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Superoxide poisons mononuclear iron enzymes by causing mismetallation.

ABSTRACT: Superoxide (O(2)(-)) is a primary agent of intracellular oxidative stress. Genetic studies in many organisms have confirmed that excess O(2)(-) 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?O(2)(-) also poisons a broader cohort of non-redox enzymes that employ ferrous iron atoms as catalytic cofactors. These enzymes were inactivated by O(2)(-) both in vitro and in vivo. Although the enzymes are known targets of hydrogen peroxide, the outcome with O(2)(-) differs substantially. When purified enzymes were damaged by O(2)(-) in vitro, activity could be completely restored by iron addition, indicating that the O(2)(-) 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 O(2)(-) stress was terminated, cells gradually restored activity by extracting zinc from the proteins. The overloading of cells with zinc caused mismetallation even without O(2)(-) stress. These results support a model in which O(2)(-) 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 O(2)(-) stress.

SUBMITTER: Gu M

PROVIDER: S-EPMC3731988 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Superoxide poisons mononuclear iron enzymes by causing mismetallation.

Gu Mianzhi M Imlay James A JA

Molecular microbiology 20130607 1

Superoxide (O(2)(-)) is a primary agent of intracellular oxidative stress. Genetic studies in many organisms have confirmed that excess O(2)(-) 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 O(2)(-) also poisons a broader cohort of non-redox enzymes that employ ferrous iron atoms as catalytic cofactors. These enzymes were inactivated by O(2)(-) both in vitro and in vivo. Alt ...[more]

PMID: 23678969

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Project description:ETHE1 is a member of a growing subclass of nonheme Fe enzymes that catalyzes transformations of sulfur-containing substrates without a cofactor. ETHE1 dioxygenates glutathione persulfide (GSSH) to glutathione (GSH) and sulfite in a reaction which is similar to that of cysteine dioxygenase (CDO), but with monodentate (vs bidentate) substrate coordination and a 2-His/1-Asp (vs 3-His) ligand set. In this study, we demonstrate that GSS- binds directly to the iron active site, causing coordination unsaturation to prime the site for O2 activation. Nitrosyl complexes without and with GSSH were generated and spectroscopically characterized as unreactive analogues for the invoked ferric superoxide intermediate. New spectral features from persulfide binding to the FeIII include the appearance of a low-energy FeIII ligand field transition, an energy shift of a NO- to FeIII CT transition, and two new GSS- to FeIII CT transitions. Time-dependent density functional theory calculations were used to simulate the experimental spectra to determine the persulfide orientation. Correlation of these spectral features with those of monodentate cysteine binding in isopenicillin N synthase (IPNS) shows that the persulfide is a poorer donor but still results in an equivalent frontier molecular orbital for reactivity. The ETHE1 persulfide dioxygenation reaction coordinate was calculated, and while the initial steps are similar to the reaction coordinate of CDO, an additional hydrolysis step is required in ETHE1 to break the S-S bond. Unlike ETHE1 and CDO, which both oxygenate sulfur, IPNS oxidizes sulfur through an initial H atom abstraction. Thus, factors that determine oxygenase vs oxidase reactivity were evaluated. In general, sulfur oxygenation is thermodynamically favored and has a lower barrier for reactivity. However, in IPNS, second-sphere residues in the active site pocket constrain the substrate, raising the barrier for sulfur oxygenation relative to oxidation via H atom abstraction.

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Superoxide poisons mononuclear iron enzymes by causing mismetallation.

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Superoxide poisons mononuclear iron enzymes by causing mismetallation.

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