H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

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Abstract
The stoichiometry of H+ ejection by [rat liver] mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) was reexamined in the light of the recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. Earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scalar H+ production. A modified method for determination of this ratio is described which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and Pi, and 1 H+ returns through the combined action of the Pi and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to 1/3 of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 H+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.