Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 10⁵ and 2.3 × 10⁶ m⁻¹ s⁻¹ for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k₃ = 0.31 s⁻¹) than Prx1 (k₃ = 14.9 s⁻¹). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.

2-Cys peroxiredoxins (Prxs) rapidly reduce H₂O₂, thereby acting as antioxidants and also as sensors and transmitters of H₂O₂ signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H₂O₂ levels. Under these conditions, H₂O₂ oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (C_PSOH) to the sulfinate (C_PSO₂⁻) and sulfonated (C_PSO₃⁻) forms, redirecting the C_PSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the C_PSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H₂O₂ and was selected here to investigate the effect of a physiological concentration of HCO₃⁻/CO₂ (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO₃⁻/CO₂ increases Prx1 hyperoxidation and inactivation both in the presence of excess H₂O₂ and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO₃⁻/CO₂ was due to HCO₄⁻, a peroxide present in equilibrated solutions of H₂O₂ and HCO₃⁻/CO₂. Indeed, additional experiments and calculations uncovered that HCO₄⁻ oxidizes C_PSOH to C_PSO₂⁻ with a second-order rate constant 2 orders of magnitude higher than that of H₂O₂ ((1.5 ± 0.1) × 10⁵ and (2.9 ± 0.2) × 10³ m⁻¹·s⁻¹, respectively) and that HCO₄⁻ is 250 times more efficient than H₂O₂ at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO₃⁻/CO₂ pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.

Protein disulfide isomerase (PDI) is a ubiquitous dithiol−disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell damaging events. PDI can become dysfunctional by post−translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox−active Cys residues (Cys ⁵³ and Cys³⁹⁷) by hydrogen peroxide (k = (17.3 ± 1.3) M⁻¹ s⁻¹ at pH= 7.4, 25°C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerable faster (k= (6.9 ± 0.2) × 10⁴ M⁻¹ s⁻¹), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox−active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides (i.e. Cys⁵³ −Cys⁵⁶ and Cys³⁹⁷− Cys ⁴⁰⁰). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active−site residues (Trp⁵², Trp³⁹⁶, and Tyr³⁹³). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI.