ENDOGENOUS SUPEROXIDE-DISMUTASE LEVELS REGULATE IRON-DEPENDENT HYDROXYL RADICAL FORMATION IN ESCHERICHIA-COLI EXPOSED TO HYDROGEN-PEROXIDE

Aerobic organisms contain antioxidant enzymes, such as superoxide dism utase (SOD) and catalase, to protect them from both direct and indirec t effects of reactive oxygen species, such as O-2(.-) and H2O2. Previo us work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but al so contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. cull cells are exposed to increased l evels of hydroxyl radical ((OH)-O-.) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strai n AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1- oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating (OH)-O-. production. When the isogenic E. call mutant J1132, lacking bath Fe-and Mn-containing SODs , was exposed to H2O2 in a similar manner, the magnitude of (OH)-O-. s pin trapped was significantly greater than with the control strain, Pr eincubation of the bacteria with the iron chelator deferoxamine marked ly inhibited the magnitude of (OH)-O-. spin trapped, Exogenous SOD fai led to inhibit (OH)-O-. formation, indicating the need for intracellul ar SOD, Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain, T hese studies (i) extend recent data from others demonstrating increase d levels of il on in E. coli SOD mutants and (ii) support the hypothes is that a resulting increase in (OH)-O-. formation generated by Fenton chemistry: is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in thes e mutants lacking SOD.