Kinetic Evidence for Two Sites in the Inhibition by Diuron of the Electron Transport in the bc1 Segment of the Respiratory Chain in Saccharomyces cerevisiae

Abstract

A kinetic method based on the measurement of NADH oxidase activity in yeast submitochondrial particles incubated in the simultaneous presence of an inhibitor of high affinity such as antimycin and an inhibitor of low affinity such as 3(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron) permits estimation of two distinct dissociation constants for diuron, Kd.1= 3.2 μM and Kd,2= 21.5 μM. To take into account the fact that these inhibitors do not act on the rate‐limiting step of the NADH oxidase activity, the Kröger and Klingenberg kinetic model [Eur. J. Biochem. (1973) 39, 313–323] has been applied using a new method for estimating the maximal activities of the NADH: Q reductase activity (Vred= 280 nmol O × min−1× mg −1) and of the QH2: O2 oxidase activity (Vox= 850 nmol O × min−1× mg−1) (Q = ubiquinone). These latter values permit the estimation of two distinct dissociation constants for another weakly bound inhibitor, n‐heptyl‐hydroxyquinoline‐N‐oxide: Kh,1= 0.23 μM and Kh,2= 5 μM. Only one dissociation constant, Kd= 23 μM for diuron is sufficient to characterize the ‘extra‐reduction’ of cytochrome b566. Even though diuron does not induce the ‘red‐shift’ of cytochrome b562, the combined addition of suboptimal concentrations of n‐heptyl‐hydroxyquinoline‐N‐oxide and diuron, permits estimation of a single dissociation constant which is lower than 6.6 μM diuron for a site which can bind either diuron or n‐heptyl‐hydroxyquinoline oxide but which will yield a ‘red‐shift’ of cytochrome b562 when occupied by n‐heptyl‐hydroxyquinoline‐N‐oxide only. Copyright © 1978, Wiley Blackwell. All rights reserved