Quantitative relationship between protonophoric and uncoupling activities of analogs of SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol)

Uncoupling activity with rat liver mitochondria and protonophoric activity across the lecithin liposomal membranes were measured for a series of non-classical uncouplers related to the most potent uncoupler known until now, SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol). The correlation between uncoupling and protonophoric activities for a number of uncouplers, both non-classical and classical (simply substituted phenols), was examined quantitatively. Correlation was excellent when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which are structurally different, were correlated in a way that resembled the correlation of phenolic compounds, so we think that the mode of action of weakly acidic uncouplers was the same regardless of the structural type. Our findings were evidence for the shuttle-type mechanism of uncoupling action.     

Quantitative relationship between protonophoric and uncoupling activities of analogs of SF6847 (2,6-di-t-butyl-4-(2′,2′-dicyanovinyl)phenol)

Uncoupling activity with rat liver mitochondria and protonophoric activity across the lecithin liposomal membranes were measured for a series of non-classical uncouplers related to the most potent uncoupler known until now, SF6847 (2,6-di-t-butyl-4-(2′,2′-dicyanovinyl)phenol). The correlation between uncoupling and protonophoric activities for a number of uncouplers, both non-classical and classical (simply substituted phenols), was examined quantitatively. Correlation was excellent when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which are structurally different, were correlated in a way that resembled the correlation of phenolic compounds, so we think that the mode of action of weakly acidic uncouplers was the same regardless of the structural type. Our findings were evidence for the shuttle-type mechanism of uncoupling action.     

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.     

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 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.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile; a new powerful uncoupler of respiratory-chain phosphorylation

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) was found to be one of the most powerful uncouplers of respiratory-chain phosphorylation ever reported. Structure-activity studies of the derivatives indicated the role of three functional groups in SF 6847: (1) the malononitrile group as an electron withdrawing group, which may cause uncoupling by interaction with a primary energy conservation site, (ii) a free hydroxyl group as an electron donator, and (iii) bulky tertiary butyl groups of hydrophobic character located at a certain spacial distance from the electron withdrawing center.     

Uncouplers enhance photosynthetic electron transport from water to NADP+ in the presence of plastoquinone inhibitors

Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP⁺ by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP⁺ reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H₂O to NADP⁺) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.     

On the stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile).

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies. Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S13) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

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.     

Quantitative analyses of the uncoupling activity of substituted phenols with mitochondria from flight muscles of house flies

Uncoupling activity with flight-muscle mitochondria from house flies was measured for a series of weakly acidic uncouplers (substituted phenols) and compared with the protonophoric potency across lecithin liposomal membranes. The activity was linearly related to the protonophoric potency when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Relationships of the flight-muscle activity with activities measured previously with rat-liver mitochondria and spinach chloroplasts were linear. Our findings were further evidence for the shuttle-type mechanism of the uncoupling action of weakly acidic uncouplers.     

Effect of arylidene-cyclopentenedione radiosensitizers on ATP synthesis in mitochondria: action as potent inhibitors of phosphate transport

The effects of the arylidene-cyclopentenedione radiosensitizers, KIH-200, 201 and 202 on ATP synthesis in mitochondria were examined. In spite of the close similarity of their chemical structure to that of the most potent known weakly acidic uncoupler, SF 6847, they did not show any uncoupling activity at concentrations of up to 50 microM. However, these three compounds were found to have very potent inhibitory effects on Pi-transport into mitochondria, all causing 50% inhibition (I50%) at about 7 microM. Thus they are much more potent than the commonly used Pi-transport inhibitors N-ethylmaleimide (I50% = about 40 microM), and mersalyl (I50% = about 30 microM). They may act as SH-reagents, and inhibit Pi-transport by modifying an SH-group(s) in the Pi-transporter.     

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris

Various physicochemical and biochemical properties of the most potent uncoupler of oxidative phosphorylation known to date 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847), such as pH dependence of the uncoupling activity and binding to mitochondria, spectral properties in the presence of different types of liposomes, biopolymers and mitochondria, and effects on model membrane systems have been investigated. From the results, it is concluded that the uncoupler most likely is localized in the phospholipid part of the membrane.     

