title: Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli.
abstract: Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Lee
authors.initials: L
authors.lastname: Macomber
authors.forename: Christopher
authors.initials: C
authors.lastname: Rensing
authors.forename: James A
authors.initials: JA
authors.lastname: Imlay
mesh_terms: D002384:Catalysis; D003300:Copper / Q000032:analysis / Q000633:toxicity*; D004249:DNA Damage; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity; D017665:Hydroxyl Radical / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D010084:Oxidation-Reduction; D019897:Periplasm / Q000737:chemistry
chemical_list: D017665:Hydroxyl Radical; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron