An Atypical Catalytic Mechanism Involving Three Cysteines of Thioredoxin
2008; Elsevier BV; Volume: 283; Issue: 34 Linguagem: Inglês
10.1074/jbc.m802093200
ISSN1083-351X
AutoresCha San Koh, Nicolas Navrot, Claude Didierjean, Nicolas Rouhier, Masakazu Hirasawa, David B. Knaff, Gunnar Wingsle, Razip Samian, Jean‐Pierre Jacquot, Catherine Corbier, Éric Gelhaye,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoUnlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys4) and two (Cys58 and Cys61) in the classical thioredoxin active site (57WCGPC61). The property of a mutant in which Cys58 was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys4 is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys58 can be formed, linking it to either Cys61 or Cys4. We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys4 to convert this atypical thioredoxin h back to its active reduced form. Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys4) and two (Cys58 and Cys61) in the classical thioredoxin active site (57WCGPC61). The property of a mutant in which Cys58 was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys4 is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys58 can be formed, linking it to either Cys61 or Cys4. We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys4 to convert this atypical thioredoxin h back to its active reduced form. Thioredoxins (Trxs) 3The abbreviations used are:TrxthioredoxinGrxglutaredoxinNTRNADPH thioredoxin reductasePrxperoxiredoxinDTTdithiothreitolSeMetselenomethionineWTwild type. 3The abbreviations used are:TrxthioredoxinGrxglutaredoxinNTRNADPH thioredoxin reductasePrxperoxiredoxinDTTdithiothreitolSeMetselenomethionineWTwild type. are small molecular weight proteins found in all organisms from prokaryotes to higher eukaryotes. They are involved in many cellular processes, dealing primarily with cell redox regulation. In plants, numerous isoforms have been reported. For example, at least 20 genes coding for Trxs are present in the completely sequenced genome of Arabidopsis thaliana (1Meyer Y. Vignols F. Reichheld J.P. Methods Enzymol. 2002; 347: 394-402Crossref PubMed Scopus (118) Google Scholar). The Trxs f, m, x, and y are present in chloroplasts (2Schürmann P. Antiox. Redox Signal. 2003; 5: 69-78Crossref PubMed Scopus (65) Google Scholar, 3Gelhaye E. Rouhier N. Navrot N. Jacquot J.P. Cell Mol. Life Sci. 2004; 62: 24-35Crossref Scopus (216) Google Scholar), whereas the Trxs o are localized in mitochondria (4Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (221) Google Scholar). The Trxs h constitute a large group that includes cytosolic and mitochondrial isoforms (1Meyer Y. Vignols F. Reichheld J.P. Methods Enzymol. 2002; 347: 394-402Crossref PubMed Scopus (118) Google Scholar, 5Gelhaye E. Rouhier N. Navrot N. Jacquot J.P. Plant Physiol. Biochem. 2004; 42: 265-271Crossref PubMed Scopus (118) Google Scholar, 6Bréhélin C. Laloi C. Setterdahl A.T. Knaff D.B. Meyer Y. Photosynth. Res. 2004; 79: 295-304Crossref PubMed Scopus (25) Google Scholar, 7Gelhaye E. Rouhier N. Gerard J. Jolivet Y. Gualberto J. Navrot N. Ohlsson P.I. Wingsle G. Hirasawa M. Knaff D.B. Wang H. Dizengremel P. Meyer Y. Jacquot J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14545-14550Crossref PubMed Scopus (198) Google Scholar). Trxs h have been divided in three distinct subgroups in a classification based on their primary structure (5Gelhaye E. Rouhier N. Navrot N. Jacquot J.P. Plant Physiol. Biochem. 