Inhibition of the mitochondrial respiratory chain by alkylhydroxynaphthoquines: Reversal on discharge of the energized state

Inhibition of mitochondrial respiration by alkylhydroxynaphthoquinones may be reversed by addition of a variety of uncouplers including substituted phenols, carbonyl cyanide phenylhydrazones, divalent cations and univalent cations in the presence of ionophoretic antibiotics. A likely explanation for such reversibility is the requirement that the anionic inhibitor be transported to a site of action within the mitochondrion. Support for this view includes (1) failure to obtain reversal of inhibition with submitochondrial particles, (2) release of inhibition by a competing anion, succinate, (3) augmentation of inhibition when a divalent cation is taken up, (4) the chemical diversity of uncouplers that release inhibition and (5) inhibiton by uncoupling compounds of the uptake of labeled alkylhydroxynaphthoquinones. It is suggested that a similar explanation may apply to two other inhibitors of the cytochrome b–c region, antimycin and alkylhydroxyquinoline-N-oxides.     

Mitochondrial uncouplers with an extraordinary dynamic range

We have discovered that some weak uncouplers (typified by butylated hydroxytoluene) have a dynamic range of more than 10(6) in vitro: the concentration giving measurable uncoupling is less than one millionth of the concentration causing full uncoupling. They achieve this through a high-affinity interaction with the mitochondrial adenine nucleotide translocase that causes significant but limited uncoupling at extremely low uncoupler concentrations, together with more conventional uncoupling at much higher concentrations. Uncoupling at the translocase is not by a conventional weak acid/anion cycling mechanism since it is also caused by substituted triphenylphosphonium molecules, which are not anionic and cannot protonate. Covalent attachment of the uncoupler to a mitochondrially targeted hydrophobic cation sensitizes it to membrane potential, giving a small additional effect. The wide dynamic range of these uncouplers in isolated mitochondria and intact cells reveals a novel allosteric activation of proton transport through the adenine nucleotide translocase and provides a promising starting point for designing safer uncouplers for obesity therapy.     

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.     

Uncoupling activity and physicochemical properties of derivatives of fluazinam.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.     

Localized energy coupling during photophosphorylation by chromatophores ofRhodopseudomonas capsulata N22

The principle of the dual inhibitor titration method for testing models of electron-transport phosphorylation is outlined, and the method is applied to the study of photophosphorylation in bacterial chromatophores. It is concluded that energy coupling is strictly localized in nature in this system, in the sense that free energy released by a particular electron-transport chain may be used only by a particular H⁺-ATP synthase. Dual inhibitor titrations using the uncoupler SF 6847 and the H⁺-ATP synthase inhibitor oligomycin indicate that uncouplers act by shuttling rapidly between the localized energy-coupling sites.     

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.     

Mechanism of uncoupling by uncouples of oxidative phosphorylation

Classical uncouplers duplicate exactly the uncoupling actions of the valinomycin-nigericin ionophoric combination in presence of K⁺ — a combination that mediates cyclical transport of K⁺ driven by electron transfer or pyrophosphorolysis of ATP in mitochondria. Evidence has been presented that uncouplers have the properties essential for mediating coupled cyclical transport of cations and that uncoupling of oxidative phosphorylation can be rationalized in terms of one coupled process being displaced and replaced by another. The critical demonstrations were first that uncoupling is a cation-dependent process and that only those cations that can undergo complexation with uncouplers are effective in restoring mitochondrial uncoupler action in a cation-deficient medium. The second demonstration was that uncouplers are ionophores, not only of the nigericin type but also of the valinomycin type (electrogenic). This combination in one molecule of electrogenic as well as non-electrogenic ionophoric activity for cations endows uncouplers with the capability for duplicating the uncoupling action of the valinomycin-nigericin combination and for mediating coupled cyclical transport of cations.     

Organizational changes in phospholipid multibilayers induced by uncouplers of oxidative phosphorylation: a spin label study

A cholestane spin probe was used to study the effect of uncouplers of oxidative phosphorylation (2,4-dinitrophenol, pentachlorophenol and dicumarol) on the degree of organization of phospholipids in hydrated multibilayers. Disruptive effects were observed—their magnitude depending on pH, time and the presence of cholesterol. A correlation between changes in probe organization and ion conductivity, with maximum effects at the pH corresponding to the pK of the uncoupler, could be demonstrated in the films containing cholesterol. Egg lecithin films containing no cholesterol were disordered maximally at pH 4.0 irrespective of the uncoupler used. The effect of uncouplers on the probe disorganization varied with time after exposure. These time effects indicated that relative movement of uncoupler, probe and lipid molecules occur to produce lipid organizations differing from those after initial exposure to uncoupler. The results show that even in a simple model system uncoupler effects may be complex, and suggest that changes in bilayer lipid organization parameters may play a role in uncoupling oxidative phosphorylation.