Mitochondrial ATP synthase: fifteen years later

Mitochondrial Fo.F1-H+-ATP synthase is the main enzyme responsible for the formation of ATP in aerobic cells. An alternating binding change mechanism is now generally accepted for the operation of the enzyme. This mechanism apparently leaves no room for the participation of nucleotides and Pi other than sequential binding to (release from) the catalytic sites. However, the kinetics of ATP hydrolysis by mitochondrial ATPase is very complex, and it is difficult to explain it in terms of the alternating binding change mechanism only. Fo.F1 catalyzes both delta muH+-dependent ATP synthesis and ATP-dependent delta muH+ generation. It is generally believed that this enzyme operates as the smallest molecular electromechanochemical reversible machine. This essay summarizes data which contradict this simple reversible mechanism and discusses a hypothesis in which different pathways are followed for ATP hydrolysis and ATP synthesis. A model for a reversible switch mechanism between ATP hydrolase and ATP synthase states of Fo. F1 is proposed.     

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.     

Molecular slipping in redox and ATPase H+ pumps

The titration of the mitochondrial ATPase H+ pump with oligomycin has been compared with the titration of the redox H+ pump with antimycin. In both cases there is extensive inhibition of the pumps without significant depression of delta muH. The two pumps exhibit 'nonohmic' behavior in different ranges of delta muH. This discrepancy favors the hypothesis of nontightly coupled or 'slipping' H+ pumps with respect to that of a steep dependence of the membrane 'leak' conductance for H+ on delta muH.     

On Why Thylakoids Energize ATP Formation Using Either Delocalized or Localized Proton Gradients – A Ca2+ Mediated Role in Thylakoid Stress Responses

By the early 1970s, the chemiosmotic hypothesis of Peter Mitchell was widely accepted by bioenergetics researchers as the best conceptual scheme to explain how ATP is formed in oxidative and photosynthetic phosphorylation. At about the same time, however, work from a few laboratories suggested that some aspects of that elegant, relatively simple hypothesis required revision - not abandonment, but refinement to accommodate more complex movements of protons in the ATP formation mechanism than originally envisioned by Peter Mitchell. In some situations it appeared that protons were constrained to localized domains rather than always delocalized within an enclosed vesicle as envisioned by chemiosmosis. This minireview tells that story from my perspective, as one of the researchers involved in the experimental approaches that revealed more complex energy coupling proton flux patterns. Ionic conditions during isolated thylakoid storage were found to reversibly switch the [Formula: see text] gradient driving ATP formation between delocalized and localized energy coupling modes. Thylakoid accessible Ca(2+) ions proved to be the switching factor that was responding to the ionic conditions in the storage treatment. The mechanism of Ca(2+) was at least partially demystified when it was shown that the reversible switching between [Formula: see text] energy coupling modes involved Ca(2+) interactions with the 8 kDa CF(0) (the H(+) channel) subunit in a type of H(+) flux gating action. Other experiments showed that the Ca(2+) gating of H(+) flux into the lumen may be a critical regulatory factor in controlling the lumen pH and thereby help regulate the activity of the violaxanthin de-epoxidase enzyme, a key part of the chloroplast photoprotective response to over-energization (excess light) stress.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

Integrated functioning of the chloroplast coupling factor

This review is focused on some functional characteristics of the chloroplast coupling factor. The structure of the enzyme and the putative role of its subunits are recalled. An attempt is made to discriminate the driving force and the activator effects of the electrochemical proton gradient. Respective roles of delta pH, delta phi, external and internal pH are discussed with regard to mechanistic implications. The hypothesis of a functional switch of the enzyme between two states with better efficiency either in ATP synthesis or in ATP hydrolysis is also examined. A brief survey is made on some problems complicating quantitative studies of energy coupling, such as localized chemiosmosis, delta pH and delta phi computations, and scalar ATPases. The main data on the enzyme activation and the energy-dependent release of tightly bound nucleotides are summarized. The arguments for and against the catalytic competence of theses nucleotides are reviewed. Lastly, some prevailing models of the catalytic mechanism are presented. The relevance of nucleotides binding change events in this process is discussed.     

Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase

The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.     

Obligatory coupling between proton entry and the synthesis of adenosine 5''-triphosphate in Streptococcus lactis.

Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.     

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.     

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

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.     

DCCD sensitivity of electron and proton transfer by NADH: ubiquinone oxidoreductase in bovine heart submitochondrial particles--a thermodynamic approach

The sensitivity of the H+/2e- ratio of the redox-driven proton pumping by the NADH: ubiquinone reductase (complex I) of the submitochondrial particles to dicyclohexylcarbodiimide (DCCD) was studied by a thermodynamic approach, measuring the membrane potential and delta pH across the membrane and the redox potential difference across the complex I span of the respiratory chain. The delta Gr/delta muH+ ratio did not decrease upon additions of 50 or 100 nmol of DCCD per mg protein in the presence of oligomycin although the H+/2e- ratio has been demonstrated to decrease upon DCCD addition in kinetic experiments with mitochondria. Complex I then becomes reminiscent of the cytochrome bc1 complex, which shows DCCD sensitivity of the kinetically but not thermodynamically determined H+/2e- ratio.     

Mitochondrial transhydrogenase: general principles of functioning]

A new mechanism for the functioning of mitochondrial transhydrogenase has been proposed. This mechanism makes it possible, without additional postulates, to explain the generation of delta muH+ of different signs in the forward and reverse transhydrogenase reactions and why this generation is not accompanied by the membrane uncoupling. It is suggested that the reduced nicotinamide rings of NADH and NADPH participate in a relay transfer of H+ ions across the membrane, while the oxidized nicotinamide rings of NAD+ and NADP+ block the H+-transporting paths in the transhydrogenase.     

Double-inhibitor and uncoupler-inhibitor titrations. 1. Analysis with a linear model of chemiosmotic energy coupling

The results of double-inhibitor and uncoupler-inhibitor titrations have been simulated and analyzed with a linear model of delocalized protonic coupling using linear nonequilibrium thermodynamics. A detailed analysis of the changes of the intermediate delta muH induced by different combinations of inhibitors of the proton pumps has been performed. It is shown that with linear flow-force relationships the published experimental results of uncoupler-inhibitor titrations are not necessarily inconsistent with, and those of double-inhibitor titrations are inconsistent with, a delocalized chemiosmotic model of energy coupling in the presence of a negligible leak. Also shown and discussed are how the results are affected by a nonnegligible leak and to what extent the shape of the titration curves can be used to discriminate between localized and delocalized mechanisms of energy coupling.     

A second mechanism of respiratory control

According to the chemosmotic hypothesis, ATP is synthesized in mitochondria, bacteria and chloroplasts via the proton motive force delta p, the energy-rich intermediate of electron transport and photosynthetic phosphorylation. The general applicability of the chemosmotic hypothesis, however, was disputed until present. In particular the relationship between the rate of respiration and delta p in mitochondria was found variable, depending on the experimental conditions. Recently, a new mechanism of respiratory control was found, based on binding of ATP or ADP to subunit IV of cytochrome c oxidase, which is independent of delta p and could explain many previous results contradicting the chemosmotic hypothesis.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.     

H+/ATP stoichiometry of proton pump of turtle urinary bladder

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.     

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)