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Title:The metabolism of iron-sulfur clusters during hydrogen peroxide stress in escherichia coli
Author(s):Jang, Soojin
Director of Research:Imlay, James A.
Doctoral Committee Chair(s):Imlay, James A.
Doctoral Committee Member(s):Cronan, John E.; Kuzminov, Andrei; Slauch, James M.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Hydrogen Peroxide
iron-sulfur cluster
oxidative stress
Abstract:An Escherichia coli strain that cannot scavenge hydrogen peroxide has been used to identify the cell processes that are most sensitive to this oxidant. Low micromolar concentrations of H2O2 completely blocked the biosynthesis of leucine. The defect was tracked to the inactivation of isopropylmalate isomerase. This enzyme belongs to a family of [4Fe-4S] dehydratases that are notoriously sensitive to univalent oxidation, and experiments confirmed that other members were also inactivated. In vitro and in vivo analyses showed that H2O2 directly oxidizes their solvent-exposed clusters in a Fenton-like reaction. The oxidized cluster then degrades to a catalytically inactive [3Fe-4S] form. In vitro experiments indicate that H2O2 accepts two consecutive electrons during the oxidation event; as a consequence, hydroxyl radicals are not released, the polypeptide is undamaged, and the enzyme is competent for reactivation by repair processes. Strikingly, in scavenger-deficient mutants in which H2O2 generated as an adventitious by-product of metabolism (< 1 µM) was sufficient to damage these [4Fe-4S] enzymes. This result demonstrates that aerobic organisms must synthesize H2O2 scavengers to avoid poisoning their own pathways. However, the basal H2O2 scavengers are likely insufficient to protect organisms from exogenous H2O2 which results in several injuries including the oxidation of dehydratase [4Fe-4S] clusters. To protect itself, E. coli activates the OxyR regulon, including genes that encode the Suf iron-sulfur-cluster assembly system. E. coli normally relies on the Isc system for cluster assembly; however, when E. coli scavenger mutants were exposed to low-grade H2O2 stress, Suf was needed to maintain dehydratase activities. Experiments showed that Suf repaired damaged clusters in isopropylmalate isomerase but not in fumarase. The reason was that damaged [3Fe-4S] clusters in IPMI were degraded in vivo, possibly to an apoprotein form, and thus required de novo assembly system for reactivation. In contrast, the [3Fe-4S] clusters in fumarase were stable and could be repaired by a simpler reduction/metallation process. Surprisingly, submicromolar H2O2 poisoned the Isc system, thereby creating a requirement for Suf both to repair IPMI and to activate Fe-S enzymes in general. The IscS and IscA components are H2O2-resistant, suggesting that oxidants disrupt Isc by oxidizing nascent Fe-S clusters as they are assembled on or transferred from the IscU scaffold. Consistent with these results, organisms that are chronically exposed to oxidants depend upon Suf rather than Isc for cluster assembly.
Issue Date:2010-05-14
Rights Information:Copyright 2010 Soojin Jang
Date Available in IDEALS:2010-05-14
Date Deposited:May 2010

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