Sites of Electron Transfer to Membrane-Bound Copper and Hydroperoxide-Induced Damage in the Respiratory Chain ofEscherichia coli

Previous studies inEscherichia colias a model system for peroxide toxicity (L. Rodrı́guez-Montelongo, L. C. De la Cruz-Rodrı́guez, R. N. Farı́as, and E. M. Massa, 1993,Biochim. Biophys. Acta1144, 77–84) have shown that electron flow through the respiratory chain supports a membrane-associated Cu(II)/Cu(I) redox cycle involved in irreversible impairment of the respiratory system bytert-butyl hydroperoxide (t-BOOH). In this paper,E. colimutants deficient in specific respiratory chain components have been used to determine the sites of copper reduction and the targets inactivated by t-BOOH. Two sites of electron transfer to membrane-bound copper were identified: one in the region between NADH and ubiquinone supported by NADH as electron donor and another localized between ubiquinone and the cytochromes supported by electrons coming from NADH, succinate, orD-lactate. Electron flow through the former site in the presence of t-BOOH led to inactivation of NADH dehydrogenase II, whereas electron flow through the latter site in the presence of the hydroperoxide led to damage of ubiquinone. In agreement with the abovein vitroresults with isolated membranes, copper-dependent inactivation of NADH dehydrogenase and ubiquinone was demonstrated inE. colicells exposed to t-BOOH. It is proposed that the t-BOOH-induced damage is a consequence of t-butylalkoxy radical generation through a Fenton-type reaction mediated by redox cycling of membrane-bound copper at those two loci of the respiratory chain.