Interaction of uncouplers with the mitochondrial membrane: a high-affinity binding site

2-Nitro-4-azidocarbonylcyanide phenylhydrazone (N₃CCP), a potent water-soluble uncoupler at pH 6–8, was used to determine the nature of binding of the uncoupler to the mitochondrial membrane. Equilibrium binding studies with N₃CCP showed that isolated pigeon heart mitochondria contain 1.6 ± 0.3 high-affinity binding sites per cytochrome a. Several different types of chemical uncouplers were also found to bind to the same high-affinity site as evidenced by their observed competition with N₃CCP. The potassium ionophore valinomycin and the respiratory inhibitor antimycin A did not affect uncoupler binding to the high-affinity sites nor did active respiration of the mitochondria. The number of high-affinity binding sites was essentially unchanged by extraction of 80% of the mitochondrial phospholipids. The ability of the uncouplers to bind to the high-affinity binding sites is proportional to the uncoupler activities. These data support the idea that the high-affinity binding sites of mitochondria are protein(s) which are involved in the coupling reactions of oxidative phosphorylation and that uncoupler bound at these sites is responsible for the uncoupling activity.     

Membrane bioenergetic parameters in uncoupler-resistant mutants of Bacillus megaterium.

Mutants of Bacillus megaterium displaying malate-driven ATP synthesis resistant to uncouplers of oxidative posphorylation are further characterized. Both the pH gradient and electrical potential generated across the membrane by malate respiration are equally sensitive to uncouplers in the wild type and uncoupler-resistant mutants. The mutants possess 0 to 10% of the wild type ATPase activity which is not activated by pretreatment with heat or trypsin. Despite this inability to measure ATPase activity, the mutants demonstrate acid-pulse-driven ATPase synthesis which is sensitive to uncouplers as well as malate-driven ATP synthesis which becomes uncoupler sensitive at pH 5.5. N,N' -Dicyclohexylcarbodiimide and valinomycin plus potassium inhibition of ATP synthesis is reversed by uncouplers in the mutants but not in the wild type. The data support the existence of a specific site on the ATPase complex for uncoupler binding which, if altered by mutation, affects uncoupler binding to the complex. The retention of malate-driven ATP synthesis in the absence of a significant pH gradient or electrical potential suggests that an alternative intermediate is involved in coupling oxidation to phosphorylation.     

Energy conservation and uncoupling in mitochondria

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of M_r = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.     

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.     

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N3CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membrane of Paracoccus denitrificans and Tetrahymena pyriformis. The N3CCP uncouples respiration in P. denitrificans and T. pyriformis cells with U1/2 values of 1.05 microM and 0.24 microM, respectively. Binding studies show the presence of 0.65 +/- 0.05 high affinity sites per cytochrome alpha with Kd of 0.5 +/- 0.1 microM in P. denitrificans membranes and 1.4 +/- 0.2 sites per cytochrome alpha 2 with a Kd of 0.4 +/- 0.1 microM in T. pyriformis membranes. Irradiation of [3H]-N3CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10--15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647--656).     

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.

The maximal capacities of the cytochrome and alternate paths, V_cyt and V_alt, respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V_alt decreases slightly with ripening.

The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.

Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

     

Electron transport pathways for the oxidation of endogenous substrate(s) in Acidithiobacillus ferrooxidans

Oxidation of endogenous substrate(s) of Acidithiobacillus ferrooxidans with O2 or Fe3+ as electron acceptor was studied in the presence of uncouplers and electron transport inhibitors. Endogenous substrate was oxidized with a respiratory quotient (CO2 produced/O2 consumed) of 1.0, indicating its carbohydrate nature. The oxidation was inhibited by complex I inhibitors (rotenone, amytal, and piericidin A) only partially, but piericidin A inhibited the oxidation with Fe3+ nearly completely. The oxidation was stimulated by uncouplers, and the stimulated activity was more sensitive to inhibition by complex I inhibitors. HQNO (2-heptyl-4-hydroxyquinoline N-oxide) also stimulated the oxidation, and the stimulated respiration was more sensitive to KCN inhibition than uncoupler stimulated respiration. Fructose, among 20 sugars and sugar alcohols including glucose and mannose, was oxidized with a CO2/O2 ratio of 1.0 by the organism. Iron chelators in general stimulated endogenous respiration, but some of them reduced Fe3+ chemically, introducing complications. The results are discussed in view of a branched electron transport system of the organism and its possible control.     

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.     

