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Originally published In Press as doi:10.1074/jbc.M411493200 on October 29, 2004 Originally published In Press as doi:10.1074/jbc.M411493200 on October 27, 2004

J. Biol. Chem., Vol. 280, Issue 1, 840-846, January 7, 2005
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Anti-oxidative Stress System in Cyanobacteria

SIGNIFICANCE OF TYPE II PEROXIREDOXIN AND THE ROLE OF 1-Cys PEROXIREDOXIN IN SYNECHOCYSTIS SP. STRAIN PCC 6803*

Naomi Hosoya-Matsuda{ddagger}, Ken Motohashi{ddagger}§, Hidehisa Yoshimura¶||, Akiko Nozaki{ddagger}**, Kazuhito Inoue**, Masayuki Ohmori¶{ddagger}{ddagger}, and Toru Hisabori{ddagger}§§§

From the {ddagger}Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, the §ATP System Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 5800-3 Nagatsuta-cho, Midori-ku, Yokohama 226-0026, the Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, and the **Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa 259-1293, Japan

Received for publication, October 8, 2004 , and in revised form, October 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two antioxidant proteins, SLL1621 and SLR1198, were captured in the cyanobacteria Synechocystis sp. PCC 6803 using thioredoxin affinity chromatography, which was first applied to the survey of thioredoxin target proteins in chloroplasts ( Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229[Abstract/Free Full Text]). They are annotated as AhpC/TSA family protein (SLL1621) and antioxidant protein (SLR1198) in CyanoBase ( Nakamura, Y., Kaneko, T., Hirosawa, M., Miyajima, N., and Tabata, S. (1998) Nucleic Acids Res. 26, 63-67[Abstract/Free Full Text]). Based on sequence homology analysis SLL1621 and SLR1198 are categorized into type II peroxiredoxin and 1-Cys type peroxiredoxin, respectively. In vitro interaction between SLL1621 and thioredoxin was confirmed using the recombinant proteins expressed in Escherichia coli. Furthermore, we found that SLL1621 shows remarkable glutathione-dependent peroxidase activity. Disruption of the sll1621 gene had a dramatic effect on the viability of the cyanobacterial cells even under weak light conditions (50 µmol·m-2·s-1), suggesting this peroxiredoxin is essential for this cyanobacterium. In contrast, although the peroxidase activity of SLR1198 was scarcely detected, disruption of the gene, slr1198, certainly affected the growth rate of the cells. The results indicate the physiological significance of two different peroxiredoxins as an anti-oxidative stress system in cyanobacteria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins (Prx),1 which are known as thiol peroxidases or thioredoxin peroxidases, catalyze the reduction of various hydroperoxides (1). In plants, Prx proteins are categorized into four subclasses based on their different subunit composition and the position of the conserved cysteine residues. 2-Cys Prx is a homodimeric enzyme where the two subunits are linked via a disulfide bond in the oxidized form (2-4). Chloroplastic Prx-Q, which is a homologue of the Escherichia coli bacterioferritin comigratory protein (Bcp), belongs to the second group and functions as a monomer (5, 6). Another group is the 1-Cys Prx containing single conserved catalytic cysteine (7-9). The last subgroup is named type II Prx. Members of this subfamily can use thioredoxin (Trx) as an electron donor (10) but also glutaredoxin (Grx), which is used at higher efficiency (11). This subclass has multiple isoforms localized in many subcellular compartments (12, 13).

In general, the catalytic Cys residues of Prx proteins, which are oxidized during the peroxide reduction, have to be reduced by electron donors such as Trx, Grx, or cyclophilins for the next catalytic cycle (4, 11, 14). Although broad substrate specificity has been reported for the Prx proteins (15-17), the catalytic properties of the plant Prx are not very well known.

The cyanobacterium Synechocystis sp. strain PCC 6803 is a prokaryotic photosynthetic microalgae, which is often used as a model organism for the study of photosynthesis and other important physiological processes of photosynthetic organisms. In addition, this organism was one of the first prokaryotes to be fully sequenced (18, 19). In the Synechocystis cells, the genes for 2-Cys Prx (sll0755), 1-Cys Prx (slr1198), Prx-Q (sll0221 and slr0242), and type II Prx (sll1621) were revealed by genome analysis. In addition, multiple genes for Trx and Trx-like proteins (sll0685, sll1057, sll1980, slr0233, slr0623, slr1139, and slr1796) were found. The reason for the existence of multiple genes for various Prx proteins, Trx, and their homologues in this unicellular microalgae is still unknown. So far, not very much is known on the relation between these multiple Prx and Trx proteins in cyanobacteria.

