Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition.
The interaction of rotenone with active ('pulsed') and thermally de-activated ('resting') membrane-bound Complex I (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) as revealed by inhibition of NADH-ubiquinone- and ubiquinol-NAD+ reductase activities was studied. Ki = 1 x 10(-9) M, k(on) = 5 x 10(7) M-1 min-1 and k(off) = 0.02 min-1 (inhibitory effect of rotenone on NADH oxidation) and Ki = 2 x 10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K approximately 100) after the slow strongly temperature-dependent de-activation process has completed. Rotenone partially prevents and reverses the enzyme de-activation. About two order of magnitude difference in affinity of rotenone to the active and de-activated forms of the enzyme was demonstrated. The strong difference in rotenone sensitivity of the direct and reverse reactions can not be accounted for delta mu H(+)-dependence of rotenone binding. We propose that two rotenone-specific inhibitory sites exist in Complex I: one is involved in NADH oxidation by ubiquinone and the other is operating in ubiquinol-NAD+ reductase reaction. The affinities of rotenone for both sites are strongly altered upon the slow enzyme active/inactive transition.
Grivennikova
V G
VG
Department of Biochemistry, School of Biology, Moscow State University, Russia.
Maklashina
E O
EO
Gavrikova
E V
EV
Vinogradov
A D
AD
D000818:Animals; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D012402:Rotenone / Q000378:metabolism* / Q000494:pharmacology; D013367:Submitochondrial Particles / Q000201:enzymology*
D009840:Oligomycins; D012402:Rotenone; D016660:NAD(P)H Dehydrogenase (Quinone)