Bulk phase proton fluxes during the generation of membrane potential in rat liver mitochondria

The addition of oxygen to anaerobic rat liver mitochondria incubated at 15 degrees C in the absence of permeant cations produced negligible rapid H+ ejection, monitored spectroscopically with phenol red, which corresponded kinetically to the rise in delta psi, as monitored by merocyanine 540. Slow H+ translocation was observed under these conditions during the aerobic phase, the extent of which was proportional to the amount of oxygen added and the rate dependent on the rate of counter-ion movement. Measurement of H+ disappearance in the mitochondrial matrix, as monitored by neutral red, likewise showed little or no rapid H+ change in the absence of counter-ion movements. In the presence of permeant cations, the H+ disappearance in the matrix was readily measured. This observation argues against the importance of the mitochondrial outer membrane and intermembrane space in masking H+ movements. The H+ translocation required in the generation of maximal or static head delta psi was determined by following the spectral response of merocyanine to increasing oxygen additions. The amount of oxygen giving maximal Delta psi corresponds to an extrusion of 2-3 ng ions of H+ . mg of protein-1. The absence of H+ movement of near this magnitude during the development of the Delta psi argues against the Delta psi-driven backflow of H+ ions as the sole explanation of these observations.