The mechanism by which manganese protects Escherichia coli from hydrogen peroxide

Anjem, Adil

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https://hdl.handle.net/2142/29517 Copy

Description

Title

The mechanism by which manganese protects Escherichia coli from hydrogen peroxide

Author(s)

Anjem, Adil

Issue Date

2012-02-01T00:53:35Z

Director of Research (if dissertation) or Advisor (if thesis)

Imlay, James A.

Doctoral Committee Chair(s)

Imlay, James A.

Committee Member(s)

• Cronan, John E.
• Slauch, James M.
• Kuzminov, Andrei

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• mntH
• OxyR
• peptide deformylase
• threonine dehydrogenase
• cytosine deaminase
• manganese
• hydrogen peroxide
• sulfenic
• sulfhydryl oxidation

Abstract

The goal of this study was to understand the mechanism by which manganese protects cells against reactive oxygen species, particularly H2O2. We showed while manganese transport mutants, mntH, have no growth defect, mntH mutants in strains that cannot scavenge H2O2, Hpx- mntH, failed to grow when cultured aerobically. Thus manganese import is only crucial when cells are stressed with H2O2; cells appear to use iron to metallate mononuclear enzymes otherwise. Other workers have observed that manganese improves the ability of a variety of microbes to tolerate oxidative stress, and the prevailing hypothesis is that manganese does so by chemically scavenging hydrogen peroxide and/or superoxide .We found that manganese does not protect peroxide-stressed cells by scavenging peroxide. Instead, the beneficial effects of manganese correlate with its ability to metallate mononuclear enzymes. Because iron-loaded enzymes are vulnerable to the Fenton reaction, the substitution of manganese may prevent protein damage. Accordingly, during H2O2 stress, mutants that cannot import manganese and/or are unable to sequester iron suffer high rates of protein oxidation. To directly test our hypothesis, I studied three functionally distinct mononuclear enzymes: peptide deformylase (PDF), threonine dehydrogenase (TDH) and cytosine deaminase (CDA). We showed that these enzymes use iron as their cofactor, and that manganese functionally replaces iron and therefore protects these enzymes when cells are stressed with H2O2. We believe iron is frequently overlooked due to quick oxidation of ferrous iron to the insoluble ferric form in aerobic buffers. There are over two hundred mononuclear enzymes in E. coli, quite a few of which could be using iron in non-stressed conditions and thus during H2O2-stressed conditions could be using manganese. This implies that damage to iron-loaded enzymes is a global problem for cells. As we have demonstrated before, manganese import is critical for cell survival under H2O2 stress. In fact, the key metabolic failure of Hpx- mntH cells is due to lack of PDF activity: overexpressing PDF allows these cells to grow. We also demonstrated that these enzymes use manganese as their cofactor in iron-starved but H2O2-scavenging cells, which precludes the role of manganese as a scavenger. This conclusion is also supported by the ability of cobalt to relieve the cellular dependence on manganese during H2O2 stress. In this study, we also demonstrated that apo-PDF and apo-TDH are sensitive to H2O2. Both PDF and TDH have a metal-coordinating cysteine residue. While H2O2 is known to oxidize free cysteine, it does so at a maximal rate of 2 M-1 s-1 at neutral pH (half-life of inactivation at 1 μM H2O2 ~ 96 h), which is not fast enough to be physiologically relevant. In contrast, we have demonstrated that PDF and TDH are oxidized at a physiologically relevant rate of 1000-1300 M-1 s-1 (half-life of inactivation at 1 μM H2O2 ~ 10 min). These two enzymes are the first examples of proteins with cysteine residues that react with H2O2 at such a fast rate. We believe that H2O2-senstive active site cysteine residues in these enzymes are preferentially oxidized by H2O2 as a mechanism to spare other protein residues from irreversible covalent damage. This oxidized cysteine can then be later repaired by a reductant. Consistent with this idea, we have shown that oxidized inactivated proteins can be easily restored to full activity by a reductant in vitro. We have also only been able to retrieve proteins with cysteinyl residues in the over-oxidized state, which implies that the cell repairs oxidized proteins quite rapidly in vivo.

Graduation Semester

2011-12

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Copyright 2011 Adil Anjem

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