Almitrine, a new kind of energy-transduction inhibitor acting on mitochondrial ATP synthase

At low concentrations, almitrine inhibits yeast cell multiplication by acting on oxidative metabolism. Studies on isolated mitochondria display the following features: (i) almitrine inhibits ATPase activity and decreases ATP/O ratio during oxidative phosphorylation; (ii) no direct effect on respiration can be evidenced; (iii) ATP/O value decreases without any change in the magnitude of delta p; (iv) the higher the ATP synthesis and respiratory fluxes, the larger is the decrease in ATP/O ratio induced by almitrine. These results indicate that almitrine does not act as a classical protonophoric uncoupler nor as previously studied non protonophoric uncouplers (e.g., general anesthetics). Our data show a direct inhibitory effect of almitrine on ATPase-ATP synthase complex. But, in contrast to the classical inhibitors of this complex, almitrine decreases the ATP/O ratio in a flux-dependent manner. Thus, almitrine could induce either an intrinsic uncoupling of H+/-ATPase (i.e., slip in this proton pump) or a change in the mechanistic H+/ATP stoichiometry at the ATPase level.     

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.     

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.     

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

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

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

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.     

Regulation of ATP hydrolysis in hepatoma 22a mitochondria.

A decrease in the rate of ATP hydrolysis was observed after preincubation of intact mitochondria from hepatoma 22a with an uncoupler. This effect is due both to a decrease in the rate of ATP transport and to an inactivation of the F0F1-ATPase. The former effect is shown to result from an uncoupler-induced ADP efflux. In de-energized mitochondria from hepatoma (but not from mice liver), the concentration of adenine nucleotides in the matrix equilibrates with the medium concentration via a carboxyatractyloside (CATR)-insensitive transport system. CATR-insensitive accumulation of medium ADP and stoichiometric exchange of added ATP are observed in energized hepatoma mitochondria. The dependence of the uncoupler-induced inactivation of ATPase activity on delta mu H+, pH, and ATP is consistent with the effect being caused by the natural protein inhibitor (IF1) of F0F1. ATP- and pH-dependent inactivation of the enzyme is also observed after disruption of mitochondria with the detergent Lubrol-WX. Almost all F0F1 in hepatoma mitochondria have IF1 bound in a noninhibitory manner. In the presence of uncoupler, this complex converts, via a reversible pH-dependent and an irreversible ATP-dependent process, to an inhibitory complex. The pH-dependent step can be blocked by Zn2+ and Cd2+ ions which probably bind to negatively charged residues on IF1, thereby preventing their protonation and conversion of the protein to an inhibitory conformation.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.     

H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.     

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

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

Intrinsic and extrinsic uncoupling of oxidative phosphorylation

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H(+)/e(-) stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential DeltaPsi, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential DeltaPsi and maintains high DeltaPsi values (150-200 mV). The second occurs only in mitochondria, is suggested to keep DeltaPsi at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of DeltaPsi and the production of reactive oxygen species (ROS) in mitochondria at high DeltaPsi values (150-200 mV) are discussed.     

Further comments on the logic of the application of uncoupler- inhibitor titrations for the elucidation of the mechanisms of energy coupling

Following the appearance of two papers in this journal commenting on the logic of the application of uncoupler-inhibitor titrations as a means of discriminating between 'delocalized' and 'localized' chemiosmotic mechanisms [(1984) FEBS Lett. 176,79-82; (1985) FEBS Lett. 186, 8-10], and in contrast with the arguments presented there and elsewhere, we show that in a linear model the increase in delta mu H which accompanies partial inhibition of the ATPases always leads to a relatively higher decrease of the rate of ATP synthesis by a given concentration of uncoupler in the presence of an ATPase inhibitor than in its absence. This is due to the fact that the same titre of uncoupler leads to a higher dissipative H+ flow in the presence of inhibitor, since the driving force delta mu H is higher.     

Thermodynamic control of electron flux through mitochondrial cytochrome bc1 complex.

The redox states of exogenously added ubiquinone-2 and cytochrome c, and the protonmotive force (delta p) of rat liver mitochondria were measured as the respiration rate was titrated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone. The force ratio delta Eh/delta p across the bc1 complex was close to 1:1 in State 4, indicating an H+/e- stoichiometry of 1:1 for the cytochrome bc1 complex, excluding protons moved by pool ubiquinone. Assuming a constant stoichiometry the rate of electron transport increased linearly with the disequilibrium (delta Eh - delta p) across the complex.     

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.     

Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria

Free fatty acids (FFA) are known to uncouple oxidative phosphorylation in mitochondria. However, their mechanism of action has not been elucidated as yet. In this study we have investigated in detail the patterns of uncoupling by the FFA oleate and palmitate in rat liver mitochondria and submitochondrial particles. The patterns of uncoupling by FFA were compared to uncoupling induced by the ionophores valinomycin (in the presence of K+) and gramicidin (in the presence of Na+) and the proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP). The most striking difference in the pattern of uncoupling relates to the effect on the proton electrochemical potential gradient, delta mu H. Uncoupling by ionophores, particularly valinomycin, is associated with and most likely caused by a major reduction of delta mu H. In contrast, uncoupling by FFA is not associated with a significant reduction of delta mu H, indicating another mechanism of uncoupling. We suggest the use of the term decouplers for uncoupling agents such as FFA and general anesthetics that do not collapse the delta mu H [Rottenberg, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3313-3317]. The protonophore CCCP and to some extent the ionophore gramicidin indicate a mixed mode of uncoupling since their effect on delta mu H is moderate when compared to that of valinomycin. Another distinguishing feature of uncouplers that collapse the delta mu H is their ability to stimulate ADP-stimulated respiration (state 3) further. Decouplers such as FFA and general anesthetics do not stimulate state 3 respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unique action of a modified weakly acidic uncoupler without an acidic group, methylated SF 6847, as an inhibitor of oxidative phosphorylation with no uncoupling activity: possible identity of uncoupler binding protein

The potent weakly acidic uncoupler SF 6847 was modified by methylation of its phenolic OH group, and the effect of the resulting derivative, with no acid-dissociable group, on oxidative phosphorylation in rat liver mitochondria was examined. The methylated SF 6847 did not induce uncoupling at up to 40 microM, while SF 6847 uncoupled oxidative phosphorylation completely at about 20 nM, indicating that the acid-dissociable group is essential for uncoupling. The O-methylated SF 6847 at 20 microM did, however, inhibit state 3 respiration of mitochondria, although it did not inhibit electron-flow through the respiratory chain, ATPase activated by weakly acidic uncouplers or Pi-ATP exchange. At the same concentration, it also inhibited ATP synthesis in submitochondrial particles. These features are different from those of known inhibitors of oxidative phosphorylation. Thus, O-methylated SF 6847 is a unique inhibitor of oxidative phosphorylation. The possible identity of the uncoupler binding protein is discussed on the basis of these results.