The relative proton stoichiometries of the mitochondrial proton pumps are independent of the proton motive force

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.     

The hypothesis of localized chemiosmosis is unsatisfactory.

The hypothesis of 'localized' chemiosmosis have been put to an experimental test in the system of oxidative phosphorylation by rat liver mitochondria. We find that the variation of the ratio of phosphate potential to delta muH with delta muH does not depend on how delta muH is varied. This is in conflict with hypotheses of localized chemiosmosis. Of all coupling hypotheses, only the parallel-coupling hypothesis can explain the observations, unless variation of the H+/ATP stoichiometry of the ATPase proton pump is accepted. In the latter case. 'macroscopic' chemiosmosis can explain the observations equally well as hypotheses of localized chemiosmosis. It is concluded that either variation of H+/ATP stoichiometries must be accepted, or that the parallel-coupling hypothesis should be reformulated so that it becomes open to experimental tests.     

Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli

The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli. The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant. The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate. According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV. The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used. However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25. Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Local protons and uncoupling of aerobic and artificial delta muH-driven ATP synthesis

Gramicidin D causes inhibition of ATP synthesis either in the absence or in the presence of depression of delta muH, in low-salt and in high-salt media, respectively, at concentrations 2 orders of magnitude higher in the former with respect to the latter case. When the number of active redox pumps is reduced by increasing the antimycin concentration, the P/O ratio of respiring, gramicidin-treated mitochondria either is slightly increased in low-salt media or is first decreased and then constant in high-salt media. Addition of gramicidin D in low-salt media to mitochondria synthesizing ATP by means of artificially imposed delta muH gradients results in (a) no effect on the K+ efflux ratio +/- ADP (equivalent to the aerobic respiratory control ratio) and (b) no effect on the ATP/K+ ratio (equivalent to the P/O ratio) except at the low gramicidin D concentrations where there is also a slight enhancement of the rate of ATP hydrolysis. During respiration-driven ATP synthesis, addition of valinomycin plus K+ causes depression of delta muH with little inhibition of ATP synthesis while addition of gramicidin D causes inhibition of ATP synthesis with little depression of delta muH. The view is discussed that the gramicidin-accessible protons which uncouple aerobic ATP synthesis in a delta muH-independent manner are of a different class from the gramicidin-inaccessible protons which uncouple diffusion potential driven ATP synthesis in a delta muH-dependent manner. The gramicidin-accessible protons are suggested to be pump associated and to reflect primary events in energy transduction.     

Thermodynamic limits to the ATP/site stoichiometries of oxidative phosphorylation by rat liver mitochondria

From measurements of reactants, products, and the oxidation-reduction state of cytochrome c + c1 during 3-hydroxybutyrate-supported oxidative phosphorylation by rat liver mitochondria at static head (state 4), we determined the free energy change of ATP formation from ADP and Pi (phosphorylation potential or delta GP) and the oxidation-reduction free energy changes (redox potentials or delta GR values) across Sites 1 + 2 (delta GR1 + 2), across Site 3 (delta GR3), and across Sites 1 + 2 + 3 (delta GR). At pH 7.4, -delta GR1 + 2/delta GP, -delta GR3/delta GP, and -delta GR/delta GP were maximally 1.80, 1.56, and 3.37. These can be taken as thermodynamic upper limits to the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometry of 3-hydroxybutyrate-supported oxidative phosphorylation. The theory of linear nonequilibrium thermodynamics were employed to estimate lower limits to the ATP/site stoichiometries. The lower limit is given by the expression, q2(-delta GRsite/delta GP). The degree of coupling, q, was 0.977 as determined from the dependence of respiratory rate on delta GP. Determined in this way, lower limits of the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometries were 1.67, 1.44, and 3.11, respectively. ADP addition to mitochondria incubated at static head lowered delta GP by 1.1 kcal/mol and stimulated respiration by a factor of about 2.5 but caused negligible changes in delta GR1 + 2 and delta GR3. This observation demonstrates that the respiratory reactions from substrate to cytochrome c and from cytochrome c to oxygen both move away from thermodynamic equilibrium with delta GP during the transition from resting to active oxidative phosphorylation. The findings are discussed in terms of current schemes of chemiosmotic coupling.     

Analysis of the causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers, Citellus undulatus.

