Overoxidation of 2-Cys peroxiredoxin in prokaryotes: cyanobacterial 2-Cys peroxiredoxins sensitive to oxidative stress

Abstract

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.

FIGURE 1.

Phylogenetic analysis of 2-Cys Prxs from eukaryotic and prokaryotic organisms. Amino acid sequences were obtained from the National Center for Biotechnology Information (NCBI) protein database, and accession numbers of these sequences are presented in supplemental Table 1. The tree was obtained by the MEGA4 package, and the neighbor-joining method was applied with 1000 bootstrap replications. The scale bar indicates the frequency of substitutions per site. Blue, prokaryotic organisms; red, eukaryotic organisms. The box indicates the clade formed by cyanobacterial, algal, and plant enzymes. Anabaena represents sp. PCC 7120, and Synechocystis represents sp. PCC 6803 are represented. H. pylori, Helicobacter pylori; P. profundum, Photobacterium profundum; B. aphidicola, Buchnera aphidicola; E. canis, Ehrlichia canis; R. sibirica, Rickettsia sibirica; P. falciparum, Plasmodium falciparum; T. annulata, Theileria annulata; R. rubrum, Rhodospirillum rubrum; C. parvum, Cryptosporidium parvum; R. baltica, Rhodopirellula baltica; C. pneumoniae, Chlamydophila pneumoniae; C. tepidum, Chlorobium tepidum; O. sinensis, Odontella sinensis; P. purpurea, Porphyra purpurea; C. reinhardtii, Chlamydomonas reinhardtii; A. thaliana, Arabidopsis thaliana; O. sativa, Oryza sativa; L. interrogans, Leptospira interrogans; T. vaginalis, Trichomonas vaginalis; D. discoideum, Dictyostelium discoideum; C. elegans, Caenorhabditis elegans; T. brucei, Trypanosoma brucei; S. cerevisiae, Saccharomyces cerevisiae; S. pombe, Schizosaccharomyces pombe; C. neoformas, Cryptococcus neoformas; T. cruzi, Trypanosoma cruzi; B. glabrata, Biomphalaria glabrata; D. melanogaster, Drosophila melanogaster; H. sapiens, Homo sapiens; E. histolytica, Entamoeba histolytica; D. rerio, Danio rerio; C. pasteurianum, Clostridium pasteurianum; S. thermophilum, Symbiobacterium thermophilum; Z. mobilis, Zymomonas mobilis; B. japonicum, Bradyrhizobium japonicum; M. tuberculosis, Mycobacterium tuberculosis; S. usitatus, Solibacter usitatus; C. ruddii, Candidatus Carsonella ruddii; P. furiosus, Pyrococcus furiosus; T. pallidum, Treponema pallidum; E. faecalis, Enterococcus faecalis; P. gingivalis, Porphyromonas gingivalis; B. vietnamiensis, Burkholderia vietnamiensis; N. aromaticivorans, Novosphingobium aromaticivorans; S. typhimurium, Salmonella typhimurium; B. subtilis, Bacillus subtilis.

FIGURE 2.

Overoxidation of purified plant and cyanobacterial 2-Cys Prx in vitro. Purified recombinant 2-Cys Prx (25 μg of protein) from rice (Os), Anabaena (Ana), and Synechocystis (Syn) were incubated with 10 mm DTT and 5 mm hydrogen peroxide. At the indicated times, the reaction was stopped by protein precipitation with 5% trichloroacetic acid and washed twice with ice-cold acetone. Samples (0.3 μg of protein of the rice enzyme and 5 μg of Anabaena and Synechocystis enzymes) were then subjected to SDS-PAGE under reducing conditions. Western blot analysis was performed using antibodies against overoxidized 2-Cys Prx.

FIGURE 3.

