Superoxide sensitivity of the Escherichia coli aconitase.

Abstract

Mutants of Escherichia coli lacking superoxide dismutase (SOD) activity were used to explore the sensitivity of aconitase toward O2 and O2-. The aconitase activity in SOD-free extracts was rapidly lost under aerobic conditions and exogenous SOD afforded a concentration-dependent protection. The rate of the inactivating reaction between O2- and aconitase was estimated to be of the order of 10(9) M-1 s-1. The competitive inhibitors fluorocitrate and tricarballylate provided some protection, and at saturating concentrations, they decreased the rate of the inactivating reaction by 100- and 10-fold, respectively. Aconitase was markedly less sensitive to O2 than it was to O2-. Aerobic growth on succinate involves a greater dependence upon aconitase than does growth on glucose and, as expected, the deleterious consequences of SOD deficiency were more pronounced on succinate than on glucose. Moreover, aconitase activity was lower in extracts of aerobically grown SOD mutants, than it was in the parental strain. We suppose that inactivation of aconitase by O2- involves oxidative attack on the prosthetic iron-sulfur cluster. The extreme sensitivity of aconitase to inactivation by O2- suggests that its inactivation will be an early event in the oxidative stress imposed by hyperoxia, ultraviolet irradiation or redox-cycling agents, such as viologens or quinones.