Respiration-driven proton translocation in rat liver mitochondria

1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H⁺ (or OH⁻) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the →H⁺/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H⁺/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the →H⁺/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na⁺. These apparent changes of →H⁺/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H⁺/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H⁺/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.