Superoxide poisons mononuclear iron enzymes by causing mismetallation

Abstract

Summary: Superoxide (O2-) is a primary agent of intracellular oxidative stress. Genetic studies in many organisms have confirmed that excess O2- disrupts metabolism, but to date only a small family of [4Fe-4S] dehydratases have been identified as direct targets. This investigation reveals that in Escherichia coliO2- also poisons a broader cohort of non-redox enzymes that employ ferrous iron atoms as catalytic cofactors. These enzymes were inactivated by O2- both in vitro and in vivo. Although the enzymes are known targets of hydrogen peroxide, the outcome with O2- differs substantially. When purified enzymes were damaged by O2- in vitro, activity could be completely restored by iron addition, indicating that the O2- treatment generated an apoprotein without damaging the protein polypeptide. Superoxide stress inside cells caused the progressive mismetallation of these enzymes with zinc, which confers little activity. When O2- stress was terminated, cells gradually restored activity by extracting zinc from the proteins. The overloading of cells with zinc caused mismetallation even without O2- stress. These results support a model in which O2- repeatedly excises iron from these enzymes, allowing zinc to compete with iron for remetallation of their apoprotein forms. This action substantially expands the physiological imprint of O2- stress. © 2013 John Wiley & Sons Ltd.