Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli.

We are studying the action of tert-butylhydroperoxide (t-BOOH) on Escherichia coli as a model system for peroxide toxicity. In our previous report (De la Cruz-Rodriguez, L.C., Farías, R.N. and Massa, E.M. (1990) Biochim. Biophys. Acta 1015, 510-516), the respiratory chain was identified as a major target of t-BOOH. In the present paper, we study further the effect of t-BOOH on the NADH oxidase of the E. coli respiratory chain to clarify the mechanism of damage, especially regarding the identity and role of the metal ion involved. The results are: (a) t-BOOH toxicity is mediated by membrane-bound copper ions; (b) a small pool of the membrane-bound copper is reduced from Cu(II) to Cu(I) in the presence of NADH and other respiratory substrates (succinate, D-lactate); (c) this reduction of copper occurs at 37 degrees C but not at 0 degrees C or when the membranes are inactivated by previous heating; (d) the Cu(I) generated by reduction of Cu(II) during membrane preincubation with NADH, is oxidized by t-BOOH with simultaneous inactivation of the NADH oxidase, whereas treatment with only t-BOOH (without NADH) has no effect on the oxidase. It is concluded that the effect of t-BOOH on the respiratory chain is mediated by redox cycling of copper. It is proposed that the damage results from activation of the hydroperoxide through its interaction with Cu(I) in a site-specific Fenton-type reaction.

Damage of Escherichia coli cells by t-butylhydroperoxide involves the respiratory chain but is independent of the presence of oxygen.

The action of t-butylhydroperoxide (tBOOH) on Escherichia coli cells has been studied as a model system for organic peroxide toxicity. Exposure of E. coli cells to tBOOH led to progressive and irreversible impairment of the respiratory function, an effect which was dependent on the availability of substrate. The effect of tBOOH on growth of E. coli with different carbon sources and alternative terminal electron acceptors was investigated. It was found that the sensitivity of E. coli to tBOOH under diverse growth conditions implicating a functional respiratory chain was greater than when the bacterium grew by fermentation. Also the mutant E. coli SASX76, which requires exogenous 5-aminolevulinic acid to synthesize the cytochromes, was more resistant to tBOOH when lacking a functional respiratory chain. These data point to the respiratory chain as a major target in the in vivo action of tBOOH. Experiments with isolated membranes also showed a tBOOH-induced damage of the respiratory chain monitored by impairment of the NADH oxidase. The effect of tBOOH was produced even under anaerobiosis, indicating that development of cell damage was independent of oxygen and, therefore, that neither oxygen-derived radicals nor lipid peroxidation were involved.