Characteristics of energy‐linked proton translocation in liposome reconstituted bovine cytochrome bc₁ complex

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.