Exploiting Thiol Modifications

Molecular oxygen may be necessary for life but with its beneficial properties comes formation of potentially toxic reactive oxygen species. One of the ways in which bacteria protect themselves is explained

A s the premier biological electron acceptor, molecular oxygen (O 2 ) serves a vital role in fundamental cellular functions, including the process of aerobic respiration. Nevertheless, with the benefi cial properties of O 2 comes the inadvertent formation of reactive oxygen species, including superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (•OH); these differ from O 2 in having one, two, and three additional electrons, respectively ( Figure 1). Cells also encounter elevated levels of these reactive oxygen species when they are released by animals, plants, and insects as a defense against detrimental organisms such as microbial pathogens. Reactive oxygen species can damage cells in many ways: by inactivating proteins, damaging nucleic acids, and altering the fatty acids of lipids, which leads in turn to perturbations in membrane structure and function. The accumulation of this oxidative damage underlies the formation of many disease states in humans. It is postulated that tissue injury by these reactive oxygen species accumulates over a long period of time and plays roles in the aging process and the development of heart disease, diabetes, chronic infl ammatory diseases, cancer, and several neurodegenerative diseases (Halliwell 1999).
Many organisms have evolved strategies to remove reactive oxygen species and repair damage, which have enabled them to prosper from the tremendous oxidizing potential of O 2 without succumbing to oxidative damage. Bacteria, yeast, and mammalian cells all induce the synthesis of global regulatory responses 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 O 2 − damage identifi ed in bacteria is a class of The four-electron reduction of molecular O 2 generates two molecules of H 2 O, which is O 2 in its most reduced form. While this reduction normally occurs within the enzyme cytochrome oxidase, one-electron transfers to O 2 also occur outside of cytochrome oxidase via inadvertent reactions with other reduced electron carriers, resulting in partially reduced and reactive forms of O 2 . H 2 O 2 is also produced by the enzymatic or spontaneous dismutation of O 2 − , and •OH is generated by the reaction of iron with H 2 O 2 (the Fenton reaction). In addition, the reactive oxygen intermediates are produced by a variety of organisms as a defense against microbial invasion. (Illustration: Rusty Howson, sososo design) 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 O 2 − lead to a requirement for certain amino acids in growth media (Imlay and Fridovich 1991). H 2 O 2 is well known for its role in oxidizing thiol (SH) groups of cysteinyl amino acid residues in proteins. Elevated levels of H 2 O 2 also are associated with the oxidation of other amino acids, leading to the formation of methionine sulfoxide and a variety of carbonyls. Lastly, because of its extreme reactivity, •OH targets all of the major macromolecules of cells: RNA, DNA, protein, and lipids. The extent to which membrane lipids are targets appears to depend on the presence of polyunsaturated fatty acids in lipids, which are not as prevalent in bacteria as they are in mammals.
Many enzymes that protect against oxidative damage have been identifi ed in E. coli (Imlay 2002(Imlay , 2003 There is no enzymatic mechanism for decreasing levels of •OH, produced from H 2 O 2 . Thus, levels of •OH will be directly proportional to levels of H 2 O 2 , and accordingly, catalase and alkyl hydroperoxide reductase activities are critical to oxidative stress survival. Another component to the oxidative stress response is the reduction of oxidized thiols that arises through one of the mechanisms described below. The tripeptide glutathione and the thiol reductants glutaredoxin and thioredoxin are key to the restoration of thiols to their reduced state (SH) (Fernandes and Holmgren 2004). E. coli contains three glutaredoxins that utilize the reducing power of glutathione to catalyze the reduction of disulfi de bonds (-S-S-) in the presence of NADPH and glutathione reductase. There are two thioredoxins in E. coli that also function to reduce disulfi de bonds.
Reduced thioredoxin is regenerated by thioredoxin reductase and NADPH. The fact that NADPH is required to maintain the reduced state of glutathione and thioredoxin indicates that the response to oxidative stress is coupled to the physiological status of core pathways that generate NADPH.

