The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium

Abstract

Oxic environments are hazardous. Molecular oxygen adventitiously abstracts electrons from many redox enzymes, continuously forming intracellular superoxide and hydrogen peroxide. These species can destroy the activities of metalloenzymes and the integrity of DNA, forcing organisms to protect themselves with scavenging enzymes and repair systems. Nevertheless, elevated levels of oxidants quickly poison bacteria, and both microbial competitors and hostile eukaryotic hosts exploit this vulnerability by assaulting these bacteria with peroxides or superoxide-forming antibiotics. In response, bacteria activate elegant adaptive strategies. In this Review, I summarize our current knowledge of oxidative stress in Escherichia coli, the model organism for which our understanding of damage and defence is most well developed.

Fig. 1. The generation of O_2⁻ and H₂O₂

(a) The univalent reduction series of oxygen. The standard reduction potentials (pH 7) of molecular oxygen (O₂), superoxide (O_2⁻), hydrogen peroxide (H₂O₂), and the hydroxyl radical (HO·) indicate that the last three are potentially potent univalent oxidants, in contrast to O₂. The standard concentration of oxygen is regarded as 1 M. (b) Two pathways of adventitious dihydroflavin (FADH₂) oxidation on flavoproteins. Flavin autoxidation is possible because enzymic flavins commonly have univalent reduction potentials as low as that of molecular oxygen. The pathway to the left requires an electron spin flip by either the flavosemiquinone or superoxide, allowing adduction and ultimately H₂O₂ release. The pathway on the right releases two consecutive molecules of superoxide to the bulk solution. The left pathway predominates in most enzymes studied to date.

Fig. 1. The generation of O_2⁻ and H₂O₂

(a) The univalent reduction series of oxygen. The standard reduction potentials (pH 7) of molecular oxygen (O₂), superoxide (O_2⁻), hydrogen peroxide (H₂O₂), and the hydroxyl radical (HO·) indicate that the last three are potentially potent univalent oxidants, in contrast to O₂. The standard concentration of oxygen is regarded as 1 M. (b) Two pathways of adventitious dihydroflavin (FADH₂) oxidation on flavoproteins. Flavin autoxidation is possible because enzymic flavins commonly have univalent reduction potentials as low as that of molecular oxygen. The pathway to the left requires an electron spin flip by either the flavosemiquinone or superoxide, allowing adduction and ultimately H₂O₂ release. The pathway on the right releases two consecutive molecules of superoxide to the bulk solution. The left pathway predominates in most enzymes studied to date.

Fig. 1. The generation of O_2⁻ and H₂O₂

(a) The univalent reduction series of oxygen. The standard reduction potentials (pH 7) of molecular oxygen (O₂), superoxide (O_2⁻), hydrogen peroxide (H₂O₂), and the hydroxyl radical (HO·) indicate that the last three are potentially potent univalent oxidants, in contrast to O₂. The standard concentration of oxygen is regarded as 1 M. (b) Two pathways of adventitious dihydroflavin (FADH₂) oxidation on flavoproteins. Flavin autoxidation is possible because enzymic flavins commonly have univalent reduction potentials as low as that of molecular oxygen. The pathway to the left requires an electron spin flip by either the flavosemiquinone or superoxide, allowing adduction and ultimately H₂O₂ release. The pathway on the right releases two consecutive molecules of superoxide to the bulk solution. The left pathway predominates in most enzymes studied to date.

Fig. 2. Activation of redox-sensitive transcriptional regulators in E. coli

E. coli contains two defence systems that are induced under conditions of oxidative stress: the OxyR system (which responds to H₂O₂) and the SoxRS system (which responds to redox-active compounds). (a) Activation of the OxyR systems occurs when a sensory cysteine residue on the OxyR protein reacts rapidly with H₂O₂, forming a sulphenic acid moiety that then condenses with a resolving cysteine. The resultant disulphide bond locks OxyR into a conformation that enables it to act as a positive transcription factor for regulon members, such as katG (encoding catalase G) and ahpCF (encoding Ahp), among other genes. (b) SoxR is a homodimeric transcription factor and each monomer contains an Fe-S cluster. The dimer becomes activated through the direct oxidation of these clusters by redox-active compounds, typically phenazines or quinones, which are produced by plants and bacterial competitors. Oxidized SoxR stimulates transcription of soxS, and the SoxS protein acts as a secondary transcription factor that goes on to activate expression of regulon members, including sodA (encoding superoxide dismutase) and acrAB (encoding a multi-drug efflux pump), among a large array of other genes.

Fig. 3. The role and oxidative vulnerability of dehydratase [4Fe-4S] clusters

(a) Cellular dehydratases reversibly dehydrate α,β-dihydroxyacids, releasing enol products (shown) that subsequently tautomerize to α-ketoacid products (not shown). The cluster coordinates substrates through their β-hydroxyl and carboxylate groups. Deprotonation by a nearby base (B:) triggers hydroxide abstraction by the catalytic iron atom, comprising a net dehydration. Arrows denote electron shifts towards new bonding partners. (b) The left pathway shows the exposed cluster being oxidized by O_2⁻, resulting in the formation of H₂O₂ and conversion of the cluster to an unstable [4Fe-4S]³⁺ species that then releases ferrous iron (Fe²⁺). The loss of the catalytic iron atom eliminates enzyme activity. The right pathway shows oxidation of the cluster by H₂O₂, which presumably creates a transient ferryl species that abstracts a second electron from the cluster. Ferric iron (Fe³⁺) dissociates. After damage by either oxidant, the resultant [3Fe-4S]⁺ cluster can be reactivated in vitro and in vivo by reduction and remetallation (dashed line).

