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

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Abstract
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 comparsion 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. The negligible increase in the dissipative proton influx in the presence of chloroform does not account for the stimulation either of the rate of electron transfer or of ATP hydrolysis. The classical "chemiosmotic" explanation of the uncoupling of oxidative phosphorylation does not apply to the uncoupling action of chloroform.