Regulatory Roles of Thiol Modifi cations
As mentioned above, proteins-in particular, the thiols of cysteines-are the major targets of H 2 O 2 . The reaction of cysteinyl thiolates with H 2 O 2 can lead to the formation of different modifi cations, such as sulfenic acid (-SOH), sulfi nic acid (-SO 2 H), and sulfonic acid (-SO3H), as well as disulfi de bond formation (-S-S-) and glutathione conjugation (-S-GSH) (Jacob et al. 2004;Poole et al. 2004) (Figure 2). These modifi cations often alter the structure and function of the protein. Recent progress in this fi eld points to a common chemistry in the reaction of H 2 O 2 with thiolates through the initial formation of sulfenic acid. In the case of proteins that have a nearby cysteinyl residue, a disulfi de bond forms between the two sulfur atoms. The sulfenated cysteinyl residue also can react with a cysteinyl residue on another protein or with glutathione, leading to a mixed disulfi de. If no cysteinyl residue is nearby, the sulfenated cysteine can be further oxidized to sulfi nic or sulfonic acid, or it can remain in the sulfenic acid state. All but the sulfi nic and sulfonic acid modifi cations are readily reversible by reduction, using proteins such as thioredoxin or glutaredoxin; though sulfi nic acid reductase activities have recently been identifi ed in yeast and mammalian cells (denoted sulfi redoxin and sestrin, respectively) (Biteau et al. 2003;Budanov et al. 2004).
Given the reversible nature of most forms of thiol oxidation, it has been suggested that thiol modifi cations can play roles in signal transduction that are similar to protein phosphorylation/ dephosphorylation (Sitia and Molteni 2004). In support of this model, there are several examples of proteins whose activities are modulated by thiol oxidation and reduction.
The fi rst of these examples is the OxyR transcription factor, which upregulates peroxide defenses in E. coli and a variety of other bacteria. OxyR contains two critical cysteines that are oxidized to form an intramolecular disulfi de bond when cells encounter peroxide stress (Zheng et al. 1998;Aslund et al. 1999). Disulfi de bond formation is associated with a conformational change that alters OxyR binding to DNA and allows the DOI: 10.1371/journal.pbio.0020400.g002

Figure 2. Thiol Modifi cations of Proteins
Formation of sulfenic acid from the reaction of H 2 O 2 with protein thiolates leads to different protein modifi cations, depending on the protein. In proteins without a 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 2 H) (e.g., thiolperoxidase; Tpx) and sulfonic acid (-SO 3 H) derivatives. Alternatively, if a second cysteinyl residue is in proximity within the same polypeptide (e.g., OxyR) or an associated protein (e.g., Yap1 and Orp1), a disulfi de bond can form between the two sulfur atoms (-S-S-). Lastly, the sulfenated cysteinyl residue can react with glutathione (GSH), leading to a mixed disulfi de (e.g., MetE). (Illustration: Rusty Howson, sososo design) protein to activate the transcription of genes encoding enzymes, such as catalase and the alkylhydroperoxide reductase, that destroy H 2 O 2 . Once the H 2 O 2 concentration is decreased, OxyR is reduced and the system is reset. The unusually reactive cysteine in OxyR that is oxidized by H 2 O 2 to form the sulfenic acid intermediate can clearly be nitrosylated and glutathionylated in vitro (Hausladen et al. 1996;Kim et al. 2002), but the in vivo relevance of these other modifi cations is questionable (Mukhopadhyay et al. 2004).
Two other examples of redoxregulated proteins are the E. coli chaperone protein Hsp33 (Jakob et al. 2000) and the Streptomyces coelicolor anti-sigma factor, RsrA (Li et al. 2003;Paget and Buttner 2003;Bae et al. 2004). For these proteins, the cysteine residues, which form intramolecular disulfi de bonds, are in a reduced state when coordinated to a zinc ion (Zn 2+ ), and zinc is released upon oxidation of the thiols. For both proteins, oxidation and zinc release are associated with an opening of the protein structure. For Hsp33, this structural change allows for dimerization and activates its chaperone activity (Graf et al. 2004). RsrA, on the other hand, dissociates from a promoter specifi city factor of RNA polymerase (an extracytoplasmicfunction-type alternative sigma factor) allowing the transcription of genes that permit recovery from the stress (Li et al. 2003;Bae et al. 2004). Among the target gene products is a thioredoxin, which reduces the disulfi de bonds that form within oxidized RsrA. Presumably, reduction of the disulfi de restores the binding of zinc and its inhibitory association with the sigma factor. Thus, the RsrA regulatory circuit provides another example, comparable to OxyR, in which the modifi cation of a regulatory protein thiol group can be linked to a change in the transcriptional output of genes that remediate stress.
The peroxide-sensing repressor OhrR from Xanthomonas campestris pv. phaseoli (Panmanee et al. 2002) and Bacillus subtilus (Fuangthong and Helmann 2002) can be inactivated by H 2 O 2 or by organic peroxides (ROOH) formed by the oxidation of a variety of organic molecules in the cell or in the environment. The B. subtilis OhrR transcription regulator contains only a single cysteine that forms a relatively stable sulfenic acid upon its reaction with H 2 O 2 or organic peroxides (Fuangthong and Helmann 2002). Oxidation of the single cysteine leads to the dissociation of OhrR from its DNA binding site and the derepression of the gene encoding an organic hydroperoxidase that eliminates the initial oxidizing insult.
In this issue, Hondorp and Matthews (2004) provide an example of a thiol modifi cation that protects an enzyme activity during oxidative stress. Their data suggest that when cells encounter oxidative stress, a key cysteinyl residue near the active site of methionine synthase (MetE) is glutathionylated. This modifi cation blocks access of the substrate and prevents further synthesis of methionine. This fi nding is signifi cant in that it presents a mechanism to reversibly preserve the function of a protein during oxidative challenge. By glutathionylating a single cysteinyl residue, the protein is protected from further oxidation of that cysteinyl residue to the irreversible sulfi nic and sulfonic acid forms. Once the stress is removed, the mixed disulfi de bond will be readily reduced, and access to the substrate restored.

