Non-linear inhibition curves for tight-binding inhibitors of dimeric ubiquinol-cytochrome c oxidoreductases. Evidence for rapid inhibitor mobility.
Steady-state electron flow through and electron delivery into isolated dimeric bc1 complex (ubiquinol--cytochrome c oxidoreductase) from Neurospora crassa and beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate-determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate less than 500 s-1) and of myxothiazol (rate greater than 1 s-1). Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol--O2 oxidoreductase turns over at about 300 s-1, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate approximately 100 s-1) of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophores obtained from Rhodobacter capsulatus. As recently described [Fernandez-Velasco, J. & Crofts, A. R. (1992) Biophys. J. 2, A153], cytochrome b becomes fully reoxidized within 1 s after a flash at substoichiometric concentrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimeric and interdimeric redistribution with rate constants of about 10 s-1 and 2 x 10(6) M-1 s-1, respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.
Bechmann
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Institut für Biochemie, Heinrich-Heine-Universität, Düsseldorf, Federal Republic of Germany.
Weiss
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Rich
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D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors*; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000201:enzymology; D009492:Neurospora crassa / Q000201:enzymology; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013844:Thiazoles / Q000494:pharmacology
D000880:Anthraquinones; D003573:Cytochrome b Group; D006873:Hydroquinones; D046911:Macromolecular Substances; D008689:Methacrylates; D013844:Thiazoles; C015286:funiculosin (anthraquinone); D000968:Antimycin A; C030517:myxothiazol; D003575:Cytochromes c1; D014450:Electron Transport Complex III