Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.
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.
Cocco
T
T
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Lorusso
M
M
Di Paola
M
M
Minuto
M
M
Papa
S
S
D000818:Animals; D001693:Biological Transport, Active; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D004563:Electrochemistry; D004579:Electron Transport / Q000187:drug effects; D014450:Electron Transport Complex III / Q000378:metabolism*; D004734:Energy Metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D008564:Membrane Potentials; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011188:Potassium / Q000378:metabolism; D011522:Protons; D013053:Spectrophotometry; D014634:Valinomycin / Q000494:pharmacology
D008081:Liposomes; D011522:Protons; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D014450:Electron Transport Complex III; D011188:Potassium