Among various Prx proteins in Synechocystis, the biochemical properties and the in vivo role of 2-Cys Prx, which is the gene product of sll0755, were investigated in detail by Klughammer et al. (20) and Yamamoto et al. (21). In addition, the role of the homologous peroxidase of Synechococcus sp. PCC 7942 was recently reported (22). In both strains, disruption of the gene for 2-Cys Prx certainly eliminated the tolerance against H2O2, supporting the role of this Prx as anti-oxidative stress protein, although the pathway of the reducing equivalents for 2-Cys Prx is not known.

In contrast, Muller and Buchanan (23) investigated the significance of TrxM by gene disruption in Anacystis nidulans R2, whose present nomenclature is Synechococcus sp. PCC 7942. Disruption of the gene for TrxM, which corresponds to slr0623 in Synechocystis sp. PCC 6803, gave lethality, suggesting this Trx gene is indispensable for photosynthetic growth. Gene disruption of a Trx-like protein in Synechococcus sp. PCC 7942, which corresponds to sll1980 in Synechocystis sp. PCC 6803, was also examined (24). From the results the authors suggested that this Trx-like protein is essential for the correct assembly of a photosynthetic protein complex. However, these studies did not provide information on all interacting partners for Trx or Trx-like protein.

Trx regulates the activity of various enzymes through formation or reduction of a disulfide bridge in the enzyme molecule (25-27). In the last couple of years a number of studies have been attempted to elucidate the variety of target proteins of Trxs in plant cells, in chloroplasts, and in bacteria using two-dimensional gel electrophoresis analysis (28) or Trx-affinity chromatography with immobilized mutant Trx (6, 29-31).

By using the Trx-affinity chromatography, we identified two anti-oxidative stress proteins, type II Prx, SLL1621 and 1-Cys Prx group protein, SLR1198, as major counterpart proteins of Trx in Synechocystis sp. PCC 6803. In the present study we investigated the physiological significance of these anti-oxidative stress proteins in cyanobacteria.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cyanogen bromide-activated Sepharose 4B was purchased from Amersham Bioscience. DTT, tert-butyl hydroperoxide (t-BOOH), cumene hydroperoxide (CHP), glutathione reductase purified from Saccharomyces cerevisiae, and glutaredoxin from E. coli were from Sigma. BCA Protein Assay Reagent was from Pierce. 4-Acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS) was from Invitrogen. Other chemicals were the highest grade commercially available.

Cloning, Expression, and Purification of TrxA from Synechocystis sp. PCC 6803—Genomic DNA from Synechocystis cells was used as a template for PCR. The coding region of trxA gene, slr1139, was amplified by PCR using two primers: 5'-cccggaattccatatgagtttactggaaatcaccgacg-3' (NdeI) and 5'-ccccgctcgagttaaataaaatccaattcctctttc-3' (XhoI). The restriction sites shown in parentheses are underlined in the sequences. The amplified DNA fragment was cloned into the NdeI and XhoI sites of pET-23c (Novagen), and the DNA sequence of the obtained plasmid was confirmed by DNA sequencing (Prism 310, Applied Biosystems).

The plasmid, TrxA-pET23c, was transformed into the expression host E. coli BL21(DE3), and the desired protein was overexpressed. The expressed protein was then purified at 0-4 °C as described previously (32). The concentration of the purified protein was determined by BCA Protein Assay Reagent using bovine serum albumin as standard.

Preparation of Trx Mutant—The plasmid for the TrxA mutant, TrxAC34S whose active site sequence was mutated to WCGPS, was generated using the Mega-primer method (33) with the expression plasmid of the slr1139 gene, TrxA-pET23c, as a template. The mutant protein was expressed in E. coli and purified by the method as for the wild type TrxA.

Preparation of the Trx-immobilized Resin—TrxA mutant (5 mg) in 100 mM sodium carbonate buffer (pH 8.3) containing 0.5 mM NaCl was incubated with 2 ml of CNBr-activated Sepharose 4B according to the manufacturer's instruction. More than 95% of TrxAC34S was immobilized on the resin.

Collection of the Target Protein by Immobilized Trx Mutant—To obtain the cytosol fraction, the glucose-tolerant strain of Synechocystis sp. PCC 6803 (34) was grown at 32 °C in BG-11 medium (35) under continuous illumination with white fluorescent light (30-50 µmol·m-2·s-1). The liquid culture of Synechocystis cells was bubbled with air containing 1% (v/v) CO2. Cell growth was measured by absorbance at 730 nm, and cultures whose absorbance value reached to 1.0 were collected by centrifugation. The collected cells were washed once with 50 mM Tris-HCl (pH 8.0) and re-suspended in 50 mM Tris-HCl (pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 1 mM p-aminobenzamidine-2HCl, 1 mM 6-amino-n-caproic acid, and 5 units/ml DNase. The cells were then disrupted by French Pressure Cell at 140 MPa, and the cell debris was removed by centrifugation at 10,000 x g for 15 min. The supernatant was further centrifuged at 60,000 x g for 1 h, and the cytosol fraction was obtained as the supernatant. The precipitate was washed once with buffer and used as the crude thylakoid membrane fraction.

