Glutaredoxin-dependent Peroxiredoxin from Poplar
2002; Elsevier BV; Volume: 277; Issue: 16 Linguagem: Inglês
10.1074/jbc.m111489200
ISSN1083-351X
AutoresNicolas Rouhier, Éric Gelhaye, Jean‐Pierre Jacquot,
Tópico(s)Enzyme function and inhibition
ResumoRecently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained ∼25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx. Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained ∼25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx. Peroxiredoxins (Prxs) 1The abbreviations used are: PrxperoxiredoxinGrxglutaredoxinTrxthioredoxinWTwild typeDTNB5,5′-dithiobis(nitrobenzoic acid)constitute a recently discovered family of non-heme peroxidases present in all organisms from prokaryotes to eukaryotes, and they catalyze the reduction of various hydroperoxides into the corresponding alcohol and water (1.Rhee S.G. Kang S.W. Netto L.E. Seo M.S. Stadtman E.R. Biofactors. 1999; 10: 207-209Crossref PubMed Scopus (157) Google Scholar). Currently, these proteins are the subject of numerous studies because their function seems to be particularly important in the detoxification of reactive oxygen species that can cause serious damage to the nucleic acids, proteins, and lipids (2.Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4464) Google Scholar, 3.Beckman K.B. Ames B.N. J. Biol. Chem. 1997; 272: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 4.Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2806) Google Scholar). Prx is also involved in the control of signal transduction by modulating the reactive oxygen species-mediated cellular responses and by regulating transcription factors (5.Jin D.Y. Chae H.Z. Rhee S.G. Jeang K.T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 6.Kim H. Lee T.H. Park E.S. Suh J.M. Park S.J. Chung H.K. Kwon O.Y. Kim Y.K. Ro H.K. Shong M. J. Biol. Chem. 2000; 275: 18266-18270Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 7.Zhou Y. Kok K.H. Chun A.C. Wong C.M. Wu H.W. Lin M.C. Fung P.C. Kung H. Jin D.Y. Biochem. Biophys. Res. Commun. 2000; 268: 921-927Crossref PubMed Scopus (151) Google Scholar). peroxiredoxin glutaredoxin thioredoxin wild type 5,5′-dithiobis(nitrobenzoic acid) All the Prx isoforms have in common a conserved catalytic cysteine, localized in the N-terminal part of the protein, that is converted into a sulfenic acid by hydroperoxides (8.Rhee S.G. Kim K.H. Chae H.Z. Yim M.B. Uchida K. Netto L.E. Stadtman E.R. Ann. N. Y. Acad. Sci. 1994; 738: 86-92Crossref PubMed Scopus (58) Google Scholar). This cysteine was demonstrated by site-directed mutagenesis to be essential for catalysis (9.Chae H.Z. Uhm T.B. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7022-7026Crossref PubMed Scopus (281) Google Scholar). Based mainly on the number of conserved cysteines, a classification has been proposed for the multiple existing Prxs. The 1-Cys Prxs mediate the reduction of H2O2 with the use of an unknown proton donor, which could be Trx for a mitochondrial 1-Cys Prx fromSaccharomyces cerevisiae (10.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 11.Pedrajas J.R. Miranda-Vizuete A. Javanmardy N. Gustafsson J.A. Spyrou G. J. Biol. Chem. 2000; 275: 16296-16301Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Recently, human cyclophilin A was identified as an electron donor to the mammalian Prx VI, the only mammalian 1-Cys Prx characterized, and also to all known mammalian Prxs (12.Lee S.P. Hwang Y.S. Kim Y.J. Kwon K.S. Kim H.J. Kim K. Chae H.Z. J. Biol. Chem. 2001; 276: 29826-29832Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Among the Prxs with two conserved cysteines, at least three classes can be distinguished, according to the position of the cysteines. The first class, comprising mammalian Prx V, includes monomeric enzymes that form an intramolecular disulfide bridge as a reaction intermediate (13.Knoops B. Clippe A. Bogard C. Arsalane K. Wattiez R. Hermans C. Duconseille E. Falmagne P. Bernard A. J. Biol. Chem. 1999; 274: 30451-30458Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 14.Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). The second class is formed by the homologues of the bacterioferritin co-migratory protein, which are also shown to be monomeric enzymes with an intramolecular disulfide bridge in the oxidized state (15.Jeong W. Cha M.K. Kim I.H. J. Biol. Chem. 2000; 275: 2924-2930Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 16.Kong W. Shiota S. Shi Y. Nakayama H. Nakayama K. Biochem. J. 2000; 351: 107-114Crossref PubMed Scopus (76) Google Scholar). In this second class, the spacing between the two cysteines that are part of the disulfide bridge is considerably shorter, consistently containing 4 amino acids, instead of the ∼100 amino acids for the first class. The third class, which includes mammalian Prx I to IV, consists of dimeric enzymes that form an intermolecular disulfide bridge between two identical subunits (14.Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar,17.Chae H.Z. Kim H.J. Kang S.W. Rhee S.G. Diabetes Res. Clin. Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Despite these differences, the three types of 2-Cys Prx use Trx as a proton donor. In a previous report, a poplar phloem Prx was characterized (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). This Prx is a small protein of 162 amino acids that contains only two cysteines in position 51 and 76. The primary sequence is quite different from that of the 1-Cys Prxs and most of the 2-Cys Prxs, especially because of the distance that separates the two cysteines. An unexpected finding was that this Prx could use Trx but also Grx as a proton donor for its catalysis (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). This Prx has been referred to as type C Prx, whereas the other 2-Cys Prxs, which use only Trx, were referred to as type A Prx, and 1-Cys Prxs were referred to as type B Prx. Among the biochemically well-characterized Prxs, one of the closest proteins is the mammalian Prx V. This enzyme also comprises 162 amino acids and displays 40% identity to poplar Prx at the amino acid level. A notable difference is the presence of an additional cysteine in position 152 that is linked together with Cys-51 to form an intramolecular disulfide bridge in Prx V (14.Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). A Prx from Chinese cabbage, highly homologous to poplar Prx, was also shown to be a Trx-dependent enzyme, but no attempt was made to evaluate the potential for Grx as a proton donor in this work (19.Choi Y.O. Cheong N.E. Lee K.O. Jung B.G. Hong C.H. Jeong J.H. Chi Y.H. Kim K. Cho M.J. Lee S.Y. Biochem. Biophys. Res. Commun. 1999; 258: 768-771Crossref PubMed Scopus (40) Google Scholar). To get a better understanding of the catalytic mechanism of this Grx-dependent Prx, cysteinic mutants of Grx and Prx have been created by site-directed mutagenesis. Based on kinetic measurements and the creation of heterodimers, a new mechanism for the Grx-dependent Prx activity is proposed. NADPH was obtained from Roche Molecular Biochemicals; diamide, β-mercaptoethanol, glutathione reductase, and reduced glutathione were from Sigma. Dithiothreitol, isopropyl-1-thio-β-d-galactopyranoside, kanamycin, and ampicillin were from Fermentas. The procedures for the isolation of the cDNAs and their subsequent cloning in expression plasmids are described in Refs. 18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar, 20.Rouhier N. Gelhaye E. Jacquot J.P. Prot. Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar, and 21.Behm M. Jacquot J.P. Plant Physiol. Biochem. 2000; 38: 363-369Crossref Scopus (31) Google Scholar. The mutagenesis of poplar Grx was effected as we described (31.Rouhier N. Gelhaye E. Jacquot J.P. FEBS Lett. 2002; 511: 145-149Crossref PubMed Scopus (63) Google Scholar). The Prx mutants C51A, C76A, and V152C were generated by PCR using the oligonucleotides shown below (NcoI and BamHI sites areunderlined, mutagenic bases are in bold). Cloning oligonucleotides were as follows: direct, 5′-GGGGCCATGGCCCCGATTGCTGTTGGT-3′; and reverse, 5′-GGGGGGGATCCTCAAAGATCCTTGAGGATATCCTCGGCACT-3′. Mutagenic oligonucleotides were as follows: C51A direct, 5′-GCCTTCACCCCCACCGCCAGCTTGAAGCATGTG-3′; C51A reverse, 5′-CACATGCTTCAAGCTGGCGGTGGGGGTGAAGGC-3′; C76A direct, 5′-GTTACTGAAATTTTGGCCATCAGCGTCAACGAC-3′; C76A reverse, 5′-GTCGTTGACGCTGATGGCCAAAATTTCAGTAAC-3′; V152C direct, 5′-GGGGGTGGAGAATTCACTTGCTCCAGTGCCGAGGATATC-3′; and V152C reverse, 5′-GATATCCTCGGCACTGGAGCAAGTGAATTCTCCACCCCC-3′. The mutated PCR products that contained the restriction sites were in turn cloned into the expression plasmid pET-3d, yielding the constructions pET Prx C51A, pET Prx C76A, and pET Prx V152C. The sequences of the recombinant plasmids were verified by sequencing. All procedures for the expression and purification of Arabidopsis thaliana NADPH Trx reductase, poplar Trx h1, and WT and mutant Grx are described elsewhere (20.Rouhier N. Gelhaye E. Jacquot J.P. Prot. Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar, 21.Behm M. Jacquot J.P. Plant Physiol. Biochem. 2000; 38: 363-369Crossref Scopus (31) Google Scholar, 23.Jacquot J.P. Rivera-Madrid R. Marinho P. Kollarova M. Le Marechal P. Miginiac-Maslow M. Meyer Y. J. Mol. Biol. 1994; 235: 1357-1363Crossref PubMed Scopus (129) Google Scholar, 31.Rouhier N. Gelhaye E. Jacquot J.P. FEBS Lett. 2002; 511: 145-149Crossref PubMed Scopus (63) Google Scholar). For the expression of Prx, the recombinant plasmids were used to transform Escherichia coli strain BL21(DE3), which was also co-transformed with the plasmid helper pSBET as described previously (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar, 24.Schenk P.M. Baumann S. Mattes R. Steinbiss H.H. BioTechniques. 1995; 19: 196-200PubMed Google Scholar). Because preliminary experiments indicated that Prx is susceptible to oxidation, all purification steps for the wild type and the three mutant proteins were done in the presence of 14 mm β-mercaptoethanol. The thiol content of each protein preparation was measured using the DTNB procedure. To eliminate the β-mercaptoethanol, 1 mg of each protein was incubated with 10% trichloroacetic acid for 30 min on ice. The mixture was then centrifuged for 15 min at 13,000 rpm, and the pellet was washed three times with 1 ml of 2% trichloroacetic acid. The pellet was resuspended in 100 mm Tris-HCl, pH 8, and 1 mm EDTA, and the protein concentration was determined by measuring the absorbance at 280 nm. SDS was then added to a final concentration of 1%, and the reaction was started by adding 100 μm DTNB. The reaction mixture was stored in the dark for 20 min, and the absorbance at 412 nm was measured. A second measurement was performed after a 30-min incubation in the dark. Both measurements gave identical results. Similar results were obtained when we used 80% acetone as a precipitant instead of trichloroacetic acid. The reduction of H2O2 by poplar Prx in the presence of the Trx or Grx system was followed spectrophotometrically using a Cary 50 spectrophotometer as described in Ref. 18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar. The activities were measured at a fixed concentration of Prx (2.5 μm) and at variable concentrations of Grx or Trx. 1.25 μg of Prx and Grx were mixed in the presence of TE buffer (30 mmTris-HCl, pH 8, and 1 mm EDTA) to a final volume of 10 μl. This reaction mixture was incubated at room temperature for 2–3 min before the addition of 10 mm diamide. The mixture was incubated at room temperature for 20 min and then subjected to 14% SDS-PAGE in the presence of an equivalent volume of a nonreducing sample buffer (0.5 m Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and bromphenol blue) (25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207487) Google Scholar). When needed, 10 mmdithiothreitol or 20–50 mm glutathione was added after incubation in the presence of diamide to reduce the covalent disulfide adducts between Prx and Grx. 2 μl of the mixture described above were subjected to 14% SDS-PAGE before transfer. The immunodetection using the Immune Star Goat Anti Rabbit Detection Kit from Bio-Rad was performed as described in Ref. 18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar. Rabbit polyclonal antibodies against Trx, Grx, and Prx were purified onto affinity columns according to the procedure described for Prx antibodies in Ref. 18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar. A previous study has indicated that poplar Prx differs from the other peroxiredoxins characterized thus far, essentially by the position of the conserved cysteine residues and by the