Fig. 3. The role and oxidative vulnerability of dehydratase [4Fe-4S] clusters

(a) Cellular dehydratases reversibly dehydrate α,β-dihydroxyacids, releasing enol products (shown) that subsequently tautomerize to α-ketoacid products (not shown). The cluster coordinates substrates through their β-hydroxyl and carboxylate groups. Deprotonation by a nearby base (B:) triggers hydroxide abstraction by the catalytic iron atom, comprising a net dehydration. Arrows denote electron shifts towards new bonding partners. (b) The left pathway shows the exposed cluster being oxidized by O_2⁻, resulting in the formation of H₂O₂ and conversion of the cluster to an unstable [4Fe-4S]³⁺ species that then releases ferrous iron (Fe²⁺). The loss of the catalytic iron atom eliminates enzyme activity. The right pathway shows oxidation of the cluster by H₂O₂, which presumably creates a transient ferryl species that abstracts a second electron from the cluster. Ferric iron (Fe³⁺) dissociates. After damage by either oxidant, the resultant [3Fe-4S]⁺ cluster can be reactivated in vitro and in vivo by reduction and remetallation (dashed line).

Fig. 4. The role and oxidative vulnerability of mononuclear iron enzymes

Hydrogen peroxide and superoxide also diminish the activity of mononuclear iron enzymes, which use single iron atoms as prosthetic groups. (a) Peptide deformylase is presented as an example of this enzyme class. The cationic iron atom of peptide deformylase both activates a water molecule to provide a strong hydroxyl nucleophile (left) and stabilizes the negatively charged oxygen atom of the reaction intermediate (center). BH⁺ represents the enzymic proton donor that ultimately cleaves the carbon-nitrogen bond. (b) In the left pathway, oxidation of the mononuclear iron enzyme by H₂O₂ generates a transient ferryl species (Fe⁴⁺=O) that is then quenched by a coordinating cysteine residue. Since a sulphenic species (-SOH) is the ultimate product, it seems likely that a thiyl radical electron is transferred to the departing iron atom. The right pathway shows oxidation by superoxide, which generates a ferric iron species (Fe³⁺) that dissociates. The dashed black lines show that the activity of the superoxide-generated apoprotein can be restored by simple remetallation, although mismetallation of these enzymes by zinc can progressively diminish activity. Reactivation of the H₂O₂-damaged enzyme requires sulphenic reduction prior to remetallation (dashed grey lines).

Fig. 4. The role and oxidative vulnerability of mononuclear iron enzymes

Hydrogen peroxide and superoxide also diminish the activity of mononuclear iron enzymes, which use single iron atoms as prosthetic groups. (a) Peptide deformylase is presented as an example of this enzyme class. The cationic iron atom of peptide deformylase both activates a water molecule to provide a strong hydroxyl nucleophile (left) and stabilizes the negatively charged oxygen atom of the reaction intermediate (center). BH⁺ represents the enzymic proton donor that ultimately cleaves the carbon-nitrogen bond. (b) In the left pathway, oxidation of the mononuclear iron enzyme by H₂O₂ generates a transient ferryl species (Fe⁴⁺=O) that is then quenched by a coordinating cysteine residue. Since a sulphenic species (-SOH) is the ultimate product, it seems likely that a thiyl radical electron is transferred to the departing iron atom. The right pathway shows oxidation by superoxide, which generates a ferric iron species (Fe³⁺) that dissociates. The dashed black lines show that the activity of the superoxide-generated apoprotein can be restored by simple remetallation, although mismetallation of these enzymes by zinc can progressively diminish activity. Reactivation of the H₂O₂-damaged enzyme requires sulphenic reduction prior to remetallation (dashed grey lines).

Fig. 5. Overview of E. coli damage by reactive oxygen species

The autoxidation of redox enzymes leads to continuous H₂O₂ and O_2⁻ formation. Catalases (Cat), peroxidases (Prx) and superoxide dismutases (SOD) minimize the accumulation of these two oxidants. Nevertheless, both species damage [4Fe-4S] dehydratases and mononuclear iron enzymes. The disabled enzymes are continuously repaired, so that their steady-state activities represent the balance between damage and repair processes. H₂O₂ also reacts directly with the pool of unincorporated ferrous iron, which loosely associates with biomolecules, including DNA. The resultant hydroxyl radicals damage DNA, requiring the action of repair enzymes. The basal defenses of the cell keep the rates of these injuries low enough that growth and viability are not noticeably affected. However, when H₂O₂ and/or superoxide-generating redox compounds enter the cell, the intracellular levels of these oxidants rise; consequently, the vulnerable enzymes become predominantly disabled and metabolic pathways fail. Under these conditions, the induction of OxyR- and SoxRS-directed defence regulons are essential for cell recovery.