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. K-i = 1.10(-9) M, k(on) = 5.10(7) M-1 min(-1) and k(off) = 0.02 min(-1) (inhibitory effect of rotenone on NADH oxidation) and K-i = 2.10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K similar to 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)over bar>(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.