Open access, freely available online Primer Exploiting Thiol Modifi cations Patricia J. Kiley*, Gisela Storz s the premier biological species when they are released by and plays roles in the aging process electron acceptor, molecular animals, plants, and insects as a defense and the development of heart disease, oxygen (O ) serves a vital role against detrimental organisms such as diabetes, chronic infl ammatory diseases, A 2 in fundamental cellular functions, microbial pathogens. Reactive oxygen cancer, and several neurodegenerative including the process of aerobic species can damage cells in many ways: diseases (Halliwell 1999). respiration. Nevertheless, with the by inactivating proteins, damaging Many organisms have evolved benefi cial properties of O2 comes nucleic acids, and altering the fatty strategies to remove reactive oxygen the inadvertent formation of reactive acids of lipids, which leads in turn to species and repair damage, which oxygen species, including superoxide perturbations in membrane structure have enabled them to prosper − (O2 ), hydrogen peroxide (H2O2), and function. The accumulation of from the tremendous oxidizing • and hydroxyl radical ( OH); these this oxidative damage underlies the potential of O2 without succumbing differ from O2 in having one, two, and formation of many disease states in to oxidative damage. Bacteria, yeast, three additional electrons, respectively humans. It is postulated that tissue and mammalian cells all induce the (Figure 1). Cells also encounter injury by these reactive oxygen species synthesis of global regulatory responses elevated levels of these reactive oxygen accumulates over a long period of time to survive oxidative insults. The consequences of oxidative stress and the corresponding defense responses have been extensively studied in Escherichia coli. For ease of study in the laboratory, the stress responses are often provoked by the external addition of chemical oxidants that specifi cally elevate the levels of reactive oxygen species within cells, or by the use of mutant strains that disrupt the normal “homeostatic mechanisms” for removing reactive oxygen species or the damage they do. While this primer focuses on a particular set of protective and regulatory protein modifi cations induced by oxidative stress in E. coli, it should be noted that many of the same mechanisms are present in other organisms; some specifi c examples from other species will also be described. − The major target of O2 damage identifi ed in bacteria is a class of Citation: Kiley PJ, Storz G (2004) Exploiting thiol modi- fi cations. PLoS Biol 2(11): e400. Copyright: © 2004 Kiley and Storz. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: H2O2, hydrogen peroxide; O2, molecular oxygen; O −, superoxide; •OH, hydroxyl radical DOI: 10.1371/journal.pbio.0020400.g001 2 Patricia J. Kiley is in the Department of Biomolecular Figure 1. Formation of Reactive Oxygen Species Chemistry, University of Wisconsin Medical School in Madison, Wisconsin, United States of America. The four-electron reduction of molecular O2 generates two molecules of H2O, which is O in its most reduced form. While this reduction normally occurs within the enzyme Gisela Storz is in the Cell Biology and Metabolism 2 Branch, National Institute of Child Health and Human cytochrome oxidase, one-electron transfers to O2 also occur outside of cytochrome oxidase via inadvertent reactions with other reduced electron carriers, resulting in Development, National Institutes of Health, Bethesda, Maryland, United States of America. partially reduced and reactive forms of O2. H2O2 is also produced by the enzymatic or − • spontaneous dismutation of O2 , and OH is generated by the reaction of iron with H2O2 *To whom correspondence should be addressed. (the Fenton reaction). In addition, the reactive oxygen intermediates are produced by a E-mail: [email protected] variety of organisms as a defense against microbial invasion. (Illustration: Rusty Howson, sososo design) DOI: 10.1371/journal.pbio.0020400 PLoS Biology | www.plosbiology.org 1714 November 2004 | Volume 2 | Issue 11 | e400 dehydratase enzymes that utilize [4Fe– 4S] clusters to bind their substrate (Imlay 2003; Djaman et al. 2004). Since some of these enzymes function in the citric acid cycle (also called the Krebs cycle) and in amino acid − biosynthesis, high levels of O2 lead to a requirement for certain amino acids in growth media (Imlay and Fridovich 1991). H2O2 is well known for its role in oxidizing thiol (SH) groups of cysteinyl amino acid residues in proteins. Elevated levels of H2O2 also are associated with the oxidation of other amino acids, leading to the formation of methionine sulfoxide and a variety of carbonyls. Lastly, because DOI: 10.1371/journal.pbio.0020400.g002 of its extreme reactivity, •OH targets Figure 2. Thiol Modifi cations of Proteins all of the major macromolecules of Formation of sulfenic acid from the reaction of H2O2 with protein thiolates leads cells: RNA, DNA, protein, and lipids. to different