Reversible depolarization of in situ mitochondria by oxidative stress parallels a decrease in NAD(P)H level in nerve terminals

We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

Participation of the ATP/ADP antiporter and fatty acids in oxidative phosphorylation uncoupling in squirrel liver mitochondria during winter hibernation and awakening]

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.     

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.     

Adenosine 5''-triphosphate synthesis driven by a protonmotive force in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis energized by an artificially imposed protonmotive force (delta p) in adenosine 5'-diphosphate-loaded membrane vesicles of Escherichia coli was investigated. The protonmotive force is composed of an artificially imposed pH gradient (delta pH) or membrane potential (deltapsi), or both. A delta pH was established by a rapid alteration of the pH of the assay medium. A delta psi was created by the establishment of diffusion potential of K+ in the presence of valinomycin. The maximal amount of ATP synthesized was 0.4 to 0.5 nmol/mg of membrane protein when energized by a delta pH and 0.2 to 0.3 nmol/mg of membrane protein when a delta psi was imposed. Simultaneous imposition of both a delta pH and delta psi resulted in the formation of greater amounts of ATP (0.8 nmol/mg of membrane protein) than with either alone. The amount of ATP synthesized was roughly proportional to the magnitude of the artificially imposed delta p. Although p-chloromercuribenzoate, 2-heptyl-4-hydroxyquinoline-N-oxide, or NaCN each inhibits oxidation of D-lactate, and thus oxidative phosphorylation, none inhibited ATP synthesis driven by an artificially imposed delta p. Membrane vesicles prepared from uncA or uncB strains, which are defective in oxidative phosphorylation, likewise were unable to catalyze ATP synthesis when energy was supplied by an artificially imposed delta p.     

Prostaglandin E2 production by renal inner medullary tissue slices: effect of metabolic inhibitors

Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluted for their effects on PGE2 production and ATP levels. None of the inhibitors affected PGE2 synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE2 and ATP levels. This suggests that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather than an effect on oxidative phosphorylation. When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was omitted, PGE2 levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE2 levels, with a maximal effect at 10 mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis. These results indicate that oxygen (substrate) availability can limit inner medullary PGE2 production. In view of the low pO2 in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin production in this tissue.     

Uncoupling of mitochondrial oxidative phosphorylation by hexetidine

To gain further insight into the biochemical properties of the antibacterial hexetidine, isolated rat liver mitochondria were added with this drug and investigation made of certain features related to mitochondrial bioenergetics. Hexetidine was found to cause oxidation of intramitochondrial pyridine nucleotides and stimulate the rate of oxygen uptake caused by respiratory substrates involving three, two and one site(s) of phosphorylation. Reversal of oxygen uptake inhibition by oligomycin was also determined. By investigating hexetidine effect on oxidative phosphorylation, hexetidine was found both to inhibit the rate of ATP synthesis and to cause ATP hydrolysis. Likewise, hexetidine capability to produce acidification of extramitochondrial medium and to collapse delta psi was also observed. The reported findings show that hexetidine exhibits uncoupling properties.     

Carvedilol in heart mitochondria: protonophore or opener of the mitochondrial K(ATP) channels?

Carvedilol ([1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl) amino]-propanol-(2)]) has been shown to protect cardiac mitochondria from oxidative stress. In this work we examined the mechanisms responsible for an observed depressive effect in the mitochondrial transmembrane potential (delta psi). Two possible mechanisms were considered: a protonophoretic activity and the opening of mitochondrial ATP-sensitive potassium channels. We show that carvedilol increases mitochondrial inner membrane permeability to protons, but not to potassium, causing an increase in state IV respiration in the presence and absence of oligomycin. By contrast, a K(ATP)-channel inhibitor, 5-hydroxydecanoic acid, did not affect carvedilol-induced depolarizations. Hence, our results suggest that carvedilol depresses mitochondrial delta psi by a weak protonophoretic mechanism.     

Control of respiration and oxidative phosphorylation in isolated rat liver cells

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats. Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply). From the response of respiration to the mitochondrial membrane potential (delta psi M) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate delta psi M, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.     

"Wages of fear": transient threefold decrease in intracellular ATP level imposes apoptosis

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.     

Cell de-energization prevents plasmid transformation of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: evidence for the requirement of ATP

The dependence of the yeast Saccharomyces cerevisiae transformation on energy requirement was studied. The inhibitory effect of sodium arsenate, used for the depletion of the intracellular ATP pool, was determined. Incubation of the yeast cells in 5 mM sodium arsenate diminished ATP accumulation by 50% and the transformation efficiency decreased by 65%. To discriminate between ATP produced by substrate level phosphorylation and oxidative phosphorylation, the inhibitory analysis of a mutant with defective mitochondria was performed. Sodium fluoride (10–50 mM), as inhibitor of glycolysis, elicited a concentration-dependent decrease in intracellular ATP levels in both parental and mutant cells. The equal transformation efficiency of the mitochondrial mutant and parental strain, in addition to experiments with oligomycin, demonstrated the independence of plasmid transformation on mitochondrial ATP synthesis. This is consistent with our hypothesis that yeast transformation efficiency is associated with ATP produced by substrate level phosphorylation.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles

It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na⁺ and K⁺ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions.2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin.3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value.4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis.5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes

Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Reasons causing a lag period in the oxidative phosphorylation process. Isn't ATP an internal uncoupler of ATP synthetase?]

The kinetics of oxidative phosphorylation catalyzed by bovine heart submitochondrial particles was studied in a range of MgATP and MgADP concentrations from 0.3 to 10 mM. It is shown that, at a low uncoupler concentration (0.9 microM of tetrachlorotrifluoromethylbenzimidazole, the lag period of the reaction increases from 12 s to 2-3 min, and KM for Pi increases severalfold; the value of Vmax remains practically unchanged. Increasing the [MgATP]/[MgADP] concentration ratio, with their total concentration being unchanged, leads to similar changes in the kinetics of oxidative phosphorylation. The value of delta pH generated on the membrane of AS particles at delta microH+ = 60 delta pH was measured using 9-aminoacridine. It was found that the electrochemical potential of H+ ions shows the same thermodynamic shift in the reaction of energy-dependent Pi -ATP exchange throughout the [MgATP]/[MgADP] concentration range studied, from 0.1 to 10: the synthesis on the ATP molecule is provided by the transmembrane transfer of two H+ ions. It was shown that the binding of ATP and/or ADP in the allosteric site, whose saturation is necessary for the functioning of ATP synthase, occurs with equal constants, 1-2 mM. It is concluded that the lag period in the synthesis of ATP indicates the monomolecular transition ATP hydrolase-->ATP sysnthase, which comes about by the action of transmembrane potential. The binding of MgADP or MgATP renders the enzyme structure "more coupled" or "less coupled", respectively. Structural distinctions manifest themselves in a kinetically different behavior of mitochondrial ATP synthase at [MgATP] > [MgADP] and [MgATP] < [MgADP] and do not suggest futile leakage of H+ through the membrane.