Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin

Abstract

The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

Fig. 1.

Catalytic cycle of 2-Cys Prx, overoxidation by peroxide, and proposed reaction mechanism of retroreduction by Srx. On the left part of the figure the peroxide oxidizes the N-terminal cysteine (the peroxidatic cysteine, Cys-S_PH) of one subunit to sulphenic acid (Cys-S_POH) (1), which reacts with the C-terminal cysteine (the resolving cysteine, Cys-S_RH) of the other subunit to form an intermolecular disulphide that is reduced. However, at high concentrations of H₂O₂ the peroxidatic cysteine can be overoxidized to sulphinic acid (Cys-S_PO₂H) (2) inactivating the enzyme. According to the most accepted mechanism of retroreduction the sulphinic acid is phosphorylated, through a reversible step, by a direct attack on the γ-phosphate of ATP in the presence of Srx (3). The phosphoryl ester ($\text{Prx}-{\text{SO}}_{\text{2}}-{\text{PO}}_{\text{3}}^{\text{2}-}$) converted into a thiosulphynate (Prx-SO-S-Srx) and inorganic phosphate (P_i) is released (4). A reducing agent (R-SH) (Trx, GSH or DTT) reduces this complex to release Prx-Cys-S_POH and Srx-S-S-R (5) that is subsequently reduced to Srx-SH (6).

Fig. 2.

Purification of recombinant AtSrx and confirmation of the Srx identity. (A) SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract; lane 2, after His-tag column; lane 3, pure protein after Mono Q chromatography. (B) Western blot with a specific antibody against Srx.

Fig. 3.

Assessment of the production of overoxidized forms of At-2-Cys Prx. (A) Purified 2-Cys Prx was overoxidized to the Prx-SO₂H form in the presence of H₂O₂, and then treated with AMS as described in the Materials and methods. After SDS-PAGE, reduced (2× AMS) and overoxidized (1× AMS) forms of 2-Cys Prx were visualized with Western blot with an antibody against 2-Cys Prx. The reduced form of 2-Cys Prx without AMS is also visualized as a control. (B) Western blot of reduced and oxidized forms of purified 2-Cys Prx with antibodies against hyperoxidized Prx (2-Cys Prx-SO₂H) and 2-Cys Prx.

Fig. 4.

Correlation between Srx activity measured by the determination of released phosphate and that using specific antibodies against hyperoxidized Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl₂, 250 μM ATP, 10 mM GSH, 15 μM Prx-SO₂H, and 5 μM Srx was incubated at 30 °C. (A) At the indicated times, aliquots were withdrawn and subjected to Western blot with specific antibodies against Prx-SO₂H and 2-Cys Prx (control). (B) The concentration of Prx-SO₂H reduced was determined from the corresponding Western blot band intensity (by analysed imaging) and plotted against the concentration of phosphate determined in the same samples.

Fig. 5.

Srx enzymatic activity determined by the quatification of the phosphate resulting from the hydrolysis of ATP. Reaction mixtures containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl₂, 250 μM or 1 mM ATP and various concentrations of 2-Cys Prx (reduced and overoxidized) and Srx were incubated at 30 °C. At the indicated times, aliquots of 100 μl were withdrawn and subjected to phosphate determination as described in the Materials and methods. (A) The reaction was carried out with 250 μM ATP, 5 μM Srx, and various concentrations of Prx-SO₂H, namely, 30 μM (closed circles), 20 μM (open diamonds), 15 μM (open squares); 15 μM and 10 mM GSH (closed triangles); control with 15 μM Prx-SH (open circles). (B) The reaction was carried out with 1 mM ATP, 15 μM Prx-SO₂H, and various concentrations of Srx, namely, 5 μM (open circles), 2.5 μM (closed squares), 1 μM (open diamonds); 2.5 μM and 10 mM GSH (open triangles); control without Srx (open squares). Data are means ±SD of values from three independent experiments.

Fig. 6.

Recovery of the peroxidase activity of the sulphinic form of Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl₂, 1 mM ATP, 10 mM GSH, 5 μM Prx-SO₂H, and 1 μM Srx was incubated at 30 °C. At 1, 2, and 3 h aliquots were withdrawn and peroxidase activity was determined at the indicated times, as described in the Materials and methods. Peroxidase activity of recombinant reduced 2-Cys Prx (Prx-SH) and overoxidized Prx (Prx-SO₂H) were also determined. Peroxidase activity without Prx was determined as a control.

Fig. 7.

Activity of Srx variants. (A) Activity was measured as described in Fig. 5, in the presence of 250 μM ATP, 15 μM Prx-SOOH, and 5 μM Srx, namely, AtSrx WT (open squares); R28Q Srx (closed triangles); K40Q Srx (open circles); C72S Srx (closed squares); E76A Srx (closed triangles), and R28Q/E76A Srx (open diamonds). (B) Activity was measured with the same mix of reactions, adding 10 mM of GSH. Data are means ±SD of values from three independent experiments.

Fig. 8.

FPLC analysis of Srx activity. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl₂, 100 μM ATP, 15 μM Srx, and 30 μM Prx-SO₂H (AtSrx and variants) were incubated at 30 °C. Aliquots of 200 μl were withdrawn at the indicated times and the ADP formed was separated from ATP in a Mono Q anion exchange column as indicated in the Materials and methods. (A) AtSrx activity with Prx-SO₂H after the indicated times. (B) Variants Srx activity R28Q, K40Q, and C72S with Prx-SO₂H after 3 h of reaction.

Fig. 9.

Characterization of ΔPrx B and ΔSrx and its response to oxidative stress. (A) Phenotype of untreated plants at 5 weeks old under a long cycle photoperiod. (B) Western blot of levels of total Prx and overoxidized Prx (2-Cys Prx-SO₂H) in 10 μg of total leaf protein and levels of Srx in 20 μg of total leaf protein from untreated plants and after 24 h or 48 h of 20 mM H₂O₂ treatment. (C) Fluorescence measurements (F_v/F_m) and staining with DAB of H₂O₂ and ${\text{O}}_{\text{2}}^{-}$ in leaves from untreated plants and treated for 24 h or 48 h with 20 mM H₂O₂. (D) Mutants grown on MS medium with 20 mM and 50 mM H₂O₂.