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Inactivation of Human Peroxiredoxin I during Catalysis as the Result of the Oxidation of the Catalytic Site Cysteine to Cysteine-sulfinic Acid

2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês

10.1074/jbc.m206626200

1083-351X

Kap‐Seok Yang, Sang Won Kang, Hyun Ae Woo, Sung Chul Hwang, Ho Zoon Chae, Kanghwa Kim, Sue Goo Rhee,

Heat shock proteins research

By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys51-SH to Cys51-SO2H. Thus, Cys51-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys51-SO2H, which cannot be reversed by thioredoxin. The presence of H2O2 alone was not sufficient to cause oxidation of Cys51 to Cys51-SO2H. Rather, the presence of complete catalytic components (H2O2, thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys172/Ser172 mutant Prx I required not only H2O2, but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 μm) of H2O2 indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 °C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H2O2. By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys51-SH to Cys51-SO2H. Thus, Cys51-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys51-SO2H, which cannot be reversed by thioredoxin. The presence of H2O2 alone was not sufficient to cause oxidation of Cys51 to Cys51-SO2H. Rather, the presence of complete catalytic components (H2O2, thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys172/Ser172 mutant Prx I required not only H2O2, but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 μm) of H2O2 indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 °C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H2O2. peroxiredoxin thioredoxin thioredoxin reductase dithiothreitol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry matrix-assisted laser desorption ionization time-of-flight mass spectrometry Peroxiredoxins are a family of peroxidases that reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol, respectively, with the use of reducing equivalents provided by thiol-containing proteins (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 2Rhee S.G. Kang S.W. Chang T.S. Jeong W. Kim K. International Union of Biochemistry and Molecular Biology Life. 2001; 52: 35-41Crossref Scopus (512) Google Scholar, 3Hofmann B. Hecht H.J. Flohé L. Biol. Chem. Hoppe-Seyler. 2002; 383: 347-364Crossref PubMed Scopus (768) Google Scholar). The first peroxiredoxin (Prx)1 proteins to be discovered were a 25-kDa yeast protein initially called thiol-specific antioxidant enzyme (4Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1988; 263: 4704-4711Abstract Full Text PDF PubMed Google Scholar, 5Kim I.H. Kim K. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6018-6022Crossref PubMed Scopus (131) Google Scholar, 6Chae H.Z. Kim I.H. Kim K. Rhee S.G. J. Biol. Chem. 1993; 268: 16815-16821Abstract Full Text PDF PubMed Google Scholar), and a 21-kDa Salmonella typhimurium alkyl-hydroperoxide reductase termed AhpC (7Tartaglia L.A. Storz G. Ames B.N. J. Mol. Biol. 1989; 210: 709-719Crossref PubMed Scopus (145) Google Scholar, 8Storz G. Jacobson F.S. Tartaglia L.A. Morgan R.W. Silveira L.A. Ames B.N. J. Bacteriol. 1989; 171: 2049-2055Crossref PubMed Scopus (205) Google Scholar, 9Tartaglia L.A. Storz G. Brodsky M.H. Lai A. Ames B.N. J. Biol. Chem. 1990; 265: 10535-10540Abstract Full Text PDF PubMed Google Scholar, 10Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar). Subsequently, a mammalian homolog of thiol-specific antioxidant/AhpC was purified; and it, together with thiol-specific antioxidant and AhpC, defined a family of peroxidases that now includes six mammalian isoforms (Prx I–VI) and members identified in organisms from each kingdom (3Hofmann B. Hecht H.J. Flohé L. Biol. Chem. Hoppe-Seyler. 2002; 383: 347-364Crossref PubMed Scopus (768) Google Scholar, 10Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar, 11Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar). All Prx proteins contain a conserved Cys residue, which corresponds to Cys51 in mammalian Prx I, in the N-terminal portion of the molecule (10Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar, 11Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar). The majority of Prx proteins, including four (Prx I–IV) of six mammalian peroxiredoxins, contain an additional conserved Cys residue in the C-terminal region that corresponds to Cys172 in mammalian Prx I (2Rhee S.G. Kang S.W. Chang T.S. Jeong W. Kim K. International Union of Biochemistry and Molecular Biology Life. 2001; 52: 35-41Crossref Scopus (512) Google Scholar, 3Hofmann B. Hecht H.J. Flohé L. Biol. Chem. Hoppe-Seyler. 