Despite its toxicity, H(2)O(2) is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H(2)O(2) concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H(2)O(2) around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H(2)O(2) elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress.

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The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. However, by metabolic labeling of mammalian cells with 35 S, we show that the sulfinic form of peroxiredoxin I, produced during the exposure of cells to H 2 O 2 , is rapidly reduced to the catalytically active thiol form. The mammalian cells' ability to reduce protein sulfinic acid might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated.

By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Peroxiredoxins are a family of peroxidases that reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol, respectively, with the use of reducing equivalents provided by thiol-containing proteins (1-3). The first peroxiredoxin (Prx) 1 proteins to be discovered were a 25-kDa yeast protein initially called thiol-specific antioxidant enzyme (4 -6), and a 21-kDa Salmonella typhimurium alkyl-hydroperoxide reductase termed AhpC (7-10). Subsequently, a mammalian homolog of thiol-specific antioxidant/AhpC was purified; and it, together with thiol-specific antioxidant and AhpC, defined a family of peroxidases that now includes six mammalian isoforms (Prx I-VI) and members identified in organisms from each kingdom (3, 10, 11).All Prx proteins contain a conserved Cys residue, which corresponds to Cys 51 in mammalian Prx I, in the N-terminal portion of the molecule (10, 11). The majority of Prx proteins, including four (Prx I-IV) of six mammalian peroxiredoxins, contain an additional conserved Cys residue in the C-terminal region that corresponds to Cys 172 in mammalian Prx I (2, 3, 12, 13). The Prx enzymes containing two conserved Cys residues are thus called 2-Cys Prx, in comparison with a small number of Prx proteins termed 1-Cys Prx, which contain only one conserved cysteine residue in the N-terminal domain (2, 10). In 2-Cys Prx enzymes, the N-terminal conserved cysteine is oxidized by H 2 O 2 to cysteine-sulfenic acid (Cys 51 -SOH), which then reacts with Cys 172 -SH of the other subunit to produce an intermolecular disulfide (1, 14). Reduction of the disulfide intermediate of Prx I-IV is specific in that it can be achieved by thioredoxin (Trx), but not by GSH or glutaredoxin (15, 16). Thus, the reducing equivalents for the peroxidase activity of Prx I-IV are ultimately derived from NADPH via thioredoxin reductase (TrxR) and Trx. Not all Prx enzymes containing both conserved Cys residues are reduced by Trx: the bacterial Prx AhpC is reduced by AhpF, which contains both Trx and TrxR domains (10, 17). In the absence of a physiological electron donor, the peroxidase activities of 2-Cys Prx enzymes can be supported by small thiol molecules such as dithiothreitol (DTT) and 2-mercaptoethanol, but not by GSH (1, 14, 15). In 1-Cys Prx enzymes, including mammalian Prx VI, the conserved cysteine is also the site of oxidation, but remains in a sulfenic acid state upon oxidation because there is no nearby partner cysteine to form a disulfide bond (18,19). Trx cannot reduce this sulfenic acid-containing intermediate (18,20). GSH has been proposed to be the electron donor, but these data remain co...

Peroxiredoxins (Prxs) are a family of peroxidases that reduce peroxides, with a conserved cysteine residue (the peroxidatic Cys) serving as the site of oxidation by peroxides. Peroxides oxidize the peroxidatic Cys-SH to Cys-SOH, which then reacts with another cysteine residue (typically the resolving Cys [C(R)]) to form a disulfide that is subsequently reduced by an appropriate electron donor. On the basis of the location or absence of the C(R), Prxs are classified into 2-Cys, atypical 2-Cys, and 1-Cys Prx subfamilies. In addition to their peroxidase activity, members of the 2-Cys Prx subfamily appear to serve as peroxide sensors for other proteins and as molecular chaperones. During catalysis, the peroxidatic Cys-SOH of 2-Cys Prxs is occasionally further oxidized to Cys-SO(2)H before disulfide formation, resulting in inactivation of peroxidase activity. This hyperoxidation, which is reversed by the ATP-dependent enzyme sulfiredoxin, modulates the sensor and chaperone functions of 2-Cys Prxs. The peroxidase activity of 2-Cys Prxs is extensively regulated via tyrosine and threonine phosphorylation, which allows modulation of the local concentration of the intracellular messenger H(2)O(2). Finally, 2-Cys Prxs interact with a variety of proteins, with such interaction having been shown to modulate the function of the binding partners in a reciprocal manner.

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The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.