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

Open Access
Publisher Website
Google Scholar
Abstract
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 ΔpH 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 ΔpH. This indicates that ΔpH 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.