2004; 42: 265-271Crossref PubMed Scopus (118) Google Scholar). Members of the first and second groups are reduced by NADPH in a reaction catalyzed by NTR. Members of the first and second Trxs h subgroups contain a conserved WC(G/P)PC catalytic site. The first cysteine is the one involved in the nucleophilic attack on disulfide bonds present in target proteins, leading to the formation of a disulfide bond between the target protein and Trx. This intermolecular disulfide is then reduced by the second cysteine, leading to the release of reduced target protein and oxidized Trx. thioredoxin glutaredoxin NADPH thioredoxin reductase peroxiredoxin dithiothreitol selenomethionine wild type. thioredoxin glutaredoxin NADPH thioredoxin reductase peroxiredoxin dithiothreitol selenomethionine wild type. It is only recently that members of plant Trx h subgroup 3 have been detected and characterized, with much of the evidence coming from studies on poplar (8Gelhaye E. Rouhier N. Jacquot J.P. FEBS Lett. 2003; 555: 443-448Crossref PubMed Scopus (88) Google Scholar, 9Juttner J. Olde D. Langridge P. Baumann U. Eur. J. Biochem. 2000; 267: 7109-7117Crossref PubMed Scopus (30) Google Scholar). The poplar thioredoxin PtTrxh4, which belongs to this subgroup, contains a typical WCGPC catalytic site but differs from previously characterized Trxs h in being reduced in vitro by glutaredoxins but not by NTR (8Gelhaye E. Rouhier N. Jacquot J.P. FEBS Lett. 2003; 555: 443-448Crossref PubMed Scopus (88) Google Scholar). This unique feature raised several questions about the reaction mechanism of PtTrxh4. For example, as most characterized Trxs have a redox midpoint potential of about –290 mV, whereas Grxs are more electropositive (about –200 mV), questions about the thermodynamic favorability of reduction of a Trx-like molecule by Grx naturally arise. The structural and redox properties of animal, bacterial, and some plant Trxs have been studied extensively, but little structural information about higher plant Trx h is available (10Coudevylle N. Thureau A. Hemmerlin C. Gelhaye E. Jacquot J.P. Cung M.T. Biochemistry. 2005; 44: 2001-2008Crossref PubMed Scopus (21) Google Scholar, 11Peterson F.C. Lytle B.L. Sampath S. Vinarov D. Tyler E. Shahan M. Markley J.L. Volkman B.F. Protein Sci. 2005; 14: 2195-2200Crossref PubMed Scopus (27) Google Scholar). In addition, there is no subgroup 3 Trx h structure solved to date. Because members of this subgroup exhibit an N-terminal extension containing a conserved cysteine in the fourth position that is absent in other subgroups, questions concerning the role of this extension and its additional cysteine arise. It is of particular interest to know how this extension is positioned with respect to the conserved Trx fold and more importantly to understand why this protein does not react with its traditional reducing partner, NTR. We show here that, in contrast to other Trxs, three cysteines rather than two are involved in the catalytic mechanism of PtTrxh4. Two of these cysteines are present in the classical Trx catalytic site (WC58GPC61), whereas the third one is localized in the N-terminal extension (Cys4). From the kinetic and structural data, a new catalytic mechanism is proposed for this Trx isoform. Cloning and Mutations of PtTrxh4—The procedures for cDNA isolation of PtTrxh4 and its subsequent cloning are described in Ref. 8Gelhaye E. Rouhier N. Jacquot J.P. FEBS Lett. 2003; 555: 443-448Crossref PubMed Scopus (88) Google Scholar. The PtTrxh4 mutants C4S, C58S, and C61S were generated by PCR using cloning and mutagenic oligonucleotides shown below (NcoI and BamHI sites are underlined and mutagenic bases are in bold): PtTrxh4 direct, 5′-CCCCCCATGGGACTTTGCTTGGAT-3′; and PtTrxh4 reverse, 5′-CCCCGGATCCTCATTTGTCACTAGGGGGCAA-3′; PtTrxh4C4S direct, 5′-CCCCCCATGGGACTTAGCTTGGATAAGCAT-3′; PtTrxh4C58S direct, 5′-TTCAGTGCAACATGGAGTGGTCCTTGTAGACAG-3′; PtTrxh4C58S reverse, 5′-CTGTCTACAAGGACCACTCCATGTTGCACTGAA-3′; PtTrxh4C61S direct, 5′-ACATGGTGTGGTCCTAGTAGACAGATTGCACCG-3′; and PtTrxh4C61S reverse, 5′-CGGTGCAATCTGTCTACTAGGACCACACCATGT-3′. The mutated PCR products that contained the restriction sites have been cloned into expression plasmid pET-3d, yielding constructions pET PtTrxh4C4S, pET PtTrxh4C58S, and pET PtTrxh4C61S. The mutations of the recombinant plasmids were verified by DNA sequencing. Expression and Purification of the Recombinant Proteins—All procedures for the expression and purification of Arabidopsis thaliana NTR B (AtNTRB), poplar PrxQ (PtPrxQ), and Grx (WT and mutants) are described elsewhere (12Jacquot 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 (127) Google Scholar, 13Rouhier N. Gelhaye E. Gualberto J.M. Jordy M.N. De Fay E. Hirasawa M. Duplessis S. Lemaire S.D. Frey P. Martin F. Manieri W. Knaff D.B. Jacquot J.P. Plant Physiol. 