→H+/2(e)over-bar stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation mere capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q(1). External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t(1/2) less than or equal to 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external Hi decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting --> H+/2 (e) over bar stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q(1) reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same --> H+/2 (e) over bar stoichiometry of 4, It is concluded that the transfer of two electrons from NADH to the Q(1)-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+. (C) 1999 Federation of European Biochemical Societies.