Superoxide poisons mononuclear iron enzymes by causing mismetallation

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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 inEscherichia coliO2− also poisons a broader cohort of non-redox enzymes that employ ferrous iron atoms as catalytic cofactors. These enzymes were inactivated by O2− bothin vitroandin 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.

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