2004; 134: 1027-1038Crossref PubMed Scopus (145) Google Scholar, 14Rouhier N. Gelhaye E. Jacquot J.P. J. Biol. Chem. 2002; 277: 13609-13614Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 15Rouhier N. Gelhaye E. Sautiere P.E. Jacquot J.P. Protein Expression Purif. 2002; 24: 234-241Crossref PubMed Scopus (23) Google Scholar). All the PtTrxh4 proteins have been expressed in Escherichia coli strain BL21(DE3), which was also co-transformed with the plasmid helper pSBET as described in Ref. 16Schenk P.M. Baumann S. Mattes R. Steinbiss H.H. BioTechniques. 1995; 19: 196-200PubMed Google Scholar. The [SeMet]PtTrxh4 gene was amplified from a P. tremula × tremuloides cDNA library. The gene was inserted in the pET-3d expression plasmid, between NcoI and BamHI sites. Recombinant plasmids carrying the gene of interest were electroporated into the methionine auxotrophic strain of E. coli BL21(DE3) pSBET. Bacteria were cultured in M9 medium supplemented with selenomethionine (SeMet) and protein overexpression performed as previously described (17Navrot N. Collin V. Gualberto J. Gelhaye E. Hirasawa M. Rey P. Knaff D.B. Issakidis E. Jacquot J.P. Rouhier N. Plant Physiol. 2006; 142: 1364-1379Crossref PubMed Scopus (276) Google Scholar). Mass spectrometry was performed to assess purity and to confirm the full incorporation of SeMet, all purification steps of the mutated PtTrxh4 proteins being similar to those described for PtTrxh4 in Ref. 8Gelhaye E. Rouhier N. Jacquot J.P. FEBS Lett. 2003; 555: 443-448Crossref PubMed Scopus (88) Google Scholar. Thiol Content Titration—The thiol content of each protein preparation was measured using the dithionitrobenzoate (DTNB) procedure as described in Ref. 14Rouhier N. Gelhaye E. Jacquot J.P. J. Biol. Chem. 2002; 277: 13609-13614Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar. All thiol titrations were performed in the presence of SDS on enzymes either as purified or after dithiothreitol (DTT) reduction and dialysis. Consequently, all thiols are titrated regardless of whether or not they were accessible in the protein. Glutathionylation Experiments—The reaction mixture (50 μl) containing 30 mm Tris-HCl, pH 8.0, 1 mm 1,4-DTT, and 50 μg of poplar Trx (concentration about 80 μm) was incubated for 10 min before adding 5 mm oxidized glutathione. Electrospray Mass Spectrometry—A Micromass Q-TOF Ultima (Waters Micromass MS Technologies) hybrid tandem mass spectrometer was used for the acquisition of the electrospray ionization (ESI) mass spectra. This instrument is equipped with a nanoflow electrospray source. The samples were infused into the mass spectrometer using nanoflow capillaries (Proxeon Biosystems, Denmark). The needle voltage was ∼1800 V, and the collision energy was 10 eV for the MS analyses. Samples for flow injection analyses were diluted 1:20 with a solution of 50:50 acetonitrile, 0.1% formic acid. Data analysis was accomplished with a MassLynx data system and Transform deconvolution software supplied by the manufacturer (Waters Micromass MS Technologies). Redox Potential Determination—Oxidation-reduction titrations were carried out as described previously using the fluorescence of the monobromobimane-modified form of the reduced protein to monitor the extent of the reduction of the protein (18Krimm I. Lemaire S. Ruelland E. Miginiac-Maslow M. Jacquot J.P. Hirasawa M. Knaff D.B. Lancelin J.M. Eur. J. Biochem. 1998; 255: 185-195Crossref PubMed Scopus (60) Google Scholar, 19Hirasawa M. Ruelland E. Schepens I. Issakidis-Bourguet E. Miginiac-Maslow M. Knaff D.B. Biochemistry. 