Fatty acids as uncouplers of oxidative phosphorylation

This review summarizes data on the uncoupling effect of fatty acids on oxidative phosphorylation in mitochondria of various animal and plant tissues.     

Complexes between uncouplers of oxidative phosphorylation

Summary We have examined the effects of two weak acid uncouplers of oxidative phosphorylation, 2,4-dinitrophenol and 5,6-dichloro-2-trifluoromethyl-benzimidazole, on the electrical properties of phospholipid bilayer membranes. All the effects they produce are consistent with the charged permeant species being a HA ₂ ^– complex formed between the neutral acid HA and its anion A^– and the current in the aqueous phases being carried by buffered hydrogen ions. When both uncouplers are present simultaneously, all the evidence we have obtained is consistent with the charged permeant species being a HAB^– complex formed between the neutral acid HA of one uncoupler and the anion B^– of the other. It was necessary, however, to take into account interfacial processes and the unstirred layers adjacent to the membrane, the adsorption of anions to the bilayer and the buffer level in the aqueous phases to explain the results in terms of this model. The degree to which these factors will complicate a comparison of results obtained on artificial systems and mitochondria is also discussed.     

Quantitative structure-activity relationship of carbonylcyanide phenylhydrazones as uncouplers of mitochondrial oxidative phosphorylation

The dependence of the uncoupling activity in the series of 16 carbonylcyanide phenylhydrazones on their physico-chemical properties (partition coefficient, dissociation constant and rate constant for reaction with thiols) is investigated using two physiologically based models, one for protonophoric mechanism of uncoupling and the other assuming the covalent modification of a membrane constituent to be the key step in this process. As indicated by uptake experiments, at the given conditions a lipophilic-hydrophilic equilibrium is attained without any loss of the compounds via chemical reactions. Using this fact to reduce the number of adjustable parameters, a better fit to the data on stimulation of respiration is obtained with the former (protonophoric) model.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

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.     

The monophenolic metabolites of the herbicide 2,6-dichlorobenzonitrile in animals as uncouplers of oxidative phosphorylation

1. Both monophenolic metabolites of 2,6-dichlorobenzonitrile (2,6-dichloro-3-hydroxybenzonitrile and its 4-hydroxy analogue) added to starved yeast cells incubated with a limited quantity of glucose cause a significant rise in oxygen consumption of the cells. 2. The same compounds induce adenosine-triphosphatase activity in isolated intact rat-liver mitochondria. 3. The possible role of the hydroxylation of 2,6-dichlorobenzonitrile in mammals in relation to hepatic injury is discussed.     

Mutants of Bacillus megaterium resistant to uncouplers of oxidative phosphorylation.

Mutants of Bacillus megaterium displaying malate-stimulated ATP synthesis resistant to uncouplers of oxidative phosphorylation were isolated and partially characterized. ATP synthesis in such mutants was resistant to carbonyl cyanide m-chlorophenyl hydrazone as well as to other uncouplers including 2,4-dinitrophenol, pentachlorophenol, and sodium azide. ATP synthesis in the wild type and in resistant mutants was sensitive to N,N'-dicyclohexylcarbodiimide, tributyltin, valinomycin plus potassium, and potassium cyanide. Active transport of glycine and glutamine which are sensitive to uncouplers in the wild type was also uncoupler-sensitive in the mutants.     

Physiological adaptation of Euglena gracilis to uncouplers and inhibitors of oxidative phosphorylation

Cells of Euglena gracilis which have adapted to growth in the presence of 2,4-dinitrophenol are at the same time resistant to the inhibitory effect on growth of carbonylcyanide m-chlorophenylhydrazone, 1,7-hexafluoro-2,6-dihydroxy-2,6-bis(trifluoromethyl)-4 hep-tanone (“1799”), oligomycin, and tri-n-butylchlorotin. Cells adapted to any of the latter are at the same time resistant to dinitrophenol.The adaptation is a response to the primary effects of the uncouplers, i.e., high levels of ADP, and not to the uncouplers per se, since arsenate induced uncoupler resistance while nonuncoupling substituted phenols did not.No evidence for any protein in the cells which can bind uncouplers tightly could be found, and no modification of reactions of isolated mitochondria could be detected. Substrate utilization by adapted cells was only slightly less efficient than in normal cells.The inhibitors were accumulated by and not excluded from the cells. Thus, the cross-resistance implies either the formation of a modified mitochondrial membrane, impermeable to these hydrophobic effectors, or an alternative or greatly modified pathway for ATP synthesis, insensitive to these effectors.     

The effect of uncouplers of oxidative phosphorylation on lipid bilayer membranes: Carbonylcyanidem-chlorophenylhydrazone

Summary Detailed experimental data for conductivity and membrane potentials are presented for lecithin/cholesterol/decane bilayers in the presence of the uncoupler carbonylcyanidem-chlorophenylhydrazone (CCCP). These compare favorably with a theoretical model derived to explain the mechanism of action of uncouplers on bilayers. The model assumes that the weak acid uncoupler HA and its anion A⁻ are the sole species which permeate the membrane. Its key feature is the recognition of the existence of unstirred aqueous layers on either side of the membrane. The model accounts for, among other things, a maximum in the transmembrane conductivity at a pH to the alkaline side of the uncoupler pK _a and saturating current-voltage characteristics at high pH, both phenomena being found for CCCP. From a quantitative fit of model to data, values of 2.0×10⁻³ and 11 cm/sec are deduced for the permeability coefficients of the CCCP anion and the undissociated CCCP molecule, respectively.