Prevalence of Regulatory Thiol Modifi cations?
As illustrated by the examples above, an array of chemical modifi cations obtained by oxidizing cysteinyl residues has been exploited in combating oxidative stress. Yet it is important to note that not all cysteinyl residues of proteins are readily oxidized by oxidants such as H 2 O 2 . We do not currently understand all of the features that determine the reactivity of a particular thiol to H 2 O 2 (Poole et al. 2004). The pKa of the thiolates clearly plays an important role, as thiolates are more reactive than their protonated counterparts. In addition, the contribution of protein environment to the stability of the oxidized products is also known to be a factor, but is not well understood. Given that many of the thiol modifi cations do not appear to be in equilibrium with the redox state of the cell, the features of the protein that determine the rate at which the modifi cations are formed are another important parameter.
The added complexity of the cysteine targets that compose part of a Zn binding site found for Hsp33 and RsrA raises questions about the function of the zinc. Perhaps Zn binding provides some additional control over the reactivity of the cysteine thiols, or perhaps the loss of the zinc facilitates conformational changes. Recently, the oxidative, stress-induced thioredoxin-2 from E. coli has also been shown to contain a H 2 O 2 -labile zinc site, although the loss of zinc does not change its reductase activity (Collet et al. 2003). Thus, the way this oxidatively labile Zn site affects thioredoxin function has yet to be established.
The extent of thiol oxidation within the cell remains another open question. The variety of modifi cations that arise from treatment with H 2 O 2 and the experimental challenges associated with their detection has made it diffi cult to catalog all the proteins that are modifi ed and all the types of modifi cations that exist. In this issue, Leichert and Jakob (2004) report a general method for detecting cellular proteins whose cysteinyl residues were modifi ed after imposing an oxidative stress. Such an approach will greatly enhance our understanding of targets of oxidative stress. The method described by Leichert and Jakob also will be useful in detecting transient cysteine modifi cations.
The importance of monitoring transient changes in cysteines is highlighted by the recent fi nding that oxidation of the Yap1 activator of antioxidant genes in the yeast Saccharomyces cerevisiae requires a peroxidase denoted Gpx3 or Orp1 (Delaunay et al. 2002). In this case, H 2 O 2 reacts with a cysteine in Orp1, forming an unstable sulfenic acid intermediate that then reacts with a cysteinyl residue of Yap1 to form an intermolecular disulfi de. The disulfi de undergoes an exchange with a second cysteine within Yap1 to form an intramolecular disulfi de that locks Yap1 in a confi rmation that masks the nuclear export signal (Wood et al. 2004). Thus, methods that allow the appearance of thiol modifi cations in cells to be monitored kinetically will greatly enhance our understanding of how cysteine residues become oxidized.
The examples mentioned here illustrate the versatile potential of thiol modifi cations. Given the reversibility of thiol oxidations and the wide range of structural constraints that can be imposed by the formation of a sulfenic or sulfi nic acid or a disulfi de bond, we predict there will be many more examples of regulation by thiol modifi cation.