Thyroxine Reversibly Inhibits the Uncoupling Action of Protonophores on Energy Production in Rat Thymus Lymphocytes

Earlier we reported that some thyroid and steroid hormones and also 6-ketocholestanol used in micromolar concentrations modulated the effects of protonophoric uncouplers on isolated mitochondria (Starkov et al. (1997) Biochim. Biophys. Acta, 1318, 173-183). In the present study we investigated the effects of a thyroid hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and mitochondria isolated from these cells. The resting (oligomycin-inhibited) respiration of the isolated intact lymphocytes was stimulated by the addition of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of micromolar concentrations of thyroxin decreased the rate of uncoupler-stimulated respiration and partially reversed uncoupler-induced decrease of membrane potential (DY). In experiments with mitochondria isolated from thymus lymphocytes the re-coupling effect of thyroxine was not observed. In this case thyroxine did not influence mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation of respiration and decrease induced with another uncoupler, SF6847. The data are discussed in terms of a hypothesis that aromatic uncouplers are transported into the cell by the thyroxine carrier of the plasma membrane.     

Inhibition of bacterial transport by uncouplers of oxidative phosphorylation. Effects of pentachlorophenol and analogues in Bacillus subtilis.

Analogues of the potent uncoupler of oxidative phosphorylation pentachlorophenol were tested as inhibitors of proline and glycine transport by Bacillus subtilis. These analogues included less highly substituted chlorophenols and pentachlorothiophenol. Like pentachlorophenol, they are non-competitive inhibitors of proline transport and uncompetitive inhibitors of glycine transport. However, the less highly substituted chlorophenols are weaker acids than pentachlorophenol and also weaker inhibitors. Analysis indicated that the anionic form of the uncouplers is the inhibiting species. Pentachlorothiophenol, a water-insoluble anion, is also a potent inhibitor. These results support previous studies that concluded that uncouplers of oxidative phosphorylation inhibit amino acid transport by binding at specific sites on proteins, the free energy of interaction stabilizing 'unproductive' conformations. Such specific interactions of uncoupler with protein are probably commonplace.     

Mitochondrial ATP-Pi exchange complex

An enzyme complex with high ATP-Pi exchange activity has been purified from beef heart mitochondria, using the general procedure which also yields electron transfer complexes I, II, III and IV from the same batch of mitochondria. The ATP-Pi exchange activity of the preparation, designated complex V, is inhibited by various uncouplers, rutamycin, venturicidin, dicyclohexylcarbodiimide, arsenate, azide, adenylyl imidodiphosphate, and valinomycin plus potassium. The ATP-Pi exchange activity of complex V is specific with respect to ATP; ITP, GTP and UTP are essentially ineffective. Complex V is deficient in cytochromes, but 2–3 times enriched as compared to mitochondria with respect to binding sites for the uncoupler 2-azido-4-nitrophenol. As in mitochondria, this binding is competitively inhibited by other uncouplers. Complexes I, III and IV, which in mitochondria contain the three energy coupling sites, do not bind the above uncoupler.     

Changes in interfacial potentials induced by carbonylcyanide phenylhydrazone uncouplers: possible role in inhibition of mitochondrial oxygen consumption and other transport processes

The charged and uncharged forms of carbonylcyanide phenylhydrazone uncouplers bind to phosphatidylcholine monolayers in a dose-dependent fashion, inducing changes in the interfacial potential of these model membranes. The interfacial potential change produced by the charged uncoupler is composed of a double-layer potential and an internal electrostatic potential (boundary and/or dipole). Changes in double-layer potential induced by the uncouplers in mitochondrial membranes can explain both the inhibition of oxygen consumption (QO2) caused by the uncouplers and the competition shown by succinate when mitochondria are respiring in the presence of rotenone. From these results and from dose-response curves of QO2 versus uncoupler concentrations, we conclude that 1 microM is an upper limit for free uncoupler concentration in the medium to avoid unwanted side effects during cell physiology studies that require total mitochondrial uncoupling.     

Chemical modification of mitochondria. Uncoupler binding by mitochondria in different metabolic states

At low uncoupler concentrations the binding of carbonyl-cyanide-m-chlorophenyl-hydrazone to mitochondria was found to depend sensitively on the metabolic state of mitochondria. The binding data are consistent with the assumption that at low concentrations and pH 7.4 the uncoupler is bound mainly in anionic form to the inner mitochondrial membrane and that upon energization the inner membrane undergoes conformation change, exposes buried ionizable groups and hence acquires a negative net membrane charge. Deenergization of the inner membrane by a small amount of uncoupler removes the negative net membrane charge and consequently increases the apparent binding constants. Based upon the present results on uncoupler binding and previous observations on the physiological properties of alkylating uncouplers, a possible molecular mechanism involving electron carriers and coupling factors is suggested for coupling electron transport to phosphorylation.     