overall length of the sequence (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). As detailed in the "Introduction," this class of new Prxs was called type C Prx, whereas 2-Cys Prxs were named type A Prx, and 1-Cys Prxs were called type B Prx. A number of sequences similar to type C poplar Prx have now appeared in the literature, and an amino acid comparison of some of the closest relatives is given in Fig. 1. Poplar Prx is strongly homologous to the other plant Prxs of its class (76%, 81%, and 82% amino acid identity with Oryza sativa, Brassica rapa, and A. thaliana, respectively) and also to the well-characterized PMP20 protein from Candida boidinii and to the human Prx V (42% and 40% amino acid identity, respectively) (Fig. 1). Nevertheless, an important difference is the presence of an additional cysteine in human Prx in position 152; the poplar and all other nonmammalian sequences do not possess this additional cysteine. This particularity is examined here in terms of catalytic efficiency. In a previous work, we have shown that type C Prx could use both Grx and Trx as proton donors for catalysis. In this initial work, rather high concentrations of Trx or Grx were required in the in vitromeasurement of peroxidase activity (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). Moreover, the specific activity of the enzyme remained low. We have improved this system in two respects: (i) the truncated form of poplar Grx, which was used in the earlier experiments, was replaced by a C-terminal-extended protein, which was both more stable and more active (20.Rouhier N. Gelhaye E. Jacquot J.P. Prot. Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar); and (ii) because we observed that Prx was susceptible to oxidation, its purification and subsequent storage were carried out in the presence of β-mercaptoethanol to avoid an irreversible oxidation of the catalytic cysteine. These changes resulted in a considerable enhancement of enzymatic activity, as shown in Table I. At a saturating Grx concentration (∼10 μm), the maximal specific activity of the enzyme is 8 μmol NADPH oxidized/min−1/mg protein−1. This value is similar to those reported for human peroxiredoxins and the E. coli bacterioferritin co-migratory protein (10.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 14.Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 15.Jeong W. Cha M.K. Kim I.H. J. Biol. Chem. 2000; 275: 2924-2930Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The specific activities recorded under the conditions of Table I suggest that Grx might be a better donor than Trx for poplar Prx. The maximal specific activity at a saturating Trx concentration estimated from the results in Fig. 3, 1.5 μmol NADPH oxidized/min−1/mg protein−1, also agrees with that proposal. However, these data should be treated with caution because the assays for Prx activity are indirect and involve the coupling of several components.Table IRelative activities of the various Prx preparations with Trx and GrxInitial GrxImproved GrxTrxPrx oxidizing conditions1.d.3.550.55Prx reducing conditions1.d.4.020.82Data are expressed in μmol of NADPH oxidized/min/mg protein. The measurements were effected at 30 °C with 2.5 μm Prx in the presence of 2.5 μm Trx or Grx. Initial Grx corresponds to the construction pET-Grx 1B, and improved Grx corresponds to pET-Grx 3 as described in Ref. 20.Rouhier N. Gelhaye E. Jacquot J.P. Prot. Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar. 1.d., limit of detection. Open table in a new tab Data are expressed in μmol of NADPH oxidized/min/mg protein. The measurements were effected at 30 °C with 2.5 μm Prx in the presence of 2.5 μm Trx or Grx. Initial Grx corresponds to the construction pET-Grx 1B, and improved Grx corresponds to pET-Grx 3 as described in Ref. 20.Rouhier N. Gelhaye E. Jacquot J.P. Prot. Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar. 1.d., limit of detection. Two monocysteinic mutants, called C27S and C30S, have been created to modify the active site of poplar Grx (31.Rouhier N. Gelhaye E. Jacquot J.P. FEBS Lett. 2002; 511: 145-149Crossref PubMed Scopus (63) Google Scholar). In addition, the mutants C51A, C76A, and V152C have been engineered to explore the reactivity of poplar Prx. All these cysteinic mutants were purified in the presence of β-mercaptoethanol to avoid undesired dimerizations. Fig. 2 shows that all Grx and Trx h1 preparations are highly homogeneous. As observed previously, all Prx preparations exhibited a protein doublet that cannot be eliminated by the addition of an excess of reductant (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). The reason for this behavior is unknown, but all biochemical and structural evidence gathered otherwise indicates that the protein is nevertheless highly homogeneous. Several reports in the literature indicate that the catalytic cysteine of peroxiredoxins can be transformed into sulfenic, sulfonic, or sulfinic acids, and this has been proposed as the reason for the formation of apparent protein doublets (10.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). The titration results with DTNB do not really support such a hypothesis for the poplar protein (see below). Besides, as observed previously, if such a modification occurs, it is not the result of the purification procedure because similar protein doublets were observed after direct lysis of freshly harvested bacteria (data not shown). The thiol content of all protein preparations has been estimated using the DTNB method. All titrations were made on enzymes that were freed from reductant and after the addition of SDS (see "Experimental Procedures"). Consequently, all thiols are titrated, regardless of whether or not they were accessible in the native protein. A summary of these data is shown in Table II. For Grx, the results are quite simple; nearly two thiols are titrated for the reduced WT protein, and no SH group is present in the oxidized WT protein, suggesting that the two Cys residues are indeed linked in a disulfide bridge. The monocysteinic mutants of Grx give values around 1 SH/mol protein, as expected. For Prx, the results are more complex to analyze and depend on whether the protein was isolated in the presence or absence of a reductant. When prepared under nonreducing conditions, the WT Prx shows nearly 2 SH/enzyme monomer, and it appears that the C51A and C76A mutants are partially oxidized because values lower than 1 SH/mol were recorded. The V152C mutant gives a value of 2.6 SH/mol, nearly in agreement with the expected 3 SH/mol. When the Prx preparations were made in the presence of a the monothiol reductant β-mercaptoethanol, very contrasting results were obtained. The C51A mutant showed nearly 1 SH/mol, but all other preparations had a thiol content that was reduced by about 1 SH group compared with the expected theoretical value. We interpret this as the result of a likely interaction between the thiol group of Cys-51 and β-mercaptoethanol that can give rise to a mixed disulfide that cannot be titrated with DTNB. As Prx preparation treated with the dithiol reductant dithiothreitol titrate as the enzyme prepared under oxidizing conditions, this formation of mixed disulfide is postulated to be specific for β-mercaptoethanol. Such a behavior has already been described for the bovine 1-Cys peroxiredoxin (26.Peshenko I.V. Shichi H. Free Radic. Biol. Med. 2001; 31: 292-303Crossref PubMed Scopus (149) Google Scholar). Alternatively, because the thiol titrations are not always in perfect agreement with the theoretical values, this could also indicate that a portion of the peroxiredoxin molecules is in a denatured oxidized form with an internal disulfide bridge as described by Kang et al. for human 1-Cys Prx (10.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) or that a portion of the catalytic cysteine is in an oxidized form.Table IIThiol content of Grx and Prx purified under oxidizing or reducing conditionsOxidizing conditionsReducing conditionsGrx WT0.051.67Grx C27Sn.d.0.88Grx C30Sn.d.0.93Prx WT1.70.9Prx C51A0.280.87Prx C76A0.330.19Prx V152C2.61.4Thiols were titrated using the reduction of DTNB as described under "Experimental Procedures." Data are expressed in mol SH/mol enzyme. n.d., not determined. The S.D. is typically ± 0.2 SH/mol SH. Open table in a new tab Thiols were titrated using the reduction of DTNB as described under "Experimental