EFFECT OF MEMBRANE CONDUCTANCE ON PROTON ELECTRON STOICHIOMETRY OF CYTOCHROME-C OXIDASE ACTIVITY IN PLANT-MITOCHONDRIA

Proton translocation linked to cytochrome c oxidase activity in antimycin-inhibited potato mitochondria has been investigated using the classical oxygen pulse technique. At 25°C, proton translocation took place with an apparent H+ e- ratio of 1.10 ± 0.06 with ascorbate plus N,N,N′,N′-tetramethylphenylenediamine (TMPD) and 1.19 ± 0.05 with ascorbate plus ferrocyanide. The decrease in temperature from 25°C to 5°C resulted in an increase in membrane viscosity associated with a decrease in the rate constant of the proton back-flow. Under these conditions, the extent of proton ejection was strongly enhanced and the H+ e- ratio of ascorbate/TMPD was increased up to 1.73 ± 0.06 and reached 3.65 ± 0.20 for the span succinate-O2. In the presence of increasing concentrations of the uncoupler CCCP, at 5°C the H+ e- ratio observed or extrapolated at zero time was lowered as the rate of proton back-flow was enhanced. The same relationship was obtained between the H+ e- ratio and the rate of proton back-flow after enhancement of proton conductance by either temperature or CCCP. It results that in both cases the calculated H+ e- ratio extrapolated to zero time was underestimated. Extrapolation of H+/e- ratio in potato mitochondria under conditions of zero proton back-flow provides values which approach 2 for the cytochrome c oxidase complex and 4 for the span succinate-O2. © 1990.