Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2

Abstract

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.

Keywords: hydrogen peroxide; inflammation; oxidation-reduction (redox); peroxiredoxin; urate hydroperoxide; uric acid.

Figure 1.

Oxidation of Prx1 and Prx2 by urate hydroperoxide. Pre-reduced Prx1 and Prx2 (2 μm) were incubated in 50 mm sodium phosphate buffer, pH 7.4, with 0, 2, 5, 10, 20, 50, and 100 μm urate hydroperoxide for 5 min at room temperature. After reaction, 30 mm NEM was added to prevent further oxidation. Reduced and disulfide forms of Prx run as monomers and dimers in non-reducing SDS-polyacrylamide gel, respectively. These results are representative of three independent experiments.

Figure 2.

Kinetics of the oxidation of Prx2 by urate hydroperoxide. A, pre-reduced Prx2 (2 μm) was incubated with 20 μm urate hydroperoxide in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). Reactions were monitored over time by the variation of intrinsic protein fluorescence (λ_ex = 280 nm, emission filter >330 nm) in the stopped-flow instrument. B, first rapid phase of the reaction of Prx2 (2 μm) with 20 μm urate hydroperoxide. Observed rate constants (k_obs) were calculated by single exponential equation. Inset shows the slower linear decay of Prx2 fluorescence. C, plot of k_obs of the first rapid phase of the reaction of Prx2 versus urate hydroperoxide concentration. The second-order rate constant was calculated from this slope. D, fluorescence increase during the slow phase of the reaction of Prx2 (2 μm) with 20 μm urate hydroperoxide. Observed rate constants (k_obs) were calculated by single exponential equation. E, plot for the k_obs of the slow phase of the reaction of Prx2 versus urate hydroperoxide concentration. Non-linear curve was best fitted with a hyperbolic equation. V, voltage.

Figure 3.

Kinetics of the oxidation of WTPrx1 and Prx1C83S/C173S by urate hydroperoxide. A, first rapid phase of the reaction of pre-reduced Prx1 (5 μm) incubated with 35 μm urate hydroperoxide in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). Reactions were monitored over time by the variation of intrinsic protein fluorescence (λ_ex = 280 nm, emission filter >330 nm) in the stopped-flow instrument. Observed rate constants (k_obs) were calculated by single exponential equation. B, plot of k_obs of the first rapid phase reaction of Prx1 versus urate hydroperoxide concentration. The second-order rate constant was calculated from this slope. C, fluorescence increase during the slow phase of the reaction of Prx1 (5 μm) with 240 μm urate hydroperoxide. Observed rate constants (k_obs) were calculated by single exponential equation. D, plot of k_obs of the slow phase of the reaction of Prx1 versus urate hydroperoxide. Non-linear curve was best fitted with a hyperbolic equation. E, first rapid phase of the reaction of pre-reduced Prx1 that was mutated at the resolving (Cys-173) and non-catalytic cysteine (Cys-83) (Prx1C83S/C173S, 5 μm) incubated with 65 μm urate hydroperoxide in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). Observed rate constants (k_obs) were calculated by single exponential plus straight line equation. F, plot of k_obs of the first rapid phase of the reaction of Prx1 versus urate hydroperoxide concentration. V, voltage.

Figure 4.

