Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

Energetics of ATP-driven reverse electron transfer from cytochrome c to fumarate and from succinate to NAD in submitochondrial particles

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.     

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium.

Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.     

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.     

Proton translocation in membranes of submitochondrial particles]

Effect of an electrophilous inhibitor, chlorophenacyl, on energy-dependent functions of submitochondrial particles is studied. Chlorophenacyl at concentrations up to 1 mM is found practically not to affect the generation of membrane potential under NADH and succinate oxidation and ATP hydrolysis and to be a strong inhibitor of oxidative phosphorylation and reverse electron transport. The mechanism of the inhibition of energy-dependent functions of submitochondrial particles with chlorophenacyl is different from that of electron transport inhibitor, energy transport inhibitors and classical uncoupling agents--protonophors. The data obtained are suggested to be due to the existence of two ways of proton translocation in submitochondrial particle membrane, phosphorylating and non-phosphorylating, the effect of chlorophenacyl being directed on phosphorylating way only.     

Measurement of the electrochemical proton gradient in submitochondrial particles

The pH gradient and membrane potential of submitochondrial particles from bovine heart were estimated by the uptake of [14C]ethylamine and [36Cl]perchlorate, using filtration through a glass fiber prefilter and Millipore filter without washing to separate the vesicles from the medium. An external volume probe of [3H] sucrose was also used. Internal volume of the vesicles was measured by the extent of uptake of glucose, which equilibrates slowly across the membrane. The electrochemical potential gradient of H+ (delta micro H+) calculated from uptake of ethylamine and perchlorate, assuming the ions taken up were free in solution inside the vesicles, was 23 to 24 kJ/mol of H+ (240-250 mV) during respiration in the absence of ATP. The ratio of the free energy of ATP synthesis (delta GATP) to delta micro H+ was 2.2 to 2.3 during oxidative phosphorylation and only slightly higher during ATP hydrolysis indicating that the H+-translocating ATPase is close to equilibrium under both conditions. The nonintegral ratio suggests there is a systematic error in the measurement of delta micro H+. The value of delta micro H+ calculated from ion uptake could be too high if some of the ions taken up are bound to the membrane or concentrated into the electric double layer at the inner membrane-water interface. The effects of vesicle volume (varied osmotically) and permeant ions (which affect internal ionic strength and pH) on the ratio of delta GATP to delta micro H+ suggested that ion association with the membrane in fact caused significant overestimation of delta micro H+. Association of ethylammonium and perchlorate ions with unenergized submitochondrial particles was measured by centrifugation, in the presence of a high concentration of impermeant salt to minimize association with the external surface. The results were used to estimate the extent of binding during the ion uptake assays, and delta micro H+ was recalculated taking this binding into account. The resulting values were between 19 and 20 kJ/mol of H+ (197-207 mV) during respiration in the absence of ADP, and the ratio of delta GATP to delta micro H+ was about 3 during oxidative phosphorylation.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles.

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.     

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.     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

DCCD inhibits proton translocation and electron flow at the second site of the mitochondrial respiratory chain

DCCD inhibits formation of a succinate-driven transmembrane pH gradient in submitochondrial particles, as shown by inhibition of fluorescence quenching of 9-aminoacridine, without concomitant inhibition of succinate oxidation. On the other hand ubiquinol-cytochrome c reductase activity is inhibited by DCCD. Half-inhibition of both fluorescence quenching and ubiquinol-cytochrome c reductase occur at 35 μM DCCD. The results suggest that DCCD inhibits proton pumping activity coupled to electron flow through the bc₁ complex.     

Current-voltage relationships for proton flow through the F0 sector of the ATP-synthase, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or leak pathways in submitochondrial particles

Respiring submitochondrial particles from which the F1 sector of ATP-synthase was displaced generated a membrane potential in the range of 115-140 mV. Addition of oligomycin raised the membrane potential by approximately 40 mV. The lower membrane potential in particles with F1 displaced is attributed to partial dissipation of the proton electrochemical gradient as a consequence of proton flow through the open proton channels provided by the F0 sectors of the ATP-synthase. The characteristics of proton flow through the open F0 channels were studied by varying the rate of electron transport-driven proton translocation which permitted the establishment of a range of steady-state membrane potentials. Open F0 channels appeared to have a gated response to the membrane potential such that they were inoperative when the potential fell below approximately 110 mV. The membrane potential was measured as a function of respiratory rate in intact Mg-ATP submitochondrial particles that had been treated with low concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone. In general a linear dependence of membrane potential upon respiratory rate was observed except at the lowest concentrations of protonophore and highest respiratory rates, presumably because the effect of the protonophore was then offset by an increased rate of proton translocation driven by the respiratory chain. The effect of increasing concentrations of carbonylcyanide-p-trifluoromethoxyphenylhydrazone on the membrane potential of respiring submitochondrial particles was studied. It was found that equal amounts of the protonophore lowered the membrane potential to a lesser extent at lower values of the membrane potential. Treatment of Mg-ATP submitochondrial particles with oligomycin slightly increased (by approximately 10 mV) the size of the respiration-dependent membrane potential, but did not alter the profile of membrane potential as a function of succinate oxidation rate. The latter was controlled by titration with malonate. This result indicates that the F0 sector of the ATP-synthase does not significantly contribute to leak pathways in intact submitochondrial particles.     

