A comprehensive analysis of the peroxiredoxin reduction system in the Cyanobacterium Synechocystis sp. strain PCC 6803 reveals that all five peroxiredoxins are thioredoxin dependent

Abstract

Cyanobacteria perform oxygenic photosynthesis, which gives rise to the continuous production of reactive oxygen species, such as superoxide anion radicals and hydrogen peroxide, particularly under unfavorable growth conditions. Peroxiredoxins, which are present in both chloroplasts and cyanobacteria, constitute a class of thiol-dependent peroxidases capable of reducing hydrogen peroxide as well as alkyl hydroperoxides. Chloroplast peroxiredoxins have been studied extensively and have been found to use a variety of endogenous electron donors, such as thioredoxins, glutaredoxins, or cyclophilin, to sustain their activities. To date, however, the endogenous reduction systems for cyanobacterial peroxiredoxins have not been systematically studied. We have expressed and purified all five Synechocystis sp. strain PCC 6803 peroxiredoxins, which belong to the classes 1-Cys Prx, 2-Cys Prx, type II Prx (PrxII), and Prx Q, and we have examined their capacities to interact with and receive electrons from the m-, x-, and y-type thioredoxins from the same organism, which are called TrxA, TrxB, and TrxQ, respectively. Assays for peroxidase activity demonstrated that all five enzymes could use thioredoxins as electron donors, whereas glutathione and Synechocystis sp. strain PCC 6803 glutaredoxins were inefficient. The highest catalytic efficiency was obtained for the couple consisting of PrxII and TrxQ thioredoxin. Studies of transcript levels for the peroxiredoxins and thioredoxins under different stress conditions highlighted the similarity between the PrxII and TrxQ thioredoxin expression patterns.

FIG. 1.

Synechocystis sp. strain PCC 6803 peroxiredoxins. Four micrograms of each purified recombinant peroxiredoxin was incubated in the presence (A) or in the absence (B) of 100 mM DTT for 1 h on ice. Nonreduced samples (B) were solubilized in a buffer that included 50 mM iodoacetamide, whereas reduced samples (A) were solubilized in normal sample buffer. Thereafter, proteins were separated on 12% (A) or 10% (B) SDS-PAGE gels and stained with Coomassie brilliant blue.

FIG. 2.

Analysis of the thioredoxin-peroxiredoxin interaction. Twenty (A) or 11.25 (B and C) μg of each peroxiredoxin was incubated with 8 μg TrxA(C35S) (A), 4 μg TrxB(C34S) (B), or 4 μg TrxQ(C33S) (C). Thereafter, proteins were resolved on 12% SDS-PAGE gels under reducing (lower panels) or nonreducing (upper panels) conditions. Thioredoxins were detected by Western blotting using specific antibodies at the following dilutions: anti-TrxA (α-TrxA), 1:1,000; α-TrxB, 1:10,000; α-TrxQ, 1:5,000.

FIG. 3.

Thioredoxin-dependent peroxidase activities of 2-Cys Prx, PrxII, 1-Cys Prx, PrxQ1, and PrxQ2. Reaction mixtures contained 50 mM HEPES-NaOH (pH 7.0), 0.2 mM DTT, 4 μM Trx, 100 μM H₂O₂, and 25 μg Prx in a final volume of 250 μl. This concentration corresponds to 5 μM for 2-Cys Prx, 1-Cys Prx, and PrxII and to 5.5 μM for PrxQ1 and PrxQ2. The concentration of H₂O₂ was measured at 0, 10, 20, and 30 min after hydrogen peroxide addition. As a control, for each peroxiredoxin an assay was performed in the absence of thioredoxins but in the presence of DTT (dashed lines). The values at each time point are averages from at least three independent experiments.

FIG. 4.

Substrate specificities of 2-Cys Prx, PrxII, 1-Cys Prx, PrxQ1, and PrxQ2. Reaction mixtures contained 50 mM HEPES-NaOH (pH 7.0), 0.2 mM DTT, 4 μM TrxA, 25 μg Prx, and either 100 μM H₂O₂, 100 μM t-BOOH, or 50 μM CHP in a final volume of 250 μl. The reduction of peroxides was measured as described in Materials and Methods. The values at each time point are means from at least three independent experiments. Error bars, standard deviations.

FIG. 5.

Peroxidase activities of 2-Cys Prx, PrxII, 1-Cys Prx, PrxQ1, and PrxQ2 using GSH or Synechocystis glutaredoxins as electron donors. Each reaction mixture contained 75 mM potassium phosphate (pH 7.6), 100 μM GSH, 100 μM H₂O₂, and 25 μg Prx in a final volume of 250 μl. This concentration corresponds to 5 μM for 2-Cys Prx, 1-Cys Prx, and PrxII and to 5.5 μM for PrxQ1 and PrxQ2. (A) Reduction of hydrogen peroxide by peroxiredoxins by using GSH alone as the electron donor. Concentrations of hydrogen peroxide were determined by the FOX assay. (B) NADPH oxidation coupled to peroxiredoxin activity using GSH as the electron donor and glutathione reductase-dependent regeneration. To this end, reaction mixtures were supplemented with 0.15 U glutathione reductase from yeast and 10 mM NADPH. NADPH oxidation was measured spectrophotometrically at 340 nm. (C and D) Decomposition of hydrogen peroxide using a GSH-glutaredoxin system as the electron donor for the peroxiredoxins. Reaction mixtures were supplemented with 0.15 U glutathione reductase from yeast, 10 mM NADPH for regeneration, and either 4 μM GrxA (C) or 4 μM GrxB (D). Concentrations of hydrogen peroxide were determined by the FOX assay. Controls were performed with reaction mixtures containing no Prx but still containing the respective Grx (No Prx). Controls were also performed in the presence of NADPH, glutathione reductase, and GSH but without Grx and Prx (NADPH). The values at each time point are averages for at least three independent experiments.

FIG. 6.

Effects of hydrogen peroxide and high-light (HL) treatments on the expression of thioredoxin and peroxiredoxin genes. Synechocystis cells growing photoautotrophically were either treated with hydrogen peroxide (0.5 mM) (A and C) or exposed to a high light intensity (500 μE·m⁻² s⁻¹) (B and D). At the indicated times, samples were collected, and total RNA was obtained and hybridized with specific probes for each gene. Relative mRNA levels were determined and normalized to rnpB expression levels.

FIG. 7.

Effects of heat shock (HS) and nitrogen deprivation (−N) on the expression of thioredoxin and peroxiredoxin genes. Either Synechocystis cells growing photoautotrophically were subjected to a heat shock treatment (43°C) (A and C) or the culture was transferred to a medium lacking a nitrogen source (B and D). At the indicated times, samples were collected, and total RNA was obtained and hybridized with the specific probes for each gene. Relative mRNA levels were determined and normalized to rnpB expression levels.