity after heat inactivation. Adding iodopropane also inhibited the deiodination of TIBA by CBDB1 cells, indicating the involvement of a corrinoid-enzyme in the reductive TIBA deiodination. The results further suggest that the involved electron transport is decoupled from proton translocation and therefore growth. Graphical abstract.

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS

Conformational movements play an important role in enzyme catalysis. Respiratory complex I, an L-shaped enzyme, connects electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation through its membrane arm by a coupling mechanism still under debate. The amphipathic helix across the membrane arm represents a unique structural feature. Here, we demonstrate a new way to study conformational changes by introducing a small and highly flexible nitrile infrared (IR) label to this helix to visualize movement with surface-enhanced IR absorption spectroscopy. We find that labeled residues K551CL and Y590CL move to a more hydrophobic environment upon NADH reduction of the enzyme, likely as a response to the reorganization of the antiporter-like subunits in the membrane arm.

Structural and functional characterization of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica.

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.

Structure and conformational plasticity of the intact Thermus thermophilus V/A-type ATPase.

V (vacuolar)/A (archaeal)-type adenosine triphosphatases (ATPases), found in archaea and eubacteria, couple ATP hydrolysis or synthesis to proton translocation across the plasma membrane using the rotary-catalysis mechanism. They belong to the V-type ATPase family, which differs from the mitochondrial/chloroplast F-type ATP synthases in overall architecture. We solved cryo-electron microscopy structures of the intact Thermus thermophilus V/A-ATPase, reconstituted into lipid nanodiscs, in three rotational states and two substates. These structures indicate substantial flexibility between V1 and Vo in a working enzyme, which results from mechanical competition between central shaft rotation and resistance from the peripheral stalks. We also describe details of adenosine diphosphate inhibition release, V1-Vo torque transmission, and proton translocation, which are relevant for the entire V-type ATPase family.

Current understanding of structure, function and biogenesis of yeast mitochondrial ATP synthase.

The yeast mitochondrial ATP synthase is a rotary molecular machine primarily responsible for the production of energy used to drive cellular processes. The enzyme complex is composed of 17 different subunits grouped into a soluble F1 sector and a membrane-embedded F0 sector. The catalytic head of the F1 sector and the membrane integrated motor module in the F0 sector are connected by two stalks, the F1 central stalk and the F0 peripheral stalk. Proton translocation through the F0 motor module drives the rotation of the subunit 910-ring that generates torque which is transmitted to the calaytic head through the γ subunit of the central stalk. The rotation of the γ subunit causes changes in conformation of the catalytic head which leads to the synthesis of ATP. Biogenesis of the enzyme involves modular assembly of polypeptides of dual genetic origin, the nuclear and the mitochondrial genomes. Most of the yeast ATP synthase subunits are encoded by the genome of the nucleus, translated on cytosolic ribosomes and imported into mitochondria. In the mitochondria, the enzyme forms a dimer which contributes to the formation of cristae, a characteristic of mitochondrial morphology. Substantial progress has recently been made on the elucidation of detailed stucture, function and biogenesis of yeast mitochondrial ATP synthase. The recent availability of high-resolution structure of the complete monomeric form, as well as the atomic model for the dimeric F0 sector, has advanced the understanding of the enzyme complex. This review is intended to provide an overview of current understanding of the molecular structure, catalytic mechanism, subunit import into mitochondria, and the subunit assembly into the enzyme complex. This is important as the yeast mitochondrial ATP synthase may be used as a model for understanding the corresponding enzyme complexes from human and other eukaryotic cells in physiological and diseased states.