Procedures." Data are expressed in mol SH/mol enzyme. n.d., not determined. The S.D. is typically ± 0.2 SH/mol SH. The catalytic capacity of all Prx mutants has been evaluated in the presence of the various engineered Grxs as proton donors. This has been tested in a coupled enzymatic reaction where the peroxiredoxin-catalyzed conversion of H2O2 is linked to NADPH oxidation via glutathione reductase, glutathione, and glutaredoxin. We have shown previously, using the 2-hydroethyldisulfide and dehydroascorbate reduction tests, that Cys-27 is the catalytic residue of Grx, and Cys-30 is generally dispensable in those reactions (31.Rouhier N. Gelhaye E. Jacquot J.P. FEBS Lett. 2002; 511: 145-149Crossref PubMed Scopus (63) Google Scholar). Fig. 3 shows the H2O2-dependent NADPH oxidizing activities of the various engineered peroxiredoxins as a function of Grx concentrations ranging from 0.5 to 20 μm. Only four combinations were found to promote catalysis. The best reactivity was obtained when WT Prx was associated with WT Grx. The mutation of Cys-30 of Grx (C30S) has little effect because the rate of catalysis with this mutant is nearly identical to the one obtained with WT Grx. The other associations that are catalytically competent are those between Prx C76A and Grx WT or C30S. However, the activities of the C76S mutant are reduced by approximately 75% compared with those recorded with WT Prx. Another observation is that the Prx C51A and V152C enzymes are completely inactive with Grx as a proton donor. A last piece of information is that the C27S mutant of Grx is unable to promote catalysis for all Prxs tested. Fig. 4 presents the catalytic activity of the Prx enzymes, in the presence of WT Trx as a proton donor. In this case, the Grx generating system was replaced by the Trx system, which is composed of the A. thaliana NADPH thioredoxin reductase and poplar Trx h1 (18.Rouhier N. Gelhaye E. Sautière P.E. Brun A. Laurent P. Tagu D. Gerard J. de Fay E. Meyer Y. Jacquot J.P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (190) Google Scholar). The reduction of poplar Trx by theArabidopsis NADPH thioredoxin reductase was previously shown to be functional using the DTNB reduction test (21.Behm M. Jacquot J.P. Plant Physiol. Biochem. 2000; 38: 363-369Crossref Scopus (31) Google Scholar). In general, similar results were obtained with the Trx system: the WT Prx was the most active catalyst, the mutation C51A abolished catalysis, and C76A had a depressing effect. The most striking difference is that the V152C protein, which was inactive with Grx as a proton donor, was catalytically active with Trx (Fig. 4, ♦). No saturation of Prx V152C enzyme activity could be recorded with Trx up to concentrations of 50 μm (data not shown). From the data in Figs. 3 and 4, Km values can be determined to characterize interactions between Prx and Grx or Trx. Similar values were obtained for Grx and Trx (2.5 and 3 μm, respectively). These values are in good agreement with other parameters published in the literature for Trx (14.Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). The mutation of the cysteine that is potentially involved in the breaking of the mixed disulfide intermediates stabilizes the heterodimers between Trx or Grx and their interacting partners (27.Verdoucq L. Vignols F. Jacquot J.P. Chartier Y. Meyer Y. J. Biol. Chem. 1999; 274: 19714-19722Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). We have taken advantage of that property to create heterodimers between the peroxiredoxin and glutaredoxin molecules. The covalent heterodimer formation was greatly improved using the oxidant diamide. The four different Prx preparations (WT, C51A, C76A, and V152C) were incubated with three different versions of the Grx (WT, C27S, and C30S). Fig. 5shows the results of these associations. In all assays, the Grx polypeptide is present with an apparent molecular mass of 15 kDa and the Prx is present with the usual doublet at ∼18 kDa. By running the proteins individually and based on the molecular mass determinations, we could assess the additional polypeptides as follows: the 30-kDa polypeptide is likely a Grx di
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