-->H+/2e- 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 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 (t1/2 < or = 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+.
Galkin
A S
AS
Department of Biochemistry, School of Biology, Moscow State University, Russian Federation.
Grivennikova
V G
VG
Vinogradov
A D
AD
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I; D004594:Electrophysiology; D007476:Ionophores / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011522:Protons; D012402:Rotenone / Q000494:pharmacology; D013367:Submitochondrial Particles / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D007476:Ionophores; D011522:Protons; D014475:Uncoupling Agents; D012402:Rotenone; D014634:Valinomycin; D000246:Adenosine Diphosphate Ribose; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I