1. The causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers have been analysed. 2. The decrease of the ATP synthesis rate and suppression of the energy-dependent K(+)- and Ca(2+)-transport into mitochondria during hibernation has been found to be mainly related to a delta psi decrease in mitochondria of hibernating gophers. 3. The increase delta psi upon incubation of the mitochondria of hibernating animals in a hypotonic medium results in an essential acceleration of ATP synthesis and energy-dependent cationic transport.     

Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier.

The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor-bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source.     

Studies on the relationship between ATP synthesis and transport and the proton electrochemical gradient in rat liver mitochondria

The effect of ATP synthesis on delta mu H in rat liver mitochondria has been analyzed by separating the steps of adenine nucleotide translocation and ATP synthesis in the matrix. Either exchange of ATP, synthesized by substrate level phosphorylation in the matrix of oligomycin-treated mitochondria, for external ADP, or activity of the membrane-bound ATP synthase complex results in delta mu H depression with respect to resting state levels. This depression appears to be more pronounced, under strictly comparable conditions, when arsenate is used to stimulate ATP synthase activity than when the ornithine-citrulline conversion reaction is used for the same purpose.     

Citrate effects on the Ca2+-loading capacity of isolated rat liver mitochondria: interaction of citrate and ATP

The maximal amounts of Ca2+ being accumulated (delta Ca2+max) and H+ emitted (delta H+max) by Ca2+-loading mitochondria, with succinate (+rotenone) as respiratory substrate, were evaluated. delta Ca2+max was increased by providing either citrate or ATP to a Pi- and Mg2+-free medium. With citrate, delta H+max was only scarcely increased, so that the effect of the proton-carrying anion resulted essentially from an increase in the Ca2+/H+ ratio, i.e., from preservation of membrane potential. With ATP (+/- oligomycin), the Ca2+/H+ ratio was unaltered; i.e., the increase of delta Ca2+max was paralleled by a related increase in delta H+max. Mitochondria appeared to retain Ca at higher delta pH, i.e., at lower membrane potential, in the presence of ATP. With citrate and ATP together, both the Ca2+/H+ ratio and delta H+max were largely increased, and the product of these two terms, delta Ca2+max, was considerably enlarged. The effect of either citrate or ATP was markedly reinforced in the presence of the other anion. In addition to increasing the Ca2+/H+ ratio, citrate contributed to increasing delta H+max in the presence of ATP, i.e., apparently sensitized mitochondria to the action of ATP. A citrate-induced depression of Ca2+ cycling across the inner membrane, even though pronounced, did not account for the sensitization. Supraadditive effects of citrate and ATP persisted in the presence of MgCl2 and Pi, under conditions of massive Ca2+ loading, and may contribute to the high capacity of mitochondria, in situ, to retain calcium.     

Stability of membrane potential in heart mitochondria: single mitochondrion imaging

Mitochondrial membrane potential (delta psi(m)) plays an important role in cellular activity. Although delta psi(m) of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of delta psi(m). The current study is performed to investigate the factors that stabilize delta psi(m) in cells by observing delta psi(m) of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of delta psi(m) in cells.     

The effect of the natural protein inhibitor on H+-ATPase hepatoma 22a mitochondria

The uncoupler-induced inactivation of H+-ATPase in hepatoma 22a and mouse liver mitochondria has been studied. The dependence of this process on delta microH, and pH and ATP was established. The inactivated ATPase could be reactivated at alkaline pH values in the absence of ATP. These data indicate that the inactivation is apparently caused by the natural protein inhibitor. ATP- and pH-dependent decrease of ATPase activity is also observed after Lubrol-WX disruption of mitochondria. It can be proposed that practically all ATPase molecules in hepatoma mitochondria are in a catalytically active complex with the protein inhibitor. At low delta microH this complex is inactivated via reversible pH-dependent and irreversible ATP-dependent rearrangements. The pH-dependent rearrangement of the isolated protein inhibitor from hepatoma mitochondria is also observed.     

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.     

Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria

The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.     

Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces

The delta Gp/delta mu H ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting delta Gp and delta mu H which could lead to an artefactual increase of the delta Gp/delta mu H ratio above the highest accepted value for the H+/ATP stoichiometry (n = 4, synthesis + transport). In particular, to avoid overestimation of delta Gp due to inactivation of the ATPases at low delta mu H or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and delta Gp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of delta Gp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of delta Gp/delta mu H ratios. Both uncouplers and K+ transport cause a marked depression of delta mu H and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low delta mu H eventually results, especially at high Pi concentrations, in a relatively large delta Gp. The delta Gp/delta mu H ratios obtained at the lower delta mu H values exceed 4 and approach 6. Although slightly higher delta Gp/delta mu H ratios are obtained with valinomycin-treated than with uncoupler-treated mitochondria, the pattern of the rise of the force ratio as delta mu H decreases is similar in both cases. An increase of the delta Gp/delta mu H ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model.     

