SITES OF ELECTRON-TRANSFER TO MEMBRANE-BOUND COPPER AND HYDROPEROXIDE-INDUCED DAMAGE IN THE RESPIRATORY-CHAIN OF ESCHERICHIA-COLI

Previous studies in Escherichia coli as a model system of peroxide tox icity (L. Rodriguez-Montelongo, L.C. De la Cruz-Rodriguez, R. N. Faria s, E.M. Massa, 1993 Biochim. Biophys.Acta 1144, 77-84) have shown that electron flow through the respiratory chain supports a membrane-assoc iated Cu(II)/Cu(I) redox cycle involved in irreversible impairment of the respiratory system by tert-butyl hydroperoxide (t-BOOH). In this p aper, E. coli mutants deficient in specific respiratory chain componen ts have been used to determine the sites of copper reduction and the t argets inactivated by t-BOOH. Two sites of electron transfer to membra ne-bound copper were identified: one in the region between NADH and ub iquinone supported by NADH as electron donor and another localized bet ween ubiquinone and the cytochromes supported by electrons coming from NADH, succinate, or D-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 abov e in vitro results with isolated membranes, copper-dependent inactivat ion of NADH dehydrogenase and ubiquinone was demonstrated in E. coli c ells 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 th ose two loci of the respiratory chain.