Acid-base titration across the membrane system of rat-liver mitochondria. Catalysis by uncouplers.

1. Pulsed acid-base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes. 2. The fast process is the titration of the outer aqueous phase of the mitochondria, which is continuous with the suspension medium, and the slow process can be identified with the titration of the inner aqueous phase of the mitochondria, which is separated from the outer aqueous phase by the non-aqueous osmotic barrier or M phase of the cristae membrane system. 3. The buffering power of the outer and inner phases have been separately measured over a range of pH values. 4. The rate of titration of the inner aqueous phase under a known protonmotive force across the M phase has been characterized by an effective proton conductance coefficient, which, near pH7 and at 25 degrees , is only 0.45mumho/cm.(2) of the M-phase membrane. 5. The low effective proton conductance of the M phase will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis. 6. The addition of 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) at normal uncoupling concentrations causes a large increase in the effective proton conductance of the M phase of the cristae membrane. 7. The increase of the effective proton conductance of the M phase by 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) will account quantitatively for the short-circuiting effect of the uncoupling agent on the proton current and for the observed rise of the rate of respiration to that characteristic of state 3 or higher.