2000; 39: 3344-3350Crossref PubMed Scopus (46) Google Scholar). Ambient potentials (Eh) were established using mixtures of oxidized glutathione (GSSG) and reduced glutathione (GSH) and the PtTRXh4 samples were incubated at these defined Eh values for 2 h to reach redox equilibrium. The oxidation-reduction midpoint potential (Em) value was shown to be independent of the total concentration (GSSG and GSH) present in the redox equilibration buffer over the range from 2 to 5 mm. The Em was calculated by fitting the data to the Nernst equation for a two-electron process as described previously (20Masuda S. Dong C. Swem D. Knaff D.B. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7078-7083Crossref PubMed Scopus (73) Google Scholar). PrxQ Activity Measurement—The reduction of H2O2 by poplar PrxQ in the presence of PtTrxh4 was followed spectrophotometrically, using a Cary 50 spectrophotometer, by monitoring the decrease in absorbance arising from NADPH oxidation in a coupled enzyme assay system. The reaction mixture (500 μl) contained 30 mm Tris-HCl, pH 8.0, 1 mm EDTA, 200 μm NADPH, 0.5 IU glutathione reductase, 1 mm GSH, 6 μm PtGrx C4, 16 μm PtTrxh4 WT and mutants, 500 μm H2O2, and 2 μm PtPrxQ. Alternatively, H2O2 disappearance was followed directly. The reaction mixture (100 μl) contained 30 mm Tris-HCl, pH 7.0, 500 μm DTT, 4 μm PtPrxQ, and 36 μm PtTrxh4. The reaction was started by adding 500 μm H2O2. After given incubation times, 5 μl were mixed with 495 μl of FOX1 (ferrous oxidation in xylenol orange) reagent (21Deiana L. Carru C. Pes G. Tadolini B. Free Radic. Res. 1999; 31: 237-244Crossref PubMed Scopus (51) Google Scholar). The absorbance was then read at 560 nm after 1-h incubation. Crystallization—Three different samples were crystallized, one with Se-Met (WT PtTrxh4) and two with regular methionines (WT and PtTrxh4C61). Crystallization conditions were screened extensively at 20 °C with the microbatch method. Drops used for the initial crystallization trials consisted of 2 μl of the protein solution (20 mg/ml) mixed with 2 μl of various crystallization solutions. The [SeMet]PtTrxh4 crystals were grown in 1.0 m sodium/potassium phosphate buffer, pH 6.9, (Hampton SaltRx Screen 2, solution 54), whereas the WT PtTrxh4 was obtained by using the JBS screen 2 solution D2 (30% PEG 4000, 0.1 m NaHEPES, pH 7.5, 0.2 m CaCl2). For PtTrxh4C61S protein, orthorhombic crystals were obtained by using the Hampton SaltRx Screen condition 55 buffer (1.0 m sodium/potassium phosphate, pH 8.2). The drop was formed by mixing 25 mg/ml protein with crystallization solution in a 1:1 ratio. Crystals were cryoprotected and flash-cooled in liquid ethane at 100 K. Data Collection and Processing, Structure Solutions, and Refinements—Information and statistics of data collection and processing of the three crystals are presented in Table 1. The diffraction images of the different crystals were indexed, integrated, and scaled using either the HKL program (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) or the XDS program package (23Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3200) Google Scholar), then analyzed using the CCP4 software package version 6.0.2 (24CCP4 Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar).TABLE 1Data collection, phasing, and refinement statistics for the [SeMet]PtTrxh4, the WT PtTrxh4, and the PtTrxh4C61S crystalsData set[SeMet]PtTrxh4WT PtTrxh4PtTrxh4 C61SData collection and processing statistics Data collection siteBM30A ESRF-GrenobleX11 DESY-HamburgBW7A DESY-Hamburg Wavelength (Å)0.98050.81231.2400 Space groupP41212P61P212121 Unit cell dimensions (Å) (a, b, c)44.89, 44.89, 131.7447.35, 47.35, 196.0031.78, 44.10, 85.68 Asymmetric unit1 subunit2 subunits1 subunit Resolution range (Å)aThe values in parentheses are for the highest resolution bin.32.94-2.46 (2.60-2.46)50.00-2.15 (2.23-2.15)30.00-1.60 (1.66-1.60) RedundancyaThe values in parentheses are for the highest resolution bin.7.0 (3.6)28.3 (37.6)5.2 (5.3) Completeness (%)aThe values in parentheses are for the highest resolution