Three classes of inhibitors share a common binding domain in mitochondrialcomplex I (NADH : ubiquinone oxidoreductase)

We have developed two independent methods to measure equilibrium binding of inhibitors to membrane bound and partially purified NADH:ubiquinone oxidor eductase (complex I) to characterize the binding sites for the great variet y of hydrophobic compounds acting on this large and complicated enzyme. Tak ing advantage of a partial quench of fluorescence upon binding of the fenaz aquin-type inhibitor 2-decyl-4-quinazolinyl amine to complex I in bovine su bmitochondrial particles, we determined a K-d of 17 +/- 3 nM and one bindin g site per complex I. Equilibrium binding studies with [H-3]dihydrorotenone and the aminopyrimidine [H-3]AE F119209 (4(cis-4-[H-3]isopropyl cyclohexyl amino)-5-chloro-6-ethyl pyrimidine) using partially purified complex I from Musca domestica exhibited little unspecific binding and allowed reliable d etermination of dissociation constants. Competition experiments consistently demonstrated that all tested hydrophob ic inhibitors of complex I share a common binding domain with partially ove rlapping sites. Although the rotenone site overlaps with both the piericidi n A and the capsaicin site, the latter two sites do not overlap. This is in contrast to the interpretation of enzyme kinetics that have previously bee n used to define three classes of complex I inhibitors. The existence of on ly one large inhibitor binding pocket in the hydrophobic part of complex I is discussed in the light of possible mechanisms of proton translocation.