→H+/2e stoichiometry in NADH‐quinone reductase reactions catalyzed by bovine heart submitochondrial particles

Abstract

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q₁. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t _1/2≤1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H⁺ 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⁺/2e stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q₁ 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⁺/2e stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q₁-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H⁺.