protein modifi cations, depending on the protein. In proteins without a The extent to which membrane lipids second sulfhydryl, the sulfenic acid (–SOH) may be stabilized (e.g., OhrR) or may react with reactive oxygen species to generate the further oxidized sulfi nic (–SO H) are targets appears to depend on the 2 (e.g., thiolperoxidase; Tpx) and sulfonic acid (–SO3H) derivatives. Alternatively, if a presence of polyunsaturated fatty acids second cysteinyl residue is in proximity within the same polypeptide (e.g., OxyR) or an in lipids, which are not as prevalent in associated protein (e.g., Yap1 and Orp1), a disulfi de bond can form between the two bacteria as they are in mammals. sulfur atoms (–S–S–). Lastly, the sulfenated cysteinyl residue can react with glutathione Many enzymes that protect against (GSH), leading to a mixed disulfi de (e.g., MetE). (Illustration: Rusty Howson, sososo design) oxidative damage have been identifi ed in E. coli (Imlay 2002, 2003). Three Reduced thioredoxin is regenerated no cysteinyl residue is nearby, the superoxide dismutases, each of which by thioredoxin reductase and NADPH. sulfenated cysteine can be further contain a different metal center and The fact that NADPH is required oxidized to sulfi nic or sulfonic acid, or show different expression patterns and to maintain the reduced state of it can remain in the sulfenic acid state. subcellular localization, catalyze the glutathione and thioredoxin indicates All but the sulfi nic and sulfonic acid − dismutation of O2 to H2O2. While the that the response to oxidative stress is modifi cations are readily reversible − superoxide dismutases eliminate O2 , coupled to the physiological status of by reduction, using proteins such as they also are a source of endogenously core pathways that generate NADPH. thioredoxin or glutaredoxin; though produced H2O2 in E. coli. The major sulfi nic acid reductase activities have enzymes involved in reducing H O Regulatory Roles of Thiol recently been identifi ed in yeast and 2 2 Modifi cations to H2O and O2 in E. coli are catalase mammalian cells (denoted sulfi redoxin and alkyl hydroperoxide reductase. As mentioned above, proteins—in and sestrin, respectively) (Biteau et al. There is no enzymatic mechanism for particular, the thiols of cysteines—are 2003; Budanov et al. 2004). • decreasing levels of OH, produced the major targets of H2O2. The reaction Given the reversible nature of most • of cysteinyl thiolates with H O can from H2O2. Thus, levels of OH will be 2 2 forms of thiol oxidation, it has been directly proportional to levels of H2O2, lead to the formation of different suggested that thiol modifi cations can and accordingly, catalase and alkyl modifi cations, such as sulfenic acid play roles in signal transduction that hydroperoxide reductase activities are (–SOH), sulfi nic acid (–SO2H), and are similar to protein phosphorylation/ critical to oxidative stress survival. sulfonic acid (–SO3H), as well as dephosphorylation (Sitia and Molteni Another component to the oxidative disulfi de bond formation (–S–S–) and 2004). In support of this model, there stress response is the reduction of glutathione conjugation (–S–GSH) are several examples of proteins oxidized thiols that arises through one (Jacob et al. 2004; Poole et al. 2004) whose activities are modulated by thiol of the mechanisms described below. (Figure 2). These modifi cations often oxidation and reduction. The tripeptide glutathione and the alter the structure and function of the The fi rst of these examples is the thiol reductants glutaredoxin and protein. Recent progress in this fi eld OxyR transcription factor, which thioredoxin are key to the restoration points to a common chemistry in the upregulates peroxide defenses in E. coli of thiols to their reduced state (SH) reaction of H2O2 with thiolates through and a variety of other bacteria. OxyR (Fernandes and Holmgren 2004). E. coli the initial formation of sulfenic acid. In contains two critical cysteines that are contains three glutaredoxins that utilize the case of proteins that have a nearby oxidized to form an intramolecular the reducing power of glutathione to cysteinyl residue, a disulfi de bond disulfi de bond when cells encounter catalyze the reduction of disulfi de bonds forms between the two sulfur atoms. peroxide stress (Zheng et al. 1998; (–S–S–) in the presence of NADPH The sulfenated cysteinyl residue also Aslund et al. 1999). Disulfi de bond and glutathione reductase. There are can react with a cysteinyl residue on formation is associated with a two thioredoxins in E. coli that also another protein or with glutathione, conformational change that alters function to reduce disulfi de bonds. leading to a mixed disulfi de. If OxyR binding to DNA and allows the PLoS Biology | www.plosbiology.org 1715 November 2004 | Volume 2 | Issue 11 | e400 protein to activate the transcription stable sulfenic acid upon its reaction raises questions about the function of of genes encoding enzymes, such as with H2O2 or organic peroxides the zinc.