The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation

In the accompanying article (T.E. Gunter and B.D. Jensen, 1986 Arch. Biochem. Biophys. 248, 289-304), a method is described for measuring the efficiencies of individual steps of the process of oxidative phosphorylation. The results of applying this method to the case of state 3 phosphorylation in rat liver mitochondria are reported here. The rate of energy use (or power use) at the gradient generation, leakage, and phosphorylation steps are reported as efficiencies and energy use factors in tabular form. The limits of the degrees of coupling of the gradient generation and phosphorylation steps are also determined and under the current conditions of measurement these degrees of coupling are found to be quite close to unity. The data can be used to show that the only sets of the stoichiometric parameters noH (the charge/2e- ratio in this case from succinate to oxygen), nPH (the H+/ATP ratio), and nTH (number of protons translocated during substrate-product transport) which are simultaneously consistent with both the laws of thermodynamics and with the current data are 8, 3, 1, and 6, 3, 0. The The efficiency of the phosphorylation step which is independent of noH and nTH averages 80% for the control data analyzed. If noH is 8 (succinate to oxygen), the average value of the efficiency of generation of the electrochemical proton gradient is approximately 91 percent. Since very little power (energy) would then be left over to be coupled in parallel to phosphorylation through some other means of coupling, this would place the electrochemical proton gradient in the direct path of power flow and identify it as "an" intermediate in the process. This would suggest that any other intermediate should be considered as being "in series" with the electrochemical proton gradient. The agents butyrate and propionate have been employed to permit investigation over a range of pH gradient and membrane potential. Both butyrate and propionate decrease the efficiency of generation of the electrochemical proton gradient and increase proton leakage. In addition, butyrate activates electron transport whereas propionate inhibits it. By using butyrate to modify the values of pH gradient and membrane potential, it can be shown that the ratio of the efficiency with which the pH gradient is used in phosphorylation to that with which the membrane potential is used is 1.08 +/- 0.38.     

Hydrolysis of ITP generates a membrane potential in submitochondrial particles

ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.     

The relation of millisecond delayed light emission to light-induced ion accumulations in chloroplasts

Delayed fluorescence (delayed light emission) from chloroplasts is increased by ATP, ADP and, to a lesser extent, by ITP. However, neither phosphorylation nor ATP utilization seems to play any part in the phenomenon since the energy transfer inhibitor deoxyphlorizin, which is also an ATPase inhibitor, has no effect on the enhancement of delayed fluorescence. The enhancement of delayed fluorescence by these nucleotides is accompanied by an increase in the extent of proton uptake and n decrease in the nonphosphorylating (basal) electron transport.Uncouplers and ionophores such as imidazole, glycineamide, morpholine, methyl-amine, cyclohexylamine, atebrin, and gramicidin nearly abolish delayed fluorescence. However, ammonium salts are exceptional; they considerably enhance the emission although they also abolish phosphorylation and proton gradient formation. This enhancement of delayed fluorescence occurs only near or above pH 8 and seems to be specific for ammonia when relatively intact lamellae are employed. When particles prepared therefrom with digitonin are used, methylamine also enhances the delayed fluorescence. The enhancement by ammonium salts is correlated with the uptake of ammonium ions. Valinomycin, which is known to increase the permeability of membranes to ammonium ions, abolishes delayed fluorescence in the presence of ammonium salts. It is suggested that (a) ammonia uncoupling abolishes the pH component of the light-induced transmembrane electrochemical potential gradient, but that (b) at higher pH's the electrical component of the gradient (the membrane potential) is not abolished and may even increase while (c) this increased membrane potential is responsible for enhancement of the delayed fluorescence.Gradients which contribute to delayed fluorescence are not necessarily capable of supporting phosphorylation. The requirements for phosphorylation seem more stringent than the requirements for delayed fluorescence and it may be that phosphorylation, unlike the delayed light emission, has an obligatory requirement for a pH gradient.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.     

Free fatty acids decouple oxidative phosphorylation by dissipating intramembranal protons without inhibiting ATP synthesis driven by the proton electrochemical gradient

Free fatty acids (FFA) uncouple oxidative phosphorylation and reverse electron transport and inhibit ATP-Pi exchange in beef heart submitochondrial particles. In this, they resemble classical uncouplers and ionophores. However, in contrast to the latter agents, FFA do not collapse the substrate generated proton electrochemical potential and do not inhibit ATP synthesis when the latter is driven by artificially imposed delta microH. These results lend further support to the suggestion that oxidative phosphorylation depends, in part, on direct intramembranal proton transfer - a process which is specifically uncoupled by FFA and other membrane perturbing agents (e.g. general anesthetics).     

Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.

Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state. The covalent bond forming and breaking step at the catalytic site (ADP + Pi in equilibrium ATP + HOH) appears relatively insensitive to uncouplers. Thus to the extent that uncouplers dissipate transmembrane proton-motive force, it is unlikely that such a force is used to drive ATP formation by direct protonations of Pi oxygens. When only Pi and ADP are added and formation of ATP from added ADP by adenylate kinase and subsequent ATP hydrolysis are adequately blocked, no Pi in equilibrium HOH exchange can be observed, demonstrating a requirement of energization by ATP binding and cleavage for such an exchange. This uncoupler-insensitive energization is suggested to represent a conformationally energized state that can be used reversibly to develop a transmembrane protonmotive force accompanying ADP and Pi release. Rates of various exchanges as estimated by improved procedures are compatible with all oxygen exchanges occurring by dynamic reversal of ATP hydrolysis at the catalytic site.     

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.     

The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Delta mu(H(+)) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Delta mu(H(+)) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes.