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 ejected 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 tetramethylphen-ylenediamine 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 ATP4- in exchange for extramitochondrial ADP3-, via the H+/H2PO4- symporter.