Stoichiometry of vectorial H + movements coupled to electron transport and to ATP synthesis in mitochondria

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Abstract
In order to verify more directly earlier measurements showing that, on the average, close to 4 vectorial H+ are ejected/pair of electrons passing each of the 3 energy-conserving sites of the [rat liver] mitochondrial electron transport chain, direct tests of the H+/2e- ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 were examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H+/2e- ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with O2 as electron acceptor. The directly measured H+/2e- ratio for site 3 was close to 4. The H+/ATP ratio (number of vectorial H+ ejected/ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H+ ejection and ATP hydrolysis were measured in the presence of K+ + valinomycin. The H+/ATP ratio approached 3.0. A proton cycle for oxidative phosphorylation is proposed, in which 4 electrochemical H+ equivalents are ejected/pair of electrons passing each energy-conserving site; 3 of the H+ equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H+ is used to bring about the energy-requiring electrogenic expulsion of ATP4- in exchange for extramitochondrial ADP3-, via the H+/H2PO4- symporter.