Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide
01 Feb 1998-Journal of Bacteriology (American Society for Microbiology)-Vol. 180, Iss: 3, pp 622-625
TL;DR: The hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD is supported.
Abstract: Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped. Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD.
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TL;DR: A review of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection, updates the reader on these concepts and the topical questions in the field.
Abstract: Phagocyte-derived reactive oxygen and nitrogen species are of crucial importance for host resistance to microbial pathogens. Decades of research have provided a detailed understanding of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection. However, differences of opinion remain with regard to their host specificity, cell biology, sources and interactions with one another or with myeloperoxidase and granule proteases. More than a century after Metchnikoff first described phagocytosis, and more than four decades after the discovery of the burst of oxygen consumption that is associated with microbial killing, the seemingly elementary question of how phagocytes inhibit, kill and degrade microorganisms remains controversial. This review updates the reader on these concepts and the topical questions in the field.
1,476 citations
Cites background from "Endogenous Superoxide Dismutase Lev..."
...DNA damage is dependent on the presence of iron, which indicates that hydroxyl or ferryl radicals are toxic intermediates that are produced by the FENTON REACTION...
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TL;DR: It is found that regulators of the Escherichia coli responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake, which demonstrates that iron metabolism is coordinately regulated with the oxidative stress defenses.
Abstract: The cytotoxic effects of reactive oxygen species are largely mediated by iron. Hydrogen peroxide reacts with iron to form the extremely reactive and damaging hydroxyl radical via the Fenton reaction. Superoxide anion accelerates this reaction because the dismutation of superoxide leads to increased levels of hydrogen peroxide and because superoxide elevates the intracellular concentration of iron by attacking iron-sulfur proteins. We found that regulators of the Escherichia coli responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake. A transcript encoding Fur was induced by hydrogen peroxide in a wild-type strain but not in a DeltaoxyR strain, and DNase I footprinting assays showed that OxyR binds to the fur promoter. In cells treated with the superoxide-generating compound paraquat, we observed the induction of a longer transcript encompassing both fur and its immediate upstream gene fldA, which encodes a flavodoxin. This polycistronic mRNA is induced by paraquat in a wild-type strain but not in a DeltasoxRS strain, and SoxS was shown to bind to the fldA promoter. These results demonstrate that iron metabolism is coordinately regulated with the oxidative stress defenses.
396 citations
Cites background from "Endogenous Superoxide Dismutase Lev..."
...In addition, recent work has demonstrated that superoxide toxicity is mainly due to its role in accelerating the Fenton reaction (20, 24)....
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References
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01 Jan 1990
TL;DR: It is shown that the typical microliter laboratory syringe can introduce significant iron into solutions and these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.
Abstract: Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example. the concentration of iron can be determined by the metal-dependent rate ...
89 citations
TL;DR: Impact of oxidative stress, whether by addition of paraquat or by mutational deletion of superoxide dismutases, diminished fumarases A + B while elevating fumarase C, and results are consistent with a predominantly ferric state of the available iron in the presence of O2 and of the ferrous state in the absence of O 2.
72 citations
TL;DR: It is hypothesized that binding of MPO to bacteria could alter the magnitude and site of HO, and a possible role for MPO-derived HO in the augmentation of bacterial killing by this enzyme is hypothesized.
38 citations
"Endogenous Superoxide Dismutase Lev..." refers methods in this paper
...coli strains to O2 /H2O2 leads to the formation of OH, as detected with a 4-POBN–ethanol spin-trapping system (1)....
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TL;DR: Results support the involvement of sulphydryl groups in NADH-AFR reductase and point out the idea that a balance between reduced sulfhydryls and oxidized disulfides is required for the optimal function of this activity, considered as part of the transplasma membrane electron transport system.
28 citations
"Endogenous Superoxide Dismutase Lev..." refers methods in this paper
...0; 5 mM N-ethylmaleimide (NEM) was added to the bacteria 5 min before ascorbate exposure to inhibit ascorbate reductase activity (19)....
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