Complex I inhibitors as insecticides and acaricides.

Structurally diverse synthetic insecticides and acaricides had been shown to inhibit the proton-translocating NADH:ubiquinone oxidoreductase (complex I) activity. In addition, secondary metabolites from microbial and plant sources known to act on complex I exhibited biological activity against agricultural and environmental insect pests. Mechanistic studies indicated that these compounds interfered with ubiquinone reduction most likely at the same site(s) as the classical complex I inhibitors rotenone and piericidin A. Two approaches to characterize the mechanism of insecticidal/acaricidal complex I inhibitors were followed: enzyme kinetic studies and binding studies with radiolabeled inhibitors. Enzyme kinetic experiments were sometimes controversially interpreted regarding a competitive or non-competitive inhibitor mechanism with respect to the electron acceptor. In general, radioligand binding data with submitochondrial membranes were in line with the enzymological results but due to methodological drawbacks, saturation kinetic analyses were impossible. The main problems underlying many studies of inhibitor interaction with complex I were (i) the use of membrane-bound enzyme preparations and (ii) the physicochemical properties of the amphiphilic inhibitors with their strong tendency to accumulate in the membrane phase. A more recent approach to characterize inhbibitor interaction sites in complex I was the isolation of piericidin-resistant mutants of photosynthetic bacteria which produce a simpler homologue of mitochondrial NADH:Q oxidoreductase.

Mutants of luminous bacteria selected for bioluminescent toxicity tests.

Mutants of the luminescent bacterial strain NRRL B-11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS-PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs-mutants was altered. QSAR analysis showed that the inhibitory effect of chloro-substituted phenols on bioluminescence of the ahs-mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs-mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.

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

We have developed two independent methods to measure equilibrium binding of inhibitors to membrane-bound and partially purified NADH:ubiquinone oxidoreductase (complex I) to characterize the binding sites for the great variety of hydrophobic compounds acting on this large and complicated enzyme. Taking advantage of a partial quench of fluorescence upon binding of the fenazaquin-type inhibitor 2-decyl-4-quinazolinyl amine to complex I in bovine submitochondrial particles, we determined a Kd of 17 +/- 3 nM and one binding site per complex I. Equilibrium binding studies with [3H]dihydrorotenone and the aminopyrimidine [3H]AE F119209 (4(cis-4-[3H]isopropyl cyclohexylamino)-5-chloro-6-ethyl pyrimidine) using partially purified complex I from Musca domestica exhibited little unspecific binding and allowed reliable determination of dissociation constants. Competition experiments consistently demonstrated that all tested hydrophobic inhibitors of complex I share a common binding domain with partially overlapping sites. Although the rotenone site overlaps with both the piericidin 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 been used to define three classes of complex I inhibitors. The existence of only one large inhibitor binding pocket in the hydrophobic part of complex I is discussed in the light of possible mechanisms of proton translocation.