Anomalous response of oxonol-V to membrane potential in mitochondrial proton pumps

The response of the fluorescent membrane potential probe oxonol-V (bis[3-phenyl 5 oxoisoxazol-4-yl]pentamethine oxonol) in submitochondrial particles (SMP) was dependent upon whether the potential (inside positive) was generated by active proton pumps or by valinomycin-aided passive K+ influx. The fluorescence intensity showed a decrease in the former case and an increase in the latter situation. This anomalous behavior was not observed with other similar anionic probes. Gradual inhibition of proton pumping activity showed that the difference in the response of oxonol-V is not due to possible difference in the magnitude of membrane potential generated in these two situations. In the presence of membrane permeant anions such as TPB- (tetraphenyl boron) or chlorate, the direction of response of oxonol-V fluorescence was the same in both situations. Time-resolved fluorescence of the oxonol-V-SMP system showed three populations: one free form (fluorescence lifetime approximately 60 ps) and two SMP-bound forms (lifetimes of 0.45 ns and 1.4 ns). A fourth population was created during the action of proton pumps. The shorter lifetime (approximately 250 ps) of this new bound form suggest this population to be an aggregated form. This population was absent during the action of proton pumps in the presence of TPB- or chlorate. These results suggest the creation of a charge separated state during the action of proton pumps. The decrease in fluorescence intensity could be the result of aggregation of oxonol-V around the positive end of a proton pump existing in a dipole or charge separated state.