Proton transfer through the membrane-water interfaces in uncoupled mitochondria

Increase in maximal respiration rate of uncoupled mitochondria in response to increase in concentration of non-penetrating buffer has been demonstrated. This phenomenon did not depend on chemical structure of uncouplers and composition of the non-penetrating buffer. Use of covalently attached pH probe, FITC, revealed that at low buffer concentration (3 mM) the H(+)-pump functioning results in local increase in proton concentration on the outer surface of the inner mitochondrial membranes. In other words, local H(+) gradient was generated. Increase in buffer concentration up to 20 mM caused sharp decrease in this gradient, which occurred in parallel to increase in the respiration rate. It is concluded that both effects described here may be attributed to the process of proton transfer through the interfaces of the mitochondrial membrane: the rate of respiratory H(+) pumps of uncoupled mitochondria under conditions of low buffer capacity of medium is limited by the stage of proton release from outer surface of the coupling membrane. The inhibition mechanism of respiration by high concentrations of uncouplers is also discussed.