An ATP/2e-stoichiometry of 1 1/2 is thermodynamically possible for site 3 of oxidative phosphorylation

Free energy changes for ATP synthesis (delta GP) and 2e(-)-transfer across Site 3 (delta GR) were determined during oxidative phosphorylation by rat liver mitochondria. At static head, -delta GR/delta GP ranged narrowly between 1.55 and 1.59 with five different respiratory substrates. Thus, an ATP/2e- of 1 1/2 at Site 3 is thermodynamically possible with regards to overall reactants and products. Using nonequilibrium thermodynamics, phenomenological stoichiometries were close to 1 1/2 for all substrates suggesting that ATP/2e- at Site 3 is, in fact, 1 1/2. An ATP/2e- of 1 1/2 can only be possible if H+/O is 4 for cytochrome oxidase.     

The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. An analysis of ADP-induced oxygen jumps by linear nonequilibrium thermodynamics

Uncertainty exists as to the proton stoichiometries of mitochondrial oxidative phosphorylation and consequently as to the ATP stoichiometries. In rat liver mitochondria, ADP/O ratios were determined from the total and extra oxygen consumed during ADP-stimulated respiration under conditions of quantitative conversion of ADP to ATP. For succinate, glutamate plus malate, 3-hydroxybutyrate, and 2-oxoglutarate, respectively, ADP/total O was 1.71, 2.71, 2.61, and 3.45. ADP/extra O was 2.03, 3.04, 3.23, and 4.15. The results were interpreted in terms of linear nonequilibrium thermodynamics. It was shown that ADP/extra O = Z/q where Z is the phenomenological stoichiometry and q is the degree of coupling. q was determined from the dependence of respiratory rate on delta Gp, the phosphorylation potential, and was about 0.98 for all substrates. The results were consistent with ideal ATP/O stoichiometries of 2 for succinate, 3 for glutamate plus malate, 3 or 3 1/4 for 3-hydroxybutyrate, and 4 for 2-oxoglutarate. Taking into account the oxidation-reduction free-energy changes measured across Sites 1 + 2 at static head (J.J. Lemasters, R. Grunwald, and R.K. Emaus J. Biol. Chem. 259, 3058-3063), an ideal ATP/O stoichiometry of 3 1/4 for 3-hydroxybutyrate is proposed. The lower ATP/O for glutamate plus malate is then accounted for by proton translocation linked to glutamate/aspartate exchange. The data suggest a new 13-proton scheme of chemiosmotic coupling in which proton stoichiometries are 3 for the F1Fo-ATPase, 1 for the exchange of ATP for ADP and Pi, and 5, 4, and 4 for Sites 1, 2, and 3.     

Determination of the P/2e- stoichiometries at the individual coupling sites in mitochondrial oxidative phosphorylation. Evidence for maximum values of 1.0, 0.5, and 1.0 at sites 1, 2, and 3

P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis

The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1. The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed. Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites. When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta. This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP). Hybrid constructs as CF1(-delta)+ E. coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E. coli delta does the same on EF0. Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1. Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E. coli delta on EF0. The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E. coli delta (36% conserved residues including conservative replacements). An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta.     

A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.     

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.     

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.     

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.     

Generation of adenosine triphosphate in cytochrome-deficient mutants of Neurospora.

The fungus Neurospora crassa is known to possess a branched respiratory system consisting of the standard cytochrome chain and a cyanide-insensitive alternate oxidase. In the present experiments, the physiological function of the alternate oxidase has been analyzed by taking advantage of a number of cytochrome-deficient mutants, particularly poky f. Respiration, cellular ATP levels, and growth have been examined under the influence of three classes of inhibitors: inhibitors of the cytochrome chain (antimycin, cyanide), an inhibitor of the laternate oxidase (salicyl hydroxamic acid), and an uncoupling agent (carbonyl cyanide m-chlorophenylhydrazone). The results indicate that the over-all efficiency of the alternate oxidase in producing ATP and supporting growth is much less than that of the cytochrome chain. Depending upon the amount of oxidative phosphorylation at Sites II and III in the cytochrome chain, which varies from strain to strain, the efficiency of the alternate oxidase relative to that of the cytochrome chain ranges from 13% in wild type Neurospora to 18 to 21% in poky f, 35% in mi-3, and 57% in cyt-2. A comparison of the short term effects of cyanide and carbonyl cyanide m-chlorophenylhydrazone on cellular ATP in poky f suggests that, during respiration through the alternate oxidase, ATP can be produced both by substrate-level phosphorylation (accompanying glycolysis and the oxidation of alpha-ketoglutarate) and by oxidative phosphorylation at Site I. When cells are grown on sucrose, as much as 22% of ATP synthesis in the presence of cyanide occurs at Site I. When cells are grown on acetate to diminish the rate of glycolysis, the contribution of Site I becomes proportionately larger. Both the growth experiments and the short term inhibitor experiments reveal that ATP levels in Neurospora are kept high be a feedback process which depresses ATP breakdown (and growth) very quckly after ATP synthesis is inhibited. Thus, poky f grows more slowly that wild type Neurospora and is inhibited still further when either the cytochrome chain or the alternate oxidase is blocked. Under all of these conditions, however, cellular ATP in poky f is maintained at a high level (about 3 mmol per kg of cell water, slightly above the values measured in the wild type strain). Continue.     