2002; 383: 347-364Crossref PubMed Scopus (768) Google Scholar, 12Matsumoto A. Okado A. Fujii T. Fujii J. Egashira M. Niikawa N. Taniguchi N. FEBS Lett. 1999; 443: 246-250Crossref PubMed Scopus (130) Google Scholar, 13Seo 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 (378) Google Scholar). The Prx enzymes containing two conserved Cys residues are thus called 2-Cys Prx, in comparison with a small number of Prx proteins termed 1-Cys Prx, which contain only one conserved cysteine residue in the N-terminal domain (2Rhee S.G. Kang S.W. Chang T.S. Jeong W. Kim K. International Union of Biochemistry and Molecular Biology Life. 2001; 52: 35-41Crossref Scopus (512) Google Scholar, 10Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar). In 2-Cys Prx enzymes, the N-terminal conserved cysteine is oxidized by H2O2 to cysteine-sulfenic acid (Cys51-SOH), which then reacts with Cys172-SH of the other subunit to produce an intermolecular disulfide (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 14Chae H.Z. Uhm T.B. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7022-7026Crossref PubMed Scopus (280) Google Scholar). Reduction of the disulfide intermediate of Prx I–IV is specific in that it can be achieved by thioredoxin (Trx), but not by GSH or glutaredoxin (15Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 16Jin 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 (385) Google Scholar). Thus, the reducing equivalents for the peroxidase activity of Prx I–IV are ultimately derived from NADPH via thioredoxin reductase (TrxR) and Trx. Not all Prx enzymes containing both conserved Cys residues are reduced by Trx: the bacterial Prx AhpC is reduced by AhpF, which contains both Trx and TrxR domains (10Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar, 17Jacobson F.S. Morgan R.W. Christman M.F. Ames B.N. J. Biol. Chem. 1989; 264: 1488-1496Abstract Full Text PDF PubMed Google Scholar). In the absence of a physiological electron donor, the peroxidase activities of 2-Cys Prx enzymes can be supported by small thiol molecules such as dithiothreitol (DTT) and 2-mercaptoethanol, but not by GSH (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 14Chae H.Z. Uhm T.B. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7022-7026Crossref PubMed Scopus (280) Google Scholar, 15Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). In 1-Cys Prx enzymes, including mammalian Prx VI, the conserved cysteine is also the site of oxidation, but remains in a sulfenic acid state upon oxidation because there is no nearby partner cysteine to form a disulfide bond (18Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 19Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (330) Google Scholar). Trx cannot reduce this sulfenic acid-containing intermediate (18Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 20Fujii T. Fujii J. Taniguchi N. Eur. J. Biochem. 2001; 268: 218-225Crossref PubMed Google Scholar). GSH has been proposed to be the electron donor, but these data remain controversial (18Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 20Fujii T. Fujii J. Taniguchi N. Eur. J. Biochem. 2001; 268: 218-225Crossref PubMed Google Scholar, 21Fisher A.B. Dodia C. Manevich Y. Chen J.W. Feinstein S.I. J. Biol. Chem. 1999; 274: 21326-21334Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 22Singh A.K. Shichi H. J. Biol. Chem. 1998; 273: 26171-26178Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23Peshenko I.V. Novoselov V.I. Evdokimov V.A. Nikolaev Y.V. Kamzalov S.S. Shuvaeva T.M. Lipkin V.M. Fesenko E.E. Free Radic. Biol. Med. 1998; 25: 654-659Crossref PubMed Scopus (48) Google Scholar). The crystal structures of 2-Cys and 1-Cys Prx enzymes reveal that the catalytic cysteine is located in a small pocket formed by the N- and C-terminal domains of the two subunits (19Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (330) Google Scholar, 24Hirotsu S. Abe Y. Okada K. Nagahara N. Hori H. Nishino T. Hakoshima T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12333-12338Crossref PubMed Scopus (234) Google Scholar, 25Schröder E. Littlechild J.A. Lebedev A.A. Errington N. Vagin A.A. Isupov M.N. Struct. Fold. Des. 2000; 8: 605-615Abstract Full Text Full Text PDF Scopus (275) Google Scholar, 26Alphey M.S. Bond C.S. Tetaud E. Fairlamb A.H. Hunter W.N. J. Mol. Biol. 2000; 300: 903-916Crossref PubMed Scopus (141) Google Scholar, 27Wood Z.A. Poole L.B. Hantgan R.R. Karplus P.A. Biochemistry. 2002; 41: 5493-5504Crossref PubMed Scopus (293) Google Scholar). The reactive cysteine is thus protected from larger oxidant molecules that contain disulfide linkages. The structures also show that the N-terminal conserved cysteine is surrounded by positively charged amino acid residues, which stabilize the thiolate (Cys-S−) anion. The thiolate anion is more readily oxidized by peroxides than its protonated thiol counterpart (Cys-SH) (28Kim J.R. Yoon H.W. Kwon K.S. Lee S.R. Rhee S.G. Anal. Biochem. 