H+/2e- stoichiometry of the nadh:ubiquinone reductase reaction catalyzed by submitochondrial particles.
Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio H+/2e- (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q1 gives the value of n = 4. Thermally induced deactivation of Complex I [1, 2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q1-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q1-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.
Galkin
A S
AS
Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119899, Russia.
Grivennikova
V G
VG
Vinogradov
A D
AD
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002384:Catalysis; D002417:Cattle; D004579:Electron Transport / Q000502:physiology; D042967:Electron Transport Complex I; D004789:Enzyme Activation / Q000502:physiology; D005033:Ethylmaleimide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000166:cytology; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011522:Protons; D013367:Submitochondrial Particles / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism*
D011522:Protons; D014451:Ubiquinone; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D005033:Ethylmaleimide