On the extent of localization of the energized membrane state in chromatophores from Rhodopseudomonas capsulata N22.

1. The principle of the double-inhibitor titration method for assessing competing models of electron transport phosphorylation is expounded. 2. This principle is applied to photophosphorylation by chromatophores from Rhodopseudomonas capsulata N22. 3. It is found that, in contrast to the predictions of the chemiosmotic coupling model, free energy transfer is confined to individual electron transport chain and ATP synthase complexes. 4. This conclusion is not weakened by arguments concerning, the degree of uncoupling in the native chromatophore preparation or the relative number of electron transport chain and ATP synthase complexes present. 5. Photophosphorylation is completely inhibited by the uncoupler SF 6847 at a concentration corresponding to 0.31 molecules per electron transport chain. 6. The apparent paradox is solved by the proposal, consistent with the available evidence on the mode of action of uncouplers, that uncoupler binding causes a co-operative conformation transition in the chromatophore membrane, which leads to uncoupling and which is not present in the absence of uncoupler.     

Uncoupler-resistant mutants of bacteria.

The chemiosmotic model of energy transduction offers a satisfying and widely confirmed understanding of the action of uncouplers on such processes as oxidative phosphorylation; the uncoupler, by facilitating the transmembrane movement of protons or other compensatory ions, reduces the electrochemical proton gradient that is posited as the energy intermediate for many kinds of bioenergetic work. In connection with this formulation, uncoupler-resistant mutants of bacteria that neither exclude nor inactivate these agents represent a bioenergetic puzzle. Uncoupler-resistant mutants of aerobic Bacillus species are, in fact, membrane lipid mutants with bioenergetic properties that are indeed challenging in connection with the chemiosmotic model. By contrast, uncoupler-resistant mutants of Escherichia coli probably exclude uncouplers, sometimes only under rather specific conditions. Related phenomena in eucaryotic and procaryotic systems, as well as various observations on uncouplers, decouplers, and certain other membrane-active agents, are also briefly considered.     

Reduction in excess sludge production by addition of chemical uncouplers in activated sludge batch cultures

AIMS: To investigate the possibility of reducing excess sludge production in activated sludge processes by the addition of chemical uncouplers to greatly dissociate anabolism from catabolism. METHODS AND RESULTS: Ortho-chlorophenol (oCP), 2,4-dichlorophenol (DCP), 3,3',4',5-tetrachlorosalicylanilide (TCS), para-dinitrophenol (pNP) and 2,4-dinitrophenol (DNP) were chosen for short-term tests for their ability to reduce sludge yield by shaking bottle test. The most effective chemicals, DNP and pNP, together with TCS were tested for various uncoupler concentrations and biomass concentrations. TCS was tested in a lab-scale completely mixed activated sludge batch culture. The model (demonstrated by Liu) was verified with experimental data in completely mixed activated sludge batch test, but was inconsistent with the results from the shaking bottle batch test. The observed growth yield (Yobs) decreased with increasing of the ratio of initial uncoupler concentration to initial biomass concentration (Cu/X0). CONCLUSIONS: We suggest that the uncouplers oCP, DCP, TCS, pNP and DNP can cause a significant decrease in sludge production, the metabolism of which can explain the decline in sludge yield. SIGNIFICANCE AND IMPACT OF THE STUDY: The real strength of chemical uncoupler imposing on biomass should be Cu/X0, not initial uncoupler concentration (Cu) alone. Chemical uncouplers can be used to develop the activated sludge processes for minimizing excess sludge production.     

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.     

Interaction of respiration and luminescence in a common marine bacterium

Investigators have proposed for some time that bacterial luciferase forms a shunt around the pathway of respiratory electron transport. Certain physiologic evidence for coupling between luminescence and respiration has supported such a view. In this study, Vibrio harveyi cells were monitored for luminescent responses to artificial manipulation of respiratory electron flow. The effects of cyanide under aerobic and anaerobic conditions confirmed that luminescence and respiration compete for oxygen. The effects of an uncoupler of oxidative phosphorylation indicated that luminescence and respiration compete for a common reductant. Treatment with uncoupler also induced aldehyde deficiency in vivo.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Tris tris(hydroxymethyl) aminomethane