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 x 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 H₂O₂, as dimerization was observed after exposure of 5 × 10⁶ cells/mL to 0.5 μM H₂O₂. 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 H₂O₂, which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H₂O₂ noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H₂O₂ concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress.

Typical 2-Cys peroxiredoxins (Prxs) react rapidly with H₂O₂ to form a sulfenic acid, which then condenses with the resolving cysteine of the adjacent Prx in the homodimer or reacts with another H₂O₂ to become hyperoxidized. Hyperoxidation inactivates the Prx and is implicated in cell signaling. Prxs vary in susceptibility to hyperoxidation. We determined rate constants for disulfide formation and hyperoxidation for human recombinant Prx2 and Prx3 by analyzing the relative proportions of hyperoxidized and dimeric products using mass spectrometry as a function of H₂O₂ concentration (in the absence of reductive cycling) and in competition with catalase at a fixed concentration of H₂O₂. This gave a second order rate constant for hyperoxidation of 12,000 m⁻¹ s⁻¹ and a rate constant for disulfide formation of 2 s⁻¹ for Prx2. A similar hyperoxidation rate constant for Prx3 was measured, but its rate of disulfide formation was ∼10-fold higher, making it is more resistant than Prx2 to hyperoxidation. There are two active sites within the homodimer, and at low H₂O₂ concentrations one site was hyperoxidized and the other present as a disulfide. Prx with two hyperoxidized sites formed progressively at higher H₂O₂ concentrations. Although the sulfenic acid forms of Prx2 and Prx3 are ∼1000-fold less reactive with H₂O₂ than their active site thiols, they react several orders of magnitude faster than most reduced thiol proteins. This observation has important implications for understanding the mechanism of peroxide sensing in cells.