Superoxide poisons mononuclear iron enzymes by causing mismetallation

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 coli O₂^? 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.