Reduction of 1-Cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing another function of vitamin C

Abstract

Peroxiredoxins (Prx) are widely distributed peroxidases that can be divided into 1-Cys and 2-Cys Prx groups based on the number of conserved cysteine residues that participate in their catalytical cycle. Prx have been described to be strictly dependent on thiols, but here, we show that ascorbate (vitamin C) also reduces 1-Cys Prx, but not 2-Cys Prx, from several taxonomic groups. Reduction by ascorbate is partly related to the fact that the oxidized form of 1-Cys Prx is a stable sulfenic acid (Cys-SOH) instead of a disulfide. In addition, a histidine residue in the active site is required. In fact, we engineered a 2-Cys Prx with these two features, and it displayed ascorbate peroxidase activity. These data represent a breakthrough in the thiol-specific antioxidant paradigm. Ascorbate may be the long-sought-after biological reductant of 1-Cys Prx. Because ascorbate is present in high amounts in cells, the ascorbate/protein sulfenic acid pair represents an aspect of redox biochemistry that has yet to be explored in vivo.

Fig. 1.

Characterization of yPrx1 ascorbate-dependent peroxidase activity. (A–C) Glutamine synthetase (GS) protection from inactivation by metal-catalyzed oxidation systems containing DTT (10 mM) (gray bars) or ascorbate (10 mM) (white bars) in 15-min incubations at 37°C. GS activity was considered 100% when it was not preincubated with metal-catalyzed oxidation systems. (A) GS protection by increasing yPrx1 concentrations. (B) Inability of other yeast Prx enzymes to use ascorbate. Tsa1, gi: 464970; Tsa2, gi: 2499475; or Ahp1, gi: 1709682. (C) Inhibition of yPrxI protective effects by inactivation of cysteine residues. yPrxI was modified by N-ethylmaleimide (NEM) and by site-specific mutagenesis as described in Experimental Procedures. Afterward, a GS protection assay was performed as described above by using yPrx1 at 3 μM or in excess of 70 μM, indicated by ∗. (D) Deconvoluted MS spectra of yPrxI in different oxidation states treated with dimedone (140 amu). Samples of reduced yPrx1 (initial), treated with H₂O₂ and dimedone (oxidized) or treated with H₂O₂, ascorbate, and dimedone (ascorbate) were prepared and analyzed as described in Experimental Procedures. Molecular masses were calculated by the Transform algorithm of the BioLynx Data Analysis package of MassLynx Software (Micromass). Data were acquired in an ESI-Q-TOF (Micromass) mass spectrometer under positive ionization mode. Calculated molecular masses are indicated at the right side of the corresponding spectra. These spectra were obtained by using a recombinant yPrx1 (100 μM) without the 40 first amino acids residues and containing only one cysteine residue, which is Cys_p (see supporting information on the PNAS web site for more details). All excess reagents were washed away by ultrafiltration before activity analysis.

Fig. 2.

The 2-Cys Prx engineered for acquiring the ascorbate-dependent peroxidase activity. (A) GS protection assay was used to measure DTT- (gray bars) and ascorbate-dependent (white bars) peroxidase activity of the Tsa1 mutants. Each enzyme (50 μM) was incubated as described in the Fig. 1 legend. (B) Analysis of the oxidation state of cysteine residues from Tsa1 and the mutants C170S, I40H, and C170S-I40H by the NBD-Cl assay. Treatments and assays were performed as described in Experimental Procedures. Sulfhydryl and sulfenic acid adducts peak at 420 nm and 347 nm, respectively, with similar extinction coefficients (46).

Fig. 3.

Enzymatic and biological investigations of the ascorbate-dependent peroxidase activity of rat Prdx6. (A–C) Enzymatic activity was monitored by ascorbate consumption at 265 nm in incubations containing Prdx6 (0.5 nM), H₂O₂ and ascorbate at the concentrations specified in the figures. (D) The ability of rat homogenates to support the peroxidase activity of Prdx6 was investigated before and after treatment with ascorbate oxidase as described in Experimental Procedures. The 100% value means the amount of H₂O₂ consumed during 15 min at 37°C by each homogenate in the absence of ascorbate oxidase pretreatment. The bars represent the percentage of H₂O₂ consumed when homogenates were pretreated with ascorbate oxidase.