Escherichia coli proton-translocating F0F1-ATP synthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation-reduction under fermentation

The Escherichia coli proton-translocating F0F1-ATP synthase has a priority in H+ circulation through the membrane in maintaining proton-motive force in the context of ATP synthesis and hydrolysis. Recent advances in the study of this complex under fermentative growth have led to hypothesis that, in the absence of oxidative phosphorylation, F0F1 is implicated as an essential part of H+ movement and ATP hydrolysis, associated with solute secondary transporters and/or enzymes of anaerobic oxidation-reduction. These associations can result from a protein-protein interaction by dithiol-disulfide interchange. In such associations F0F1 has novel functions in bacterial cell physiology.     

Correlation of the turnover number of the ATP synthase in liposomes with the proton flux and the proton potential across the membrane

The fluorescent indicator pyranine was used for recording the internal pH of liposomes. The proton permeability was deduced from the velocity of the internal pH increase which was caused by shifting the external pH from 7 to 9. From valinomycin titration of the proton permeability in the presence of internal and external KCl (0.1 M), the permeability coefficient of H+ (PH) was obtained as 10(-4) cm/s at 22 degrees C. The coefficient was twice this value with the ATP synthase isolated from Wolinella succinogenes present in the liposomal membrane (10 mg protein/g phospholipid). ADP and phosphate had no effect on the latter PH. The protonophore TTFB (5 mumol/g phospholipid) increased the PH by 3 orders of magnitude. The permeability coefficients of H+ and K+ were used for calculating the delta uH and the proton flux associated with the phosphorylation which was driven by gradients of H+ and K+. For the conditions of limiting permeability of K+, the following conclusions were drawn. (1) In the steady state of rapid ion flux, the electrical potential across the liposomal membrane as calculated according to the Goldman equation, is directed opposite to the corresponding Nernst potential which is calculated from the K+ gradient. (2) The maximum turnover numbers of phosphorylation require a delta uH of 200-220 mV across the liposomal membrane. These values of delta uH and the corresponding turnover numbers are close to those brought about by the bacterial electron transport and the coupled phosphorylation. (3) The velocity of phosphorylation is linearly related to the proton flux. The slope of the line can be explained on the basis of an H+/ATP ratio of approx. 3.     

Chloride-Dependent Uncoupling of Oxidative Phosphorylation by Triethyllead and Triethyltin Increases Cytosolic Free Calcium in Guinea Pig Cerebral Cortical Synaptosomes

Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 microM TEL and 20 microM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (delta psi p) by only 3-4 mV occurs, a drop which is insufficient to open the voltage-sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (delta psi m) upon addition of TEL and TET is evident, as monitored with safranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation by TEL and TET, [corrected] a process that is dependent on external chloride, is the main mechanism by which they [corrected] increase [Ca2+]c.     

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.     

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.     

ATP synthesis in Methanobacterium thermoautotrophicum coupled to CH4 formation from H2 and CO2 in the apparent absence of an electrochemical proton potential across the cytoplasmic membrane

Methanogenic bacteria are considered to couple methane formation with the synthesis of ATP by a chemiosmotic mechanism. This hypothesis was tested with Methanobacterium thermoautotrophicum. Methane formation from H2 and CO2 (2.5 - 3 mumol X min-1 X mg cells-1) by cell suspensions of this organism resulted in the formation of an electrochemical proton potential (delta mu H +) across the cytoplasmic membrane of 230 mV (inside negative) and in the synthesis of ATP up to an intracellular concentration of 5 - 7 nmol/mg. The addition of ionophores at concentrations which completely dissipated delta mu H + without inhibiting methane formation did not result in an inhibition of ATP synthesis. It thus appears that delta mu H + across the cytoplasmic membrane is not the driving force for the synthesis of ATP in M. thermoautotrophicum.     

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.