Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Abstract

ABSTRACT Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O 2 ·− and H 2 O 2 . Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H 2 O 2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical ( · OH) as a consequence of the reaction of H 2 O 2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H 2 O 2 in the presence of an α-(4-pyridyl-1-oxide)- N -tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN– · CH(CH 3 )OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating · OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H 2 O 2 in a similar manner, the magnitude of · OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of · OH spin trapped. Exogenous SOD failed to inhibit · OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in · OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H 2 O 2 -mediated killing seen in these mutants lacking SOD.