Binding Characteristics of Methoxyiminoacetamide Derivatives to Cytochrome c Reductase

The binding characteristics of a methoxyiminoacetamide derivative, (E)-2-methoxyimino-N-methyl-2 (2-phenoxyphenyl) acetamide (compound I), to cytochrome c reductase were studied by fluorescence quench titration using 2-n-heptyl-4-hydroxyquinoline N-oxide for the Q(1) site and E-beta-methoxyacrylate-stilbene for the Q(0) site as fluorescence inhibitors. The experiments on the binding competition between inhibitors revealed that compound 1 was able to bind to the Q(0) site of the mitochondrial cytochrome c reductase from bovine heart. The first-order rate constants for reactivation of methoxyiminoacetamide derivative (MOIA)-inhibited cytochrome c reductase were estimated from the progress of regeneration of active enzyme after lowering the concentration of MOIAs. Compound 1-inhibited cytochrome c reductase was rapidly reactivated after lowering the concentration of the inhibitor. Substitution of the hydrogen on the phenoxy ring of compound 1 by chlorine or a methyl group had no effect on the rate of reactivation. In contrast, substitution with hydrophobic groups such as a phenoxy, phenoxymethyl, or benzyloxy groups markedly inhibited the regeneration of active enzyme and increased the inhibitory potency against cytochrome c reductase, indicating a close correlation between binding stability and inhibitory potency of MOIAs. The enhancement of binding stability and inhibitory potency seemed to be due to the hydrophobic interaction between the meta substituents on the phenoxy ring and the domain of the end of helix C in the Q(0) site of cytochrome c reductase. (C) 1997 Academic Press.