The obtained cytosol fraction containing 25 mg of protein was incubated with 2 ml of TrxAC34S-immobilized resin, and the resin was washed completely as described (6, 30). The resin was then suspended in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 10 mM DTT and was incubated for 30 min at 25 °C under gentle stirring. The eluted proteins were separated from the resin by centrifugation. SDS-PAGE was performed with 15% (w/v) acrylamide gel, and the separated protein bands were visualized by Coomassie Brilliant Blue.

NH2-terminal Sequence—Each of the captured proteins separated by SDS-PAGE was transferred onto a polyvinylidene difluoride membrane, and the NH2-terminal sequences of the proteins were determined by Edman degradation method with a peptide sequencer model PPSQ-21(Shimadzu).

Cloning, Expression, and Purification of Type II Prx and 1-Cys Prx—The coding region of type II Prx (sll1621) and that of 1-Cys Prx (slr1198) were amplified by a PCR method using the following combination of the primers: for type II Prx, 5'-cccggaattccatatgacccccgaacgagttcccagtg-3' (NdeI) and 5'-ccccgctcgagttagccgacaaaagctttaacgggc-3' (XhoI); and for 1-Cys Prx, 5'-cccggaattccatatggccttacaactcggtgatgttg-3' (NdeI) and 5'-ccccgctcgagttacttattgggttggggggtcaaa-3' (XhoI). The amplified DNA fragments were then cloned into NdeI and XhoI sites of pET-23c (Novagen), and the DNA sequences were confirmed. The obtained plasmids, type II Prx-pET23c and 1-Cys Prx-pET23c, were transformed into the expression host E. coli BL21(DE3), and the desired proteins were overexpressed. The expressed proteins were purified basically by the same method used for the purification of TrxA, although the desired peak fractions eluted at different salt concentrations.

Peroxidase Activity—Trx-dependent peroxidase activity was measured by the coupled oxidation of NADPH using NADPH-Trx-reductase from Arabidopsis thaliana (AtNTR) and TrxA at 25 °C (6). For the measurement of the activity of type II Prx, a reaction mixture containing 50 mM Hepes-NaOH (pH 7.3), 20 µM TrxA, 5 µM AtNTR (expressed in E. coli as recombinant protein), and 0.5 mM NADPH was used. After the addition of 3.33 µM of type II Prx, the reaction was initiated by the addition of H2O2, t-BOOH, or CHP at the final concentrations of 0.5 mM, and the decrease of the absorbance at 340 nm was monitored. For 1-Cys Prx, a reaction mixture containing 50 mM Hepes-NaOH (pH 7.3), 1 µM TrxA, 1 µM AtNTR, and 0.5 mM NADPH was used.

To measure the glutathione (GSH)-dependent peroxidase activity, a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM NaN3, 0.1 mM EDTA, 0.23 unit/ml NADPH-glutathione reductase, 30 µM NADPH, and the indicated concentration of GSH was used (36). Reaction was initiated by the addition of 250 µM H2O2, t-BOOH or CHP, and the decrease of the absorbance at 340 nm was monitored.

In Vitro Reduction of Prx Proteins—The oxidized forms of SLL1621 protein (type II Prx) was incubated with the combination of DTT and Trx, or GSH as indicated in the figure legends. Then the redox states of SLL1621 protein (type II Prx) were determined as follows. Briefly, the protein was precipitated with 10% (w/v) trichloroacetic acid (final concentration) and collected by centrifugation. The obtained protein precipitates were then washed with acetone and dissolved in freshly prepared solution containing 1% (w/v) SDS, 50 mM Tris-HCl (pH 7.5), and 10 mM AMS. Labeled proteins were then separated by nonreducing SDS-PAGE using 15% (w/v) polyacrylamide gel. The protein bands were visualized by Coomassie Brilliant Blue staining.

In the case of SLR1198 protein (1-Cys Prx), the oxidized form protein was incubated with the combination of NADPH, AtNTR, and Trx as indicated in the figure legends. Then the redox states of SLR1198 protein (1-Cys Prx) were directly assessed by their difference in the mobility on nonreducing SDS-PAGE using 15% (w/v) polyacrylamide gel.