Proton/electron stoichiometry of mitochondrial complex I estimated from the equilibrium thermodynamic force ratio.

The thermodynamic forces on electrons (delta Eh) and protons (delta p) across mitochondrial complexes I, III and IV were measured in isolated mitochondria respiring on succinate. The force ratio (delta Eh/delta p) across complex I close to equilibrium was found to be about 2. The equilibrium force ratio across complex I was measured during sulphite oxidation and was again close to 2. These results indicate that the proton/electron stoichiometry of complex I is 2, in conditions of high protonmotive force.     

ATP synthase-mediated proton fluxes and phosphorylation in rat liver mitochondria: dependence on delta mu H

The dependence of the proton flux through the ATP synthases of rat liver mitochondria on a driving force composed mainly of a potassium diffusion potential was determined and compared with the relationship between rate of phosphorylation and delta mu H given by titrations with the respiratory inhibitor malonate. The two functions are in good agreement in the lower part of the delta mu H range covered. However, the maximal proton fluxes through the ATP synthases are much lower than needed to account for the rate of State 3 phosphorylation sustained by the same mitochondria oxidizing succinate. Possible reasons for this behavior are discussed.     

Membrane electricity as a convertible energy currency for the cell

The role of transmembrane electric potential difference (delta psi) in mitochondria, chloroplasts, and bacteria has been considered. Since the electric capacitance of membranes is much lower than the pH buffer capacitance of water phases, delta psi proves to be the primary form of energy produced by generators of electrochemical H+ potential difference (delta mu-H). There are 11 distinct types of delta mu-H-generating systems in coupling membranes, involved in respiratory and light-dependent electron and proton transfer, as well as in ATP and PP1 hydrolysis and synthesis. Bacteriorhodopsin is the simplest delta mu-H generator. However, even in this case, the molecular mechanism of delta psi production remains obscure. Many types of work can be supported by delta mu-H with no ATP involved so that delta mu-H proves to be not only a transient intermediate of oxidative and photosynthetic phosphorylation but also a convertible energy currency for the cell. Among the delta mu-H-supported activities, mechanical work was recently demonstrated. It can be exemplified by the motility systems of (i) flagellar bacteria and (ii) blud--green algae. As was found in multicellular cyanobacteria, delta mu-H can be used for a power transmission over distances as long as 1 mm. It seems to be probable that in large cells of eukaryotes (e.g., in muscle fibers) giant mitochondria may serve as power-transmitting structures. Na+--K+ gradients can be used to stabilize delta mu-H in bacteria. It is suggested that the primary function of unequal distribution of these cations between the microbial cell and the medium is delta mu-H buffering.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Oxygen-pulse curves in rat liver mitochondrial suspensions. Some observations and deductions.

1. The inference, implicit in the chemiosmotic hypothesis, that protons move into the bulk phase during ATP synthesis was investigated. 2. Incubation of rat liver mitochondria in the presence of the cation exchanger CM-Sephadex C-50 caused alkalinization in the medium, though total ATP synthesis remained unchanged. The addition of N-ethylmaleimide prevented the alkalinization, but there was still no indication of protons passing into the medium. The expected proton movement [Mitchell & Moyle (1967) Biochem. J. 105, 1147--1162] was readily detected when as an equivalent acid pulse. 3. Analysis of delta H+ decay curves after O2 pulses (3 micrograms-atoms of O/g of protein) indicated the presence of fast and slow components of decay, with first-order rate constants (k) of 0.24s-1 and 0.032s-1. The fast decay was finite and was eliminated in the presence of N-ethylmaleimide. 4. These observations are interpreted as evidence for the development of unmasking of fixed charges on the outer surface of the mitochondrial inner membrane during energization and for the existence of proton-retentive electrical fields (rho-zones) on this surface. The charge concentration is calculated as about 1 charge/10nm2. 5. A cycle of changes in a single fixed-charge molecule is proposed which mediates both Ca2+ uptake and the first step in the utilization of the rho-zone protonmotive force, delta p rho.