bin.93.4 (65.7)99.8 (100.0)98.5 (98.4) I/δIaThe values in parentheses are for the highest resolution bin.17.85 (3.10)22.98 (2.60)16.03 (6.20) RmergeaThe values in parentheses are for the highest resolution bin., bRmerge = Σi|Ii — bIN|/Σ|bIN|, where I is the intensity for the ith measurement of an equivalent reflection with the indices h, k, l.0.089 (0.588)0.053 (0.392)0.092 (0.266) Phasing power (acentric/centric)1.283/0 Rcullis (isomorphous/anomalous)0/0.754 Figure of merit (acentric/centric)0.34824/0.06270Refinement statistics Resolution range (Å)41.0-2.1530.0-1.6 Reflections used12,72515,470 RcrystcRcryst = Σ|Fo — Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. (Rfree)dThe Rfree value was calculated from 5% of all data that were not used in the refinement.20.97 (28.53)19.06 (19.63) Protein/waters/PO42 × (111 residues)/63/0129 residues/115/1 Mean B factor (Å2)Main chain55.2019.40Side chain57.6721.52Water59.2932.47All56.5321.69 Rms deviation from ideal geometryBond lengths (Å)0.0300.010Bond angles (°)2.51.4Dihedral angles (°)26.324.1Improper angles (°)5.591.46Ramachandran plot Residues in most favored regions (%)91.592.8 Residues in additionally allowed regions (%)7.57.2 Residues in generously allowed regions (%)1.00.0a The values in parentheses are for the highest resolution bin.b Rmerge = Σi|Ii — bIN|/Σ|bIN|, where I is the intensity for the ith measurement of an equivalent reflection with the indices h, k, l.c Rcryst = Σ|Fo — Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.d The Rfree value was calculated from 5% of all data that were not used in the refinement. Open table in a new tab A SeMet-labeled protein was prepared and a single-wavelength anomalous dispersion dataset was collected. Although three selenium atoms per protein were expected (including the one associated to the first methionine residue), only two were found (SeMet79 and SeMet134) using SHELXL97 (25Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1867) Google Scholar). Subsequent mass spectroscopy analysis allowed us to identify a SeMet-labeled protein that is presumed to be truncated (113 instead of 139 amino acids; absence of the N-terminal part) during its production in the E. coli system. The solution with the highest correlation coefficient in the heavy atom position determination was fed into SHARP (26de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1796) Google Scholar) for further refinement of SeMet-sulfur positions and phasing. After refinement, SHARP reported that occupancies of heavy atoms were 1.0, respectively, and the calculated experimental phase had an overall figure of merit of 0.35 (acentric reflections) and 0.06 (centric reflections) for 35-2.5-Å diffraction data. The phase improvement was made using SOLOMON (27Abrahams J.P. Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1138) Google Scholar) where the figure of merit gradually increased to 0.79 and the electron density map became interpretable. This map was submitted to the automatic model building program ARP/wARP, version 6.1 (28Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar), and 95 residues of the expected 113 were built. The initial model was further improved by manual building with Turbo-Frodo (29Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1989: 77-78Google Scholar) or Coot (30Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4098) Google Scholar) interspersed with refinements using both CNS, version 1.1 (31Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar), and REFMAC5 (24CCP4 Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). Models of the wild type and the PtTrxh4C61S mutant were solved by molecular replacement (MOLREP) (32Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22534) Google Scholar) using [SeMet]PtTrxh4 as the search model. The final structure of the WT PtTrxh4 was obtained after manual rebuilding of many parts of the structure (mainly near the active site and the α-helix 3 regions) and refinements using