Disruption of sll1621 and slr1198 Genes in Synechocystis—The DNA fragments encoding sll1621 and slr1198 genes were amplified by PCR using genomic DNA from Synechocystis sp. PCC 6803 as a template. The primers were as follows: for sll1621, 5'-gacggtaatttcataacgttccg-3' and 5'-ttcctcgggctacagcgggttgg-3'; for slr1198, 5'-cgcctctcggtggcctttttcaa-3' and 5'-ttcgtcctgccacagttgccacg-3'. The PCR products were cloned into pGEM-T Easy (Promega) according to the manufacturer's instruction. Cloning of the DNA fragment was verified by the nucleotide sequence. The resulting plasmid for sll1621 was digested with BamHI, which was introduced into the coding region of sll1621 by PCR, and a 2.0-kbp BamHI fragment encoding a streptomycin/spectinomycin-resistant cassette derived from plasmid pRL453 was ligated. In the case of slr1198, kanamycin-resistant cassette was inserted into the coding region (37). Wild-type strain cells of Synechocystis sp. PCC 6803 were transformed with the plasmid according to the method of (38). Transformants were selected on a BG11 plate containing 20 µg·ml-l spectinomycin or 20 µg·ml-l kanamycin. Cultivation of the mutant cells was performed under weak light conditions (10 µmol·m-2·s-1). Complete segregation was confirmed by PCR with the above combination of the primers using the total DNA extracted from the transformants as a template.

Growth Experiment under Stress Conditions—Growth experiments under the stress conditions were performed in BG11 medium. The absorbance at 750 nm of the each culture aliquot was set as 0.05, and the growth rate was measured using Abs750. The culture flask was set on a shaker and incubated at 20 °C under the light in the presence of 1% (v/v) CO2 in air.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Acquisition of Trx Target Candidates by Trx Affinity Chromatography—By using the immobilized TrxAC34S, we successfully captured potential target proteins in the cytosol fraction of Synechocystis sp. PCC 6803 (Fig. 1). Similar results were obtained when the TrxA isoform, SLR0623, was used as an immobilized protein for Trx affinity chromatography (data not shown).



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  		FIG. 1.
SDS-PAGE profile of the captured proteins by Trx-affinity chromatography. The possible target proteins in the cytosol fraction were captured by the immobilized TrxAC34S. The obtained protein samples were separated by 15% (w/v) SDS-PAGE. The protein bands (I-V) identified by NH2-terminal Edman sequencing are indicated with arrows and numbers and are listed in Table I.

 
By NH2-terminal Edman sequencing, we analyzed the NH2-terminal sequences of five protein bands, and their annotations were surveyed. We assigned these five proteins as shown in Table I. In CyanoBase (39), sll1621 is annotated as the gene for an AhpC/TSA family protein, and slr1198 is the gene for an antioxidant protein, respectively. From the comparison of the amino acid sequences, the gene product of sll1621 is categorized into a family of type II Prx and those of slr1198 into a family of 1-Cys Prx (Fig. 2).


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  		TABLE I
List of the target protein candidates of TrxA captured in the present study

 



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  		FIG. 2.
Unrooted phylogenic tree of Prx proteins. Phylogenic relationships were analyzed with the DIALIGNE multiple sequence alignment algorithm (52), and the tree was written using the software PHYLIP version 3.6 by J. Felsenstein. On the tree, the Prx isoforms from Synechocystis sp. PCC 6803 are indicated with white letters. The accession numbers of the sequence of 1-Cys Prx are: A. thaliana putative, NP_175247 [GenBank] ; Hordeum vulgare (Barley), P52572 [GenBank] ; Brassica napus, Q8GZT7; and Synechocystis sp. PCC 6803 (for SLR1198), NP_440699 [GenBank] . Those of 2-Cys Prx are: A. thaliana putative, AAF02131 [GenBank] and AAG41453 [GenBank] Nostoc sp. PCC 7120, NP_488681 [GenBank] ; Spinacia oleracea, O24364 [GenBank] ; Pisum sativum, Q93X25; Chlamydomonas reinhardtii, Q9FNS2; Bos tauras Prx-IV, Q9BGI2; Synechocystis sp. PCC 6803 (for SLL0755), NP_442066 [GenBank] , for Prx-Q; Sedum lineare, BAA90524 [GenBank] A. thaliana, NP_189235 [GenBank] ; E. coli BCP, P23480 [GenBank] ; Synechocystis sp. PCC 6803 (for SLL0221), NP_440046 [GenBank] ; and Synechocystis sp. PCC 6803 (for SLR0242), NP_440024 [GenBank] . Those of type II Prx are: Saccharomyces cerevisiae, P38013 [GenBank] ; Plasmodium falciparum, NP_704217 [GenBank] ; Agrobacterium tumefaciens, Q8UHA4; A. thaliana, NP_176773 [GenBank] ; Oryza sativa, Q9FR35; Nostoc sp. PCC 7120, NP_485581 [GenBank] ; and Synechocystis sp. PCC 6803 (for SLL1621), NP_441096 [GenBank] .

