Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H 2 O 2 . Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 ؋ 10 7 M ؊1 s ؊1 for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H 2 O 2 , as dimerization was observed after exposure of 5 ؋ 10 6 cells/mL to 0.5 M H 2 O 2 . In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/ sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H 2 O 2 , which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H 2 O 2 noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H 2 O 2 concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress. ( IntroductionThe peroxiredoxins (Prxs) constitute a family of homodimeric peroxidases that reduce H 2 O 2 and alkyl hydroperoxides to water and alcohol, respectively. They rely on a conserved cysteine residue to catalyze peroxide reduction. There are 6 known mammalian isoforms (Prx1-6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and number of cysteines involved during catalysis. 1 When peroxiredoxin 2 (Prx2; a typical 2-Cys Prx) reacts with peroxide, the peroxidatic cysteine at the active site on one subunit is oxidized to a sulfenic acid. A second conserved cysteine at the C-terminal end of the other subunit (the resolving cysteine) then reacts with the sulfenic acid to form a disulfide bridge. Reduction of the disulfide by thioredoxin (Trx) regenerates Prx2 and completes the cycle. Trx is in turn regenerated by thioredoxin reductase (TrxR), with reducing equivalents derived from NADPH. 2 An intriguing feature of mammalian 2-Cys Prxs is that in the presence of high levels of peroxide, the peroxidatic Cys becomes overoxidized to the sulfinic (SO 2 H) or sulfonic (SO 3 H) acid form. 3 This abolishes the enzyme's peroxidase activity, although overoxidized Prx can be slowly reverted to the reduced state by sulfiredoxin. 4 It has been suggested that overoxidation allows intracellular accumulation of H 2 O 2 , which can then function as a signal transducer for various pathways. 5,6 Compared with other somatic cells, erythrocytes are exposed to oxidative stress from a wide variety of sources. They contain high levels of O 2 and hemoglobin (Hb), which continually autoxidizes to produce O 2 Ϫ and H 2 O 2 . They also have membranes...

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Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (SOD) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, SOD 15 and SOD 3 with 2.3-fold and 3.6-fold increased Cu,Zn-SOD activities, respectively, and SOCAT 3 with a 3-fold higher catalase activity and 1.7-fold higher Cu,Zn-SOD activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone SOD 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-SOD mRNA in the mouse recipient cells. According to immunoblots the content of Mn-SOD was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in SOD 3 and SOD 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-SOD overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)