CNS and REFMAC5. Concerning the PtTrxh4C61S mutant, 82% of the model was built automatically using ARP/wARP. The final mutant model was obtained using the same procedure used for the WT protein. Throughout the model building and refinement processes, qualities of all models were assessed using the program PRO-CHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Refinement statistics are summarized in Table 1. Figures were prepared with PyMOL (34DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar). Structure Superimpositions—Superimpositions of the present structures with their homologous structures obtained from the Protein Data Bank were performed using the LSQMAN program from the DEJAVU package (35Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 179-185Crossref PubMed Scopus (94) Google Scholar), and the Lsqkab (superpose) program of the CCP4 suite with default parameters proposed by the authors. Protein Data Bank Accession Codes—Atomic coordinates and structure factors have been deposited in the Protein Data Bank. Accession codes are 3D21 (WT PtTrxh4) and 3D22 (PtTrxh4C61S). PtTrxh4 belongs to the third Trx h subgroup and exhibits the classical WCGPC active site. In addition to these two cysteinyl residues, members of this subgroup contain one additional conserved cysteine found in the fourth position in an N-terminal extension (comprising the 24 most N terminus residues) (Fig. 1). Sequence analysis using several localization prediction programs suggests that the N-terminal extension does not correspond to a signal peptide and that this protein is likely to be located in the cytosol. Thiol Content Determination—To investigate the putative role of the three conserved cysteines in the catalytic mechanism of PtTrxh4, site-directed mutagenesis has been used to produce the full-length recombinant proteins in E. coli: PtTrxh4C4S, PtTrxh4C58S, and PtTrxh4C61S. Thiol content of these proteins has been determined both in reducing and non-reducing conditions. A summary of these data is shown in Table 2. Nearly three thiols per protein are titrated for the reduced WT protein, whereas approximately one SH group is present in the unreduced WT protein. Under reducing conditions, thiol contents of the PtTrxh4C4S, PtTrxh4C58S, and PtTrxh4C61S mutants were all close to 2 SH/mol in good agreement with the expected theoretical values. In the absence of reductant, the PtTrxh4C61S mutant is fully oxidized (no titrated thiol group), whereas PtTrxh4C4S and PtTrxh4C58S mutants are only partially oxidized (i.e. thiol content values lower than 1 SH/mol were measured).TABLE 2Thiol content of PtTrxh4 under nonreducing or reducing conditionsNonreducing conditionsReducing conditionsWT PtTrxh40.752.88PtTrxh4C4S0.931.64PtTrxh4C58S0.641.93PtTrxh4C61S0.051.55 Open table in a new tab SDS-PAGE was performed under reducing and non-reducing conditions (see supplementary data Fig. S1). In the absence of DTT, both monomers and dimers were detected for PtTrxh4 WT and C61S with monomers being the dominant species for both proteins. Although thiol titration results are most consistent with the formation of an intramolecular disulfide bond, the presence of reducible dimers for the C61S mutant indicates that Cys58 and/or Cys4 could be also involved in intermolecular disulfide bonds. Only monomers were observed for the C4S mutant in the absence of DTT, suggesting that an intramolecular disulfide bond is formed between Cys58 and Cys61. In the case of the C58S mutant, oligomeric forms (disappearing under reducing conditions) were present in high abundance, which is consistent with the presence of intermolecular disulfide bonds involving Cys4 and/or Cys61. Redox Potential Determination—The redox titrations of PtTrxh4 and of its three C/S variants have been carried out at pH 7.0 over the potential range of –50 to –250 mV. Each result represents the average of at least two determinations and the average deviations suggest that the experimental uncertainty