 
In addition, we captured two phycocyanin family proteins by this procedure, although the redox-related response of phycocyanin is not known at all (Table I). In the case of SSL2245, which is annotated as an "unknown protein" in CyanoBase, none of the similar protein was found in the whole protein databases. Because SSL2245 has only one cysteine, this protein may dimerize and form the intermolecular disulfide bond, which is a requirement to be captured by this method.

Localization and Expression Levels of Prx Proteins in Synechocystis—Antibodies against the two Prx proteins were prepared using their recombinant proteins. Both Prx proteins were localized in the cytosol but not in the membrane fraction (Fig. 3, A and B). The amount of the proteins in the cytosol fraction was estimated from their intensities of the bands on the Western blot. They were ~6 µg/mg cytosol proteins for type II Prx (SLL1621) and ~5 µg/mg cytosol proteins for 1-Cys Prx (SLR1198). Changes in the amount of both Prx proteins in the cell were examined in the presence of 0.5 mM H2O2 (Fig. 3C). The same results were obtained when 1 mM H2O2 was used instead. Kobayashi et al. (37) recently reported that the amount of transcripts for sll1621 in the cell drastically increased when the cells were cultivated in the presence of 10 µM methylviologen, a strong reactive oxygen species inducer, under light conditions. However, we could not detect any remarkable changes in the amount of the Prx proteins under these conditions.



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  		FIG. 3.
Localization and expression levels of Prx proteins in cyanobacteria detected by antibodies. A and B, localization of Prx proteins in the cell was examined using the antibodies raised against the recombinant Prx proteins, type II Prx (A) and 1-Cys Prx (B). 30 µg (A) or 20 µg (B) proteins of the cytosol fraction (Cytosol) or the crude thylakoid membrane fraction (Membrane) were electrophoresed. The blots were incubated with rabbit anti-type II Prx serum or anti-1-Cys Prx serum, respectively, and stained with anti-rabbit immunoglobulin conjugated to alkaline phosphatase. Marker sizes (kDa) were indicated at the left of panel B. C, changes in the amount of type II Prx and 1-Cys Prx in the cell under the oxidative stress conditions were quantified. The cells were collected at 0 (lane 1), 2 (lane 2), 4 (lane 3), 8 (lane 4), and 24 (lane 5) h after the addition of 0.5 mM H2O2 into the culture solution. 15 µg (type II) or 20 µg of proteins (1-Cys) were electrophoresed and visualized using their antibodies.

 
Peroxidase Activities of Prx Proteins—Next, we investigated the biochemical properties of the recombinant Prx proteins expressed in E. coli. First, the peroxidase activities were examined. Type II Prx, SLL1621 could reduce H2O2, t-BOOH, and CHP coupled with oxidation of NADPH in the presence of TrxA and AtNTR, although the activity was considerably low (Table II). Therefore, we tested other electron donors for this Prx, and found that GSH is the best electron donor (Table II). In this case, t-BOOH was a much better substrate than H2O2. Recently, Rouhier et al. (11, 40) did extensive characterization of type II Prx from poplar and showed that this protein could use either Trx or Grx as an electron donor. However, the acceleration of the peroxidase activity by the addition of Grx was only 10-20% in our case (Fig. 4 and Table II). Even in the absence of Grx, our SLL1621 exhibited a significant peroxidase activity (Table II) compared with the activity reported (see Table II of Ref. 11), suggesting this protein is an actual GSH-dependent peroxidase.


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  		TABLE II
Peroxidase activity of the recombinant type II Prx

The peroxidase activity of the recombinant type II Prx was measured using the coupling assay in the presence of NADPH, AtNTR, and TrxA, or in the presence of NADPH, glutathione reductase, and GSH. The activity was measured as a decrease of the absorbance at 340 nm due to the oxidation of NADPH.

 



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  		FIG. 4.
GSH-dependent peroxidase activity of type II Prx. Time course of the reduction of t-BOOH by type II Prx was monitored as the coupled NADPH oxidation at 340 nm. 250 µM t-BOOH was added at time 0 (downward arrow), and the activity was monitored in the complete assay mixture containing 0.03 mM NADPH, 0.23 unit/ml glutathione reductase, 0.72 mM GSH, and 1 µM type II Prx (solid line). The activity in the presence of 0.26 µM Grx (dotted line) or in the absence of glutathione reductase (dashed and dotted line), GSH (line with long dashes), or type II Prx (line with shorter dashes) was also measured.

 
In contrast, the recombinant SLL1198 did show a very low peroxidase activity (0.2 nmol/mg of protein/min) in the presence of NADPH, TrxA, and AtNTR, using H2O2 as a substrate. The combination of glutathione reductase and GSH was not applicable for the activity measurement of this protein.