Kinetics of the oxidation of Prx1 by hydrogen peroxide. Pre-reduced Prx1 (5 μm) was incubated with sub-stoichiometric concentrations of hydrogen peroxide (0.1–2.5 μm) in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). The reaction was monitored over time by the variation of intrinsic protein fluorescence (λ_ex = 280 nm, emission filter >330 nm) in the stopped-flow instrument. A, initial rates were calculated from the slope within 10% total fluorescence decay and plotted against hydrogen peroxide concentration. B, observed rate constants (k_obs) of the fluorescence increase in the slow phase of the reaction of Prx1 (5 μm) with hydrogen peroxide (2–30 μm) were calculated by single exponential equation and plotted against hydrogen peroxide concentration. C, competition kinetics for the oxidation of Prx1 by hydrogen peroxide. Horseradish peroxidase (HRP, 8 μm) was incubated with sub-stoichiometric concentration of hydrogen peroxide (4 μm) in 50 mm phosphate buffer (pH 7.4; 22 °C) plus 100 μm DTPA. The inhibition in the formation of compound I was dependent on the concentration of Prx1 (pre-reduced Prx1 ∼3.1 μmol of SH·μmol of protein⁻¹). D, linear plot of (F/1 − F)·k_HRP·[HRP] versus [Prx1] (slope = k_Prx1 = 3.5 ± 0.10 × 10⁷ m⁻¹s⁻¹). Each bar represents the mean ± S.E.M. of three experiments. V, voltage.

Model 1

Model 2

Figure 5.

Fitting of Prx fluorescence changes during the reaction with urate hydroperoxide or hydrogen peroxide simulated as Model 1 or Model 2. The reaction mechanisms proposed in Model 1 (A, C, and E) and Model 2 (B, D, and F) are depicted under “Results.” GEPASI (3.0) was used to perform the simulation and fitting of the experimental data. Experimental (black lines) and simulated data (red lines) are represented for the reaction of 2 μm Prx2 with 33 μm urate hydroperoxide (A and B); 5 μm Prx1 with 37.5 μm urate hydroperoxide (C and D); 5 μm Prx1 with 2.5 μm hydrogen peroxide (E and F).

Figure 6.

Analysis of Prx hyperoxidation by urate hydroperoxide and hydrogen peroxide. Prx1 (5 μm) (A) and Prx2 (5 μm) (B) were incubated in 50 mm sodium phosphate buffer, pH 7.4, with 220 μm urate hydroperoxide for 5 min at room temperature. Afterward, DTT was added in 3× molar excess per thiol and incubated for 2 h at 37 °C. Thiols were quantified by DTNB before and after reduction. Statistical analyses were performed by one-way analyses of variance (ANOVA); ***, p < 0.001 followed by Bonferroni's test, when compared with control group. C, control; HOOU, urate hydroperoxide. Each bar represents the mean ± S.E. of three independent experiments. C, enzymes were incubated with different concentrations of peroxides in 50 mm sodium phosphate buffer, pH 7.4, for 5 min at room temperature. The samples were separated in a non-reducing SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride, and probed with an antibody to Prx-SO_2/3. These results are representative of three experiments.

Figure 7.

Oxidation of Prx2 in erythrocytes by urate hydroperoxide or hydrogen peroxide. Human erythrocytes (1 × 10⁷) were incubated in 10 mm PBS, pH 7.4, plus 5 mm glucose with acetonitrile evaporated MP, 200 μm hydrogen peroxide, or 200 μm urate hydroperoxide for 10 min at 37 °C. Western blot analysis of Prx2 from erythrocyte homogenates (100 μg/lane) (A) and supernatant (10 μg/lane) (B) in non-reducing SDS-polyacrylamide gel. Reduced and disulfide forms of Prx ran as monomers and dimers in non-reducing SDS-polyacrylamide gel, respectively. Semi-quantitative band intensity of Prx2 monomer (C) and homodimer (D) from cell pellet was normalized by α-tubulin. E, band intensity of Prx2 homodimer in supernatants. F, absorbance of human erythrocyte supernatants (10⁷) were measured at 405 nm and compared with the positive control (0.1% SDS). The percentage of hemolysis in the samples was relative to the positive control (100%). Each bar represents the mean ± S.D. of three independent experiments. Statistical analyses were performed by one-way analyses of variance (ANOVA); ***, p < 0.001 followed by Bonferroni's test, when compared with control group. C, control; HOOU, urate hydroperoxide. These results are representative of three experiments.

Figure 8.

Hypothetical mechanism for the oxidation of Prx2 by urate hydroperoxide.