Overoxidation of purified Anabaena 2-Cys Prx in vitro. A, purified recombinant wild type and mutant versions of Anabaena 2-Cys Prx (4 μm final concentration) was incubated for 5 min in the absence or presence of 0.5 mm hydrogen peroxide. Thereafter, DTT was added to each sample at a final concentration of 10 mm, and samples were incubated for another 10 min and precipitated with 5% trichloroacetic acid. Samples (1 μg of protein) were subjected to two-dimensional gel electrophoresis and proteins visualized with Coomassie Brilliant Blue. Cont, control; red, reduced; ox, overoxidized. B and C, MALDI-TOF analysis of mass-to-charge ratios (m/z) of mutant versions, as indicated, of 2-Cys Prx from Anabaena. Continuous lines, control; dashed lines, H₂O₂-treated. Intens. [a.u.], intensity (arbitrary units).

FIGURE 4.

Effect of high light on Anabaena and Synechocystis 2-Cys Prx overoxidation in vivo. A, cells grown to mid-exponential phase at 50 μE m⁻² s⁻¹ were diluted to a concentration of 2.5 μg of chlorophyll ml⁻¹ and exposed to a light intensity of 800 μE m⁻² s⁻¹. After 30 min of high light exposure, chloramphenicol (200 μg/ml final concentration) was added to one aliquot and incubated for a further 30 min (Cm). Cytosolic extracts of samples from Anabaena (5 μg of protein) and Synechocystis (25 μg of protein) harvested at the times indicated were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis. The overoxidized form of the 2-Cys Prxs is distinguished as a monomer. Molecular mass markers, in kDa, are indicated on the left. mon, monomer; dim, dimer. B, extracts (15 μg of protein) from Anabaena cultures at the indicated times of high light treatment were subjected to two-dimensional gel electrophoresis and Western blot. Ox, overoxidized; Red, reduced.

FIGURE 5.

Effect of hydrogen peroxide on Anabaena and Synechocystis 2-Cys Prx overoxidation in vivo. Cultures grown at 50 μE m⁻² s⁻¹ to a concentration of 5 μg of chlorophyll ml⁻¹ were incubated for 15 min with hydrogen peroxide at the indicated concentrations. Cultures treated with 0.5 mm hydrogen peroxide were also harvested 2 and 4 h after the addition of hydrogen peroxide. Western blot analysis was performed using antibodies raised against rice and Synechocystis 2-Cys Prx for Anabaena and Synechocystis samples, respectively. Molecular mass markers, in kDa, are indicated on the left. mon, monomer; dim, dimer.

FIGURE 6.

Comparison of plant and cyanobacterial 2-Cys Prx. A, titration in vitro of the purified plant and cyanobacterial 2-Cys Prx with DTT. Samples (0.8 μg of purified protein) of 2-Cys Prx from rice, Anabaena, and Synechocystis, as indicated, were treated with increasing concentrations of DTT for 10 min. Proteins were then subjected to SDS-PAGE, and bands stained with Coomassie Brilliant Blue were quantified with the ImageJ software. B, immunological characterization of plant and cyanobacterial 2-Cys Prx. For Western blot analysis, 50 ng of the purified 2-Cys Prx from Synechocystis (Syn), Anabaena (Ana), or rice (Os) were subjected to SDS-PAGE and probed with antibodies raised against the rice (α-Os 2Cys Prx) and Synechocystis 2-Cys Prx (α-Syn 2Cys Prx), as indicated. Molecular mass markers, in kDa, are indicated on the left.

FIGURE 7.

Kinetics of hydrogen peroxide decomposition in cultures of Anabaena and Synechocystis. A, cultures were grown to mid-exponential phase, diluted to a concentration of 3.5 μg of chlorophyll ml⁻¹ and incubated under normal light conditions in the presence or absence of 0.1 mm NH₂OH, which inhibits the catalase-peroxidase activity. At time 0, hydrogen peroxide was added to a final concentration of 0.5 mm. The remaining amounts of hydrogen peroxide were monitored using the ferrous oxidation of xylenol orange assay. ▴ and △, Synechocystis in the absence (▴) or presence (△) of NH₂OH; ● and ○, Anabaena in the absence (●) or presence (○) of NH₂OH. B, effect of hydrogen peroxide on Anabaena and Synechocystis survival. Cyanobacterial cultures in mid-exponential growth phase diluted to a concentration of 2.5 μg of chlorophyll ml⁻¹ were incubated for 24 h under standard light conditions in the presence of up to 10 mm hydrogen peroxide.