In Vitro Reduction of Prx Proteins—SLL1621 has four cysteines in the molecule. Based on sequence alignments, Cys55 and Cys80 seem to be the catalytically critical cysteines. Here, oxidation or reduction of the cysteines in SLL1621 was investigated (Fig. 5). The SLL1621 protein was oxidized using 0.1 mM t-BOOH. Subsequent reduction of the protein was performed with the combination of DTT and TrxA. Reduction by GSH was also examined. The redox state of Prx was visualized using AMS modification. An intermolecular disulfide formation under oxidized conditions and Trx-dependent reduction was clearly observed (Fig. 5B). In addition, GSH could reduce the oxidized form SLL1621 efficiently (Fig. 5C) as expected from the results shown in Table II.



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  		FIG. 5.
Oxidation or reduction of type II Prx. Reduction levels of type II Prx were determined by AMS labeling. The purified type II Prx (110 µM) was incubated with 0.1 mM t-BOOH for oxidation. Then the protein was diluted 25-fold and incubated with the indicated concentrations of DTT in the absence (A) and in the presence of 4.5 µM TrxA (B) for 1 h at 25 °C. The effect of GSH was also examined (C).

 
In contrast SLR1198 has three cysteines, and Cys45 is the only conserved cysteine. According to the reported catalytic cycle of 1-Cys Prx, the catalytic cysteine is oxidized by reactive oxygen species and converted to sulfenic acid (41). Then sulfenic acid will be reduced by the reducing equivalent. When the SLR1198 protein was incubated with 1 mM H2O2, the protein band at the lower monomer position shifted to the upper monomer position even without AMS modification (Fig. 6). In addition, a new band has emerged at the dimer position. Although we did not confirm the change of the catalytic cysteine to sulfenic acid, the observed change in the mobility of the monomer might indicate the oxidation of the catalytic cysteine. The protein band at the dimer position clearly disappeared when the protein was incubated with the reduced form of Trx. These data suggest that the dimer of SLR1198 is formed by an intermolecular disulfide bridge and is specifically reduced by Trx.



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  		FIG. 6.
Oxidation or reduction of 1-Cys Prx. For oxidation of 1-Cys Prx, the protein (110 µM) was incubated with 1 mM H2O2 (lane 3). For reduction the oxidized proteins were diluted 25-fold and incubated with or without 1 mM NADPH, 100 nM AtNTR, and 6 µM TrxA for 1 h at 25 °C (lanes 4-7), or incubated with 1 mM DTT for 1 h at 25 °C (lane 2). The Prx proteins (5 µg) were electrophoresed in the presence of SDS under non-reducing conditions.

 
Growth of Wild Type, sll1621, and slr1198 Disruptants under the Stress Conditions—To evaluate the physiological significance of these Prx proteins, we prepared the disruptants of these two Prx genes of Synechocystis using insertion of an antibiotica resistance cassette into the Synechocystis genome. Growth performance of the wild type, sll1621, and slr1198 disruptant cells was investigated at 30 °C. The growth of the sll1621 disruptant was severely suppressed (Fig. 7A). Similar results were reported by Kobayashi et al. (37). In contrast, the growth of the slr1198 disruptant was partly suppressed under the normal cultivation conditions (Fig. 7B). Oxidative stress induced by the addition of 0.2 mM H2O2 or 2 µM methylviologen did not affect the growth rate remarkably (Fig. 7B).



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  		FIG. 7.
The growth of sll1621 and slr1198 disruptants. A, the growth rate of sll1621 disruptant (open circle) was compared with the wild type cell (closed circle) at a photon fluency rate of 50 µmol·m-2·s-1. B, the growth rates of the slr1198 disruptant (open circle) were compared with the wild type cell (closed circle) at a photon fluency rate of 35 µmol·m-2·s-1 in the absence or in the presence of 0.2 mM H2O2 or 2 µM methylviologen.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Significance of Trx and Prx in Cyanobacteria—The photosynthetic apparatus of cyanobacteria resembles those of chloroplasts from higher plants. However, the redox regulation system of this microorganism is considerably different from that of higher plants: the counterparts of the well known thiol enzymes in the Calvin cycle do not possess the conserved cysteines for redox regulation (42, 43). Also the important amino acid sequence, including two cysteines for regulation of ATP synthase, is missing (44). Nevertheless, Synechocystis sp. PCC 6803 has five Trx genes in total, and two of them are similar to m-type Trx of higher plants. So far, not much is known on the target proteins of cyanobacterial Trx. We therefore tried to identify potential target proteins of Trx in cyanobacteria by Trx affinity chromatography (6). The major protein category captured by the immobilized mutant TrxA was Prx (45), which is extensively studied as intracellular anti-oxidative stress system in mammals and higher plants (1). Recently, Lindahl et al. (46) reported the results of the comprehensive analysis of the target protein candidates for one of the Trx isoform SLR0623 using the C35S mutant in Synechocystis sp. PCC 6803. In their report, 18 cytosolic proteins and 8 membrane peripheral proteins were suggested as the potential candidates. They also have captured 1-Cys Prx (SLR1198) and type II Prx (SLL1621) as the targets despite the different Trx isoform used. In CyanoBase, the gene product of slr0623, which was used by Lindahl et al., and that of slr1139 in the present study are shared the annotation of TrxA. The identity of the amino acid sequence between them is 37% but the amino acid sequence around the reactive cysteines is mostly conserved. However, we cannot evaluate whether the captured protein profile is completely equivalent with those by Lindahl et al. or not, because we did not analyze the captured proteins whose molecular weights are larger than SLR1198 in this study (Fig. 1 and Table I). We could get potentially the same SDS-PAGE profile by using the immobilized SLR0623 mutant as mentioned.

