Redox Proton Pump Research Articles

Respiratory complex I: Bottleneck at the entrance of quinone site requires conformational change for its opening

The structure of the entire respiratory complex I is now known at reasonably high resolution for many species – bacteria, yeast, and several mammals, including human. The structure reveals an almost 30 angstrom tunnel-like chamber for ubiquinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. Here we characterize the geometric bottleneck forming the entrance of the quinone reaction chamber. Computer simulations of quinone/quinol passage through the bottleneck suggest that in all structures available, from bacterial to human, this bottleneck is too narrow for the quinone or quinol to pass and that a conformational change is required to open the channel. Moreover, the bottleneck is too narrow even for isoprenoid tail free passage. The closed structure can be an artifact of the crystallization packing forces, low temperature, or other unnatural conditions occurring in the structural data acquisition procedure that affect this flexible part of the enzyme. Two of the helices forming the bottleneck are in direct contact with the subunit (ND3) that was recently demonstrated to be involved in conformational changes during the redox proton pumping cycle, which indicates flexibility of that part of the enzyme. We conclude that the published structures are all locked in the unfunctional states and do not represent correctly the functional enzyme; we discuss possible ways to open the structure in the context of possible mechanisms of the enzyme.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

Comparative Investigation of O2 Delivery Pathwys in A-Type and B-Type Cytochrome C Oxidases

Cytochrome c oxidase (CcO), a redox proton pump, terminates the aerobic respiratory chain by catalyzing the reduction of O2 to water. Various isoforms of CcO enzymes with distinct biochemical properties and different organism distributions have been identified. A-type CcOs are distributed in all three kingdoms of life. B-type CcOs are found only in archaea and bacteria. CcOs rely on efficient pathways for protons, electrons, and O2, connecting their buried heme-copper catalytic site to the surface of the protein. Here, we employed molecular dynamics simulation to probe and characterize pathways for the O2 delivery to CcO's catalytic site using both explicit and implicit ligand sampling methods. A-type (CcO caa3) and B-type (CcO ba3) CcO enzymes from Thermus thermophilus were investigated. While CcO caa3 is constitutively expressed, CcO ba3 is expressed under low oxygen condition, implying possible differences between the two enzymes in their ability and efficiency to recruit O2. Although the characterized O2 pathways exhibit a close similarity between the two enzymes from a structural perspective, O2 dynamics through these pathways is quite different. In CcO ba3, O2 reached the catalytic site rather rapidly, i.e., within a few nanoseconds, while the process was much slower and required several tens of nanoseconds in CcO caa3. In each enzyme, O2 entered the protein and reached the catalytic site via a membrane-accessible hydrophobic channel observed in X-ray structures. While O2 was found to diffuse freely into CcO ba3 through the hydrophobic channel, the O2 pathway in CcO caa3 was constricted by the presence of large aliphatic and aromatic residues. The findings relate the functional and structural adaption of CcO to oxygen-stress conditions.

Hyperthyroidism and mitochondrial uncoupling.

During the past years many efforts have been made to elucidate the origin of the uncoupling mechanisms induced by hyperthyroidism in mitochondria. Two main mechanisms have been proposed: a classical protonophoric uncoupler mechanism, considering the action of thyroid hormones at the level of the lipid membrane bilayer, and a slipping mechanism with more localized effects at the level of the redox proton pumps. This short review is devoted to comparing and discussing the evidence against and in favour of these two mechanisms.

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd2+ influx via the Mn2+ porter down membrane potential (delta psi) was fast due to involvement of energy generated by two proton pumps--the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd2+ influx is suggested to be due to a series of toxic events elicited by Cd2+ accumulation down delta psi generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient (delta mu H+) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of delta mu (H+)-dependent ATP synthesis, (iii) acceleration of the initial Cd2+ influx down delta psi generated by the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd2+ of delta mu (H+)-dependent ATP synthesis and of pH gradient (delta pH)-linked [14C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of delta mu H+ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd2+ accumulation was prevented due to activity of the plasmid-coded Cd2+ efflux system. Consequently, delta mu (H+)-producing and -requiring processes were not affected by Cd2+.

Mechanism of loss of thermodynamic control in mitochondria due to hyperthyroidism and temperature.

Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.

Tracking of proton flow during transition from anaerobiosis to steady state. 1. Response of matrix pH indicators.

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non-respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions. 2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA-untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2-3 nmol H+/mg, reduced to 1-2 nmol H+/mg in EGTA-treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6-7 nmol H+/mg, it appears that 75-90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red. 3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation. 4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.

Respiration in non-phosphorylating yeast mitochondria. Roles of non-ohmic proton conductance and intrinsic uncoupling

Respiratory rate, protonmotive force and charge/O ratio were measured under two different kinds of steady state in non-phosphorylating yeast mitochondria: (i) when the electron flux was modulated by a variable limitation in electron supply or (ii) when oxygen consumption was decreased by respiratory chain inhibitor titration. We showed that the relationships between either delta p or charge/O ratio and respiratory rate are different under the two kinds of steady state, indicating different degrees of intrinsic uncoupling in respiratory chain. Moreover, we observed a non-ohmic dependence between H(+)-conductance and delta p. We concluded that the high rate of static-head respiration in yeast mitochondria was determined both by the non-ohmic proton conductance of the inner membrane and the saturation of the redox proton pump slipping.

