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

In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H(+) are rejected per pair of electrons passing each of the three energy-conserving sites of the 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 was 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 oxygen 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 per 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 was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H(+) equivalents are ejected per pair of electrons passing each energy-conserving site; three 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 ATP(4-) in exchange for extramitochondrial ADP(3-), via the H(+)/H(2)PO(4) (-) symporter.