Anti-oxidative Stress System in Cyanobacteria—In cyanobacteria, the physiological significance of catalase-peroxidase, 2-Cys Prx, and NADPH-dependent peroxidase as anti-oxidative stress system is reported (20, 21, 47, 48). As mentioned, the gene product of sll0755 in Synechocystis sp. PCC 6803, in vivo were investigated in detail by Klughammer et al. (20) and Yamamoto et al. (21). The targeted disruption of the gene for 2-Cys Prx eliminated the tolerance against H2O2, suggesting the necessity of this Prx as anti-oxidative stress protein. Based on the comparison of the oxygen evolution from the wild type cells and sll0755 disruptant cells in the presence of t-BOOH, Yamamoto et al. (21) concluded that 2-Cys Prx is responsible for the oxygen evolution coupled with the reduction of peroxides in the light. Another anti-oxidative stress system in Synechocystis cells consisting of GSH peroxidase-like proteins (Gpx-1 and Gpx-2) was reported by Gaber et al. (48). Although both Gpx-1 and Gpx-2 have significant sequence similarity to GSH peroxidase (Gpx) from mammals and higher plants, their electron donor was NADPH but not the reduced form of GSH. Hence, cyanobacteria seem to equip various anti-oxidative stress systems.

Role of type II Prx and 1-Cys Prx in Synechocystis Cells—Five Prx isoform genes were identified in Synechocystis sp. PCC 6803 by genome analysis (see Fig. 2). Recently, Kobayashi et al. (37) revealed that the transcript of the gene for sll1621 is strongly up-regulated when cells are cultured under the light conditions in the presence of methylviologen. In addition, they found a transcription regulator protein SLR1738 for the sll1621 gene. Based on the analysis of the transcripts of sll1621 under oxidative stress conditions and the phenotype analysis of the disruptant of slr1738, they concluded that type II Prx is the physiologically important anti-oxidative stress protein.

In contrast, the present study was initiated from our finding that type II Prx, SLL1621 was captured as a target protein candidate of TrxA (Fig. 1) (45). Recently, homologous type II Prx was identified as a target of Trx-h from higher plants (30). Although the amounts of type II Prx and 1-Cys Prx were not much different in the Synechocystis cells (Fig. 3, A and B), the physiological significance of these two Prx proteins seems to be very different. The disruptant of sll1621 was remarkably weak against oxidative stress (Fig. 7A). In contrast, the disruption of slr1198 gene somewhat affected the growth rate but was not lethal (Fig. 7B).

From the in vitro analysis of the recombinant proteins, we found that type II Prx exhibits remarkable GSH-dependent peroxidase activity but does not require Grx as a mediator (Fig. 4 and Table II). The observed peroxidase activity was 25-fold or more than the activity in the former report (11). In addition, our type II Prx seems to be rather specific for t-BOOH, although type II Prx from popular exhibited almost the similar activity with t-BOOH and H2O2 (11). Alteration of the specificity for the hydroperoxide species was reported by Rouhier on the mutant analysis of type II Prx from popular (49). In their case, the mutation around the active site cysteines strongly affects the specificity, although the amino acid residues introduced by their mutation are not conserved in cyanobacterial Prx. For our type II Prx, the Trx pathway was not effective. These results were inconsistent with the report by Rouhier et al. (11), which requires either Grx or Trx for the peroxidase activity. The observed GSH-dependent peroxidase activity was much higher than the Trx-dependent peroxidase activity. Hence type II Prx of Synechocystis must act as a GSH peroxidase in the cells.