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome- c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and Δ \\ ̃ gm H + , and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater Δ \\ ̃ gm H + than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase — it decreases the ATP/O value without any change in Δ \\ ̃ gm H + ; (ii) almitrine increases the mechanistic H + ATP stoichiometry of A TPase/ATPsynthase; (iii) almitrine-induced changes in H +/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters: flux value, force, pump efficiency and control coefficient.

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling.

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies.

The thermodynamic and kinetic properties associated with intrinsic uncoupling in a six-state model of a redox proton pump have been studied by computing the flow-force relations for different degrees of coupling. Analysis of these relations shows the regulatory influence of the thermodynamic forces on the extent and relative contributions of redox slip and proton slip. Inhibition has been introduced into the model in two different ways, corresponding to possible modes of action of experimental inhibitors. Experiments relating the rate of electron transfer to delta microH at static head upon progressive inhibition of the pumps have been simulated considering (1) the limiting case that the nonzero rate of electron transfer at static head is only due to intrinsic uncoupling (no leaks) and (2) the experimentally observed case that about 30% of the nonzero rate of electron transfer at static head is due to a constant proton leakage conductance in parallel with the pumps, the rest being due to intrinsic uncoupling. The same simulations have been performed for experiments in which the rate of electron transfer is varied by varying the substrate concentration rather than by using an inhibitor. The corresponding experimental results obtained by measuring delta microH and the rate of electron transfer at different succinate concentrations in rat liver mitochondria are presented. Comparison between simulated behavior and experimental results leads to the general conclusion that the typical relationship between rate of electron transfer and delta microH found in mitochondria at static head could certainly be a manifestation of some degree of intrinsic uncoupling in the redox proton pumps.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of membrane bioenergetics.

One of the first problems encountered by primitive cells was that of volume regulation; the continuous entry of ions, (eg, NaCl) and water in response to the internal colloid osmotic pressure threatening to destroy the cell by lysis. We propose that to meet this environmental challenge cells evolved an ATP-driven proton extrusion system plus a membrane carrier that would exchange external protons with internal Na+. With the appearance of the ability to generate proton gradients, additional mechanisms to harness this source of energy emerged. These would include proton-nutrient cotransport, K+ accumulation, nucleic acid entry, and motility. A more efficient system for the uptake of certain carbohydrates by vectorial phosphorylation via the PEP-phosphotransferase system probably appeared rather early in the evolution of anaerobic bacteria. The reversal of the proton-ATPase reaction to give net ATP synthesis became possible with the development of other types of efficient proton transporting machinery. Either light-driven bacterial rhodopsin or a redox system coupled to proton translocation would have served this function. Oxidation of one substrate coupled to the reduction of another substrate by membrane-bound enzymes evolved in such a manner that protons were extruded from the cell during the reaction. The progressive elaboration of this type of redox proton pump permitted the use of exogenous electron acceptors, such as fumarate, sulfate, and nitrate. The stepwise growth of these electron transport chains required the accretion of several flavoproteins, iron-sulfur proteins, quinones, and cytochromes. With modifications of these four basic components a chlorophyll-dependent photosynthetic system was subsequently evolved. The oxygen that was generated by this photosynthetic system from water would eventually accumulate in the atmosphere of the earth. With molecular oxygen present, the emergence of cytochrome oxidase would complete the respiratory chain. The proton economy of membrane energetics has been retained by most present-day microorganisms, mitochondria, chloroplasts, and cells of higher plants. A secondary use of the energy stored as an electrochemical difference of Na+ for powering membrane events probably also evolved in microorganisms. The exclusive age of the Na+ economy is distinctive of the plasma membrane of animal cells; the Na+-K+ ATPase sets up an electrochemical Na+ gradient that provides the energy for osmoregulation, Na+-nutrient co-transport, and the action potential of excitable cells.

On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in "sonic" submitochondrial particles.

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.

Proton translocation and energy transduction in mitochondria

Summary Data are presented showing that respiration-linked proton translocation in the inner mitochondrial membrane consists of a vectorial, single, electrogenic flow, mediated by system(s) different from cation carriers. The proton pump is, however, secondarily coupled through the ΔpH component of the proton gradient and at the level of a proton-cation antiporter, to flow of Na+ or K+. The redox proton pump appears to be directly coupled to oxido-reductions of respiratory carriers without the intervention of uncoupler-sensitive chemical intermediates. Two mechanism will be discussed for this direct coupling : (1) the oxido-reductase proton translocator of Mitchell ; (2) a so-called membrane Bohr effect, based on shifts of the pK of protonable groups of the apoprotein of electron carriers of the respiratory chain which accompany the redox changes of the electrony carrying metal centers.