The stoichiometry of proton translocation through H+‐ATPase of rat‐liver mitochondria

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Abstract
The ratio of the number of protons transported directly by H+-ATPase of intact mitochondria and that of the hydrolyzed ATP (the proton translocation quotient) was determined. A special kinetic method which makes possible determination of ATPase and H+-translocase activities in the same experiment was used to estimate the proton translocation quotient for mitochondrial H+-ATPase. The quotient is on average, equal to 3.31 mol/mol. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile can greatly decrease the proton translocation quotient. This supports the assumption concerning a labile coupling between H+-translocase and ATPase in mitochondria. A decrease in the translocation quotient in the presence of the protonophore is likely to be due to the ability of this uncoupler to take back the translocated protons into the mitochondrial matrix before their release into the medium. An electrostatic model of the molecular mechanism of H+-translocase and ATPase coupling in the H+-ATPase complex is discussed. The model is in agreement with the results obtained: transport of more than 2 protons per hydrolyzed ATP molecule, and variable efficiency of the process.