So far, not much is known about the biochemical property of 1-Cys Prx. In the case of 1-Cys Prx from E. coli, Fisher and co-workers (36, 50) had reported both peroxidase and phospholipase activities of the recombinant protein. In that case, H2O2 was a better substrate than other peroxides when GSH was used as an electron donor (50). Although we could observe oxidation and formation of the dimer of 1-Cys Prx in the presence of H2O2 and its reduction by the reduced form TrxA (Fig. 6), the Trx-dependent peroxidase activity of this protein was notably low. In addition, this Prx did not show any peroxidase activity in the presence of GSH and Grx, and any phospholipase activity (data not shown). Compared with other 1-Cys Prx, the SLR1198 protein contains two additional cysteines. Because the protein band on SDS-PAGE did not completely return to the position of the original reduced form after reduction of the dimer by the reduced form TrxA (Fig. 6), a part of Prx might form the intramolecular disulfide bond via these non-catalytic cysteines. Still the most efficient electron donor for the active turnover for this Prx and its physiological role is unclear, although the gene disruption of slr1198 affected the growth rate of the cells irrespective of the oxidative stress.

Merits and Demerits of Trx-affinity Chromatography—As mentioned we found that type II Prx, SLL1621, has a remarkable GSH peroxidase activity. The next question should be why type II Prx was captured by Trx affinity chromatography in our study (45) (Fig. 1) or in the former report by Lindahl et al. (46). This must be explained by the slow dithiol-disulfide exchange between type II Prx and TrxA. As shown in Fig. 5, the oxidized Prx was reduced by the reduced form of Trx when incubated for a certain period. If this is the case, type II Prx must form the stable mixed-disulfide intermediate with the mutant Trx on the resin.

This result happens to send out the warning to the comprehensive study itself using Trx affinity chromatography (6, 29, 30, 46, 51). A protein-protein interaction between Trx and a target protein should have a certain rule, and the specificity of this interaction should be maintained by the special combination of the specific superficial amino acids on both Trx and the target protein. Therefore the immobilized Trx mutant can recognize the counterpart disulfide bond on the surface of a target protein, thus efficiently collect the target proteins. However, the method could not detect a remarkable difference of the target specificity between, for example, Trx-f and Trx-m in chloroplasts (6, 29). The deficiency of this apparent specificity must be due to the rather long incubation period to complete the formation of the mixed disulfide intermediates between immobilized Trx mutant and the target candidate proteins.

Another weak point of the method may be attributed to the recognition of the disulfide bond on the surface of the target protein molecule. Although we do not have any additional evidences on the interaction between phycocyanins and Trx, these proteins were clearly captured and eluted from the gel by reduction (Fig. 1). Because phycocyanins are abundant proteins in cyanobacteria, they may be accidentally captured when they have a disulfide bond on the molecular surface even if this disulfide bond is not the actual target of Trx. Therefore the attentive biochemical research is required for each of the possible candidate proteins to reveal the Trx-regulated redox system, although the comprehensive survey is a very powerful tool to list the potential candidate proteins.

Conclusions—In the present study we clarified that two Prx proteins interact with TrxA in Synechocystis cells. Within these two Prx proteins, we found that type II Prx is the essential Prx and is indispensable for the cell. In addition, we revealed that this type II Prx has a remarkable GSH peroxidase activity. Because Synechocystis cells possess multiple Trx isoforms and several anti-oxidative stress systems, it is worth investigating the expression levels of each proteins and the efficiency of internal electron transfer to reveal the whole redox network system in cyanobacteria.


   FOOTNOTES
 
* This work was supported in part by the ATP System Project, Exploratory Research for Advanced Technology funded by the Japan Science and Technology Agency. Back

|| Present address: Center of Systems Biology and Ecology, Faculty of Science, Toho University, Funabashi, 274-8510 Chiba, Japan. Back

{ddagger}{ddagger} Present address: Dept. of Biochemistry Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-Ohkubo, Saitama, 338-8570 Saitama, Japan. Back

§§ To whom correspondence should be addressed. Tel.: 81-45-924-5234; Fax: 81-45-924-5277; E-mail: thisabor{at}res.titech.ac.jp.

1 Abbreviations used are: Prx, peroxiredoxin; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate; AtNTR, NADPH-Trx-reductase from A. thaliana; 1-Cys Prx, 1-Cys type peroxiredoxin; 2-Cys Prx, 2-Cys type peroxiredoxin; type II Prx, atypical type II peroxiredoxin; t-BOOH, tert-butyl hydroperoxide; Trx, thioredoxin; GSH, glutathione; Grx, glutaredoxin; DTT, dithiothreitol; CHP, cumene hydroperoxide. Back


   ACKNOWLEDGMENTS
 
We are grateful to H. Kato, K. Sugiyama, D. Yamazaki, H. Taguchi, and Y. Mizota for their valuable discussions and technical assistances. We thank M. Ikeuchi and M. Kobayashi for providing us the useful information concerning DNA micro-array analysis of Synechocystis sp. PCC 6803 under the oxidative stress conditions. Special thanks to G. Groth for his critically reading of the manuscript and to M. Yoshida for his continuous encouragement and desirable suggestions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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