Superoxide poisons mononuclear iron enzymes by causing mismetallation

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Superoxide (O(2)(-)) is a key mediator of intracellular oxidative stress.
• While known to affect [4Fe-4S] dehydratases, the full range of superoxide's enzymatic targets remains incompletely understood.
• Understanding superoxide's impact on diverse metalloenzymes is crucial for comprehending cellular responses to oxidative stress.

Purpose of the Study:

• To investigate the effects of superoxide (O(2)(-)) on non-redox enzymes utilizing ferrous iron (Fe2+) cofactors in Escherichia coli.
• To elucidate the mechanism of enzyme inactivation by superoxide and compare it with hydrogen peroxide-induced damage.
• To characterize the cellular response to superoxide-induced metalloenzyme dysfunction.

Main Methods:

• In vitro inactivation assays of purified enzymes with superoxide (O(2)(-)) and subsequent restoration with iron.
• In vivo studies in Escherichia coli under superoxide stress to monitor enzyme metallation status.
• Analysis of zinc (Zn2+) incorporation into enzymes during oxidative stress and recovery.

Main Results:

• Superoxide (O(2)(-)) inactivated non-redox iron-dependent enzymes both in vitro and in vivo.
• In vitro, enzyme activity was restored by iron addition, indicating apoprotein formation without polypeptide damage.
• In vivo, superoxide stress led to progressive zinc (Zn2+) mismetallation of these enzymes, with activity restored upon cessation of stress via zinc extraction.

Conclusions:

• Superoxide (O(2)(-)) acts as a potent inhibitor of a broader range of non-redox metalloenzymes than previously recognized.
• The mechanism involves repeated iron excision, allowing zinc (Zn2+) to inappropriately bind to the apoprotein.
• This expands the known physiological impact of superoxide stress on cellular metabolism and enzyme function.