in Em is between 5 and 10 mV. The data obtained for the PtTrxh4 and PtTrxh4C61S give a good fit to the curve expected for a single two-electron process with an Em value of –165 ± 10 and –178 ± 10 mV, respectively. Data from redox titrations of PtTrxh4C4S and PtTrxh4C58S could not be fitted to the Nernst equation for a single two-electron process but did give good fits for the sum of the Nernst equation for two separate two-electron processes. In the case of PtTrxh4C4S, the Em values for the two components are –140 ± 10 and –200 ± 10 mV and for PtTrxh4C58S the Em values of the two components are –130 ± 10 and –180 ± 10 mV. The two-electron nature of these redox couples, the Eh range over which they titrate and the fact that the experiments rely on a thiol-specific reagent, monobromobimane, to monitor the course of the titrations leave little doubt that the components being titrated are dithiol/disulfide couples. As the redox titration were carried out using the GSH/GSSG couple for redox buffering, it may be possible that some of the components of the redox titration arise from protein-GSH adducts. To investigate the susceptibility of PtTrxh4 to glutathionylation, the protein was reduced by DTT, incubated with a large excess of GSSG, and analyzed by quadrupole time-of-flight mass spectrometry. It showed the formation of a low-amplitude peak with a mass of 15,761.00 Da (around 35% of the total preparation) in addition to the peak corresponding to the oxidized recombinant PtTrxh4 (15,455.125 Da). This additional peak is compatible with the addition of one GSH molecule (305.5 Da). Concerning the mutants, a large amplitude additional peak (around 90% of the total preparation) corresponding to a GSH adduct was detected with PtTrxh4C58S; PtTrxh4C61S was glutathionylated to a lesser extent (around 35% of the total preparation); whereas PtTrxh4C4S showed no detectable glutathionylation. To identify the glutathionylation site(s) of the WT protein, we performed tryptic digestion of fully reduced or GSSG-oxidized protein and analyzed the tryptic fragments by mass spectrometry. The data indicated that Cys4 is the glutathionylation site on PtTrxh4, a result consistent to the absence of GSH adduct formation with PtTrxh4C4S. Due to the possibilities of extensive glutathionylation and the multiplicity of possible disulfides (both intra- and intermolecular), the potential redox values obtained are actually the average values of a mixture of disulfides. Hence, it is not yet possible to provide a unique assignment of each Em component in these two-component titrations. Nevertheless, we can conclude that the redox midpoint potential values for all of the couples involved are more positive than –200 mV, a value that is very much more electropositive than the redox potential of typical Trxs (about –300 mV). PrxQ Activity—The activity of the recombinant proteins was tested using a non-physiological PrxQ-based system involving PtPrxQ, GSH, and PtGrx (type C4 with an active site CPYC) (8Gelhaye E. Rouhier N. Jacquot J.P. FEBS Lett. 2003; 555: 443-448Crossref PubMed Scopus (88) Google Scholar). Both PtTrxh4C4S and PtTrxh4C58S are totally inactive in this system, whereas the mutant PtTrxh4C61S retained some of the activity exhibited by WT PtTrxh4 (Fig. 2a). Taking into account the data reported from previously characterized Trxs, Cys58 is most likely the catalytic residue. As sequence comparisons suggest that Cys58 and Cys61 are the active site residues (with Cys58 making the initial nucleophilic attack characteristic of Trxs), it is not surprising that converting either of these cysteine residues to serine results in loss of activity. The most interesting feature of PtTrxh4, based on the observed total loss of activity for the PtTrxh4C4S mutant, is that three cysteines seem to be involved in the catalytic mechanism. The interactions between PtTrxh4 and several PtGrx mutants have also been investig
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