2000; 283: 214-221Crossref PubMed Scopus (242) Google Scholar). This provides the mechanistic basis for the observed sensitivity of the active-site cysteine to oxidation by peroxides. Previously, we reported that Prx purified from yeast is readily inactivated during catalysis (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). We speculated that such inactivation occurs if the sulfenic acid moiety of the reaction intermediate is further oxidized by H2O2 to cysteinesulfinic acid (Cys-SO2H) before disulfide formation with Cys172 can occur (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). Sulfinic acid cannot be reduced by the Trx or DTT included in the assay mixture. Recently, Mitsumotoet al. (29Mitsumoto A. Takanezawa Y. Okawa K. Iwamatsu A. Nakagawa Y. Free Radic. Biol. Med. 2001; 30: 625-635Crossref PubMed Scopus (104) Google Scholar) used two-dimensional PAGE to compare proteins in human umbilical vein endothelial cells before and after exposure of the cells to H2O2. In H2O2-treated cells, a number of proteins (including Prx I and Prx II) demonstrated altered migration consistent with decreased pI, suggesting that such oxidative inactivation might also occur in cells. However, these acidic Prx enzymes were not characterized in detail. We have now investigated the mechanism of human Prx I inactivation by H2O2. Here, we demonstrate that the enzyme inactivation and concomitant acidic shift of Prx on two-dimensional gels are due in fact to the conversion of the active-site cysteine to Cys-SO2H. Furthermore, we observed that only those Prx molecules actively engaged in the catalytic cycle are vulnerable to oxidative inactivation. The construction of a bacterial expression vector for human Prx I (pETprxI-WT) has been described (15Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). Two Prx I mutants in which Cys51 and Cys172 were individually replaced by serine residues (C51S and C172S, respectively) were generated by standard PCR-mediated site-directed mutagenesis with pETprxI-WT as the template and complementary primers containing a single-base mismatch that converts the codon for Cys to one for Ser. The final mutated PCR products were ligated into the pET vector to generate pETprxI-C51S and pETprxI-C172S.Escherichia coli BL21(DE3) competent cells (Novagen) were transformed with pETprxI-WT, pETprxI-C51S, or pETprxI-C172S; cultured at 37 °C overnight in 100 ml of LB medium supplemented with ampicillin (100 μg/ml); and then transferred to 10 liters of fresh LB medium in a Microferm fermentor (New Brunswick Scientific). When the absorbance of the culture at 600 nm reached 0.6–0.8, expression was induced by isopropyl-β-d-thiogalactopyranoside at a final concentration of 0.4 mm. After incubation for an additional 3 h, the cells were collected by centrifugation, frozen in liquid nitrogen, and stored at −70 °C until used. The recombinant proteins were purified as described (30Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (201) Google Scholar). NADPH oxidation coupled to the reduction of H2O2 was monitored at 30 °C as a decrease inA 340 using a Hewlett-Packard Model 8453 UV-visible spectrophotometer equipped with a thermostable cell holder and a multicell transport. The reaction was initiated by addition of the indicated concentration of Prx I to a 200-μl reaction mixture containing 50 mm Hepes-NaOH (pH 7.0), 1 mmEDTA, 0.8 μm TrxR, and the indicated concentrations of H2O2, NADPH, and Trx. HeLa S3 cells were adapted to suspension growth in Spinner minimum essential medium (Quality Biological, Inc.) supplemented with 10% (v/v) fetal bovine serum (Invitrogen). The cells were grown to a density of 1 × 106 cells/ml and maintained by dilution with fresh complete medium every 4 days. HeLa S3 cells were rinsed three times with ice-cold phosphate-buffered saline and lysed in lysis buffer (8 murea, 4% CHAPS, and 40 mm Tris base) by sonication in a sonic bath three to four times for 30 s. After removal of insoluble materials by centrifugation at 14,000 × gfor 30 min, cell lysates were mixed with 10 volumes of rehydration buffer (8 m urea, 2% CHAPS, 0.5% immobilized pH gradient buffer, 20 mm DTT, and 0.005% bromphenol blue) and loaded onto immobilized pH gradient strips (pH 3–10, nonlinear). Isoelectric focusing on an IPGPhor isoelectrofocusing unit (Amersham Biosciences) and preparation (reduction and alkylation) of the immobilized pH gradient strips for the second-dimension SDS-PAGE were carried out according to the procedures recommended by the manufacturer. SDS-PAGE was conducted on 12% gels using an AmershamBiosciences SE600 vertical unit, and the protein spots were visualized by staining with silver nitrate. For immunoblot analyses of Prx enzymes, proteins on two-dimensional gels were transferred electrophoretically to a nitrocellulose membrane, and the membrane was incubated with rabbit antibodies to Prx I. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Biosciences or Pierce). Silver-stained two-dimensional gels were scanned with a Molecular Dynamics SI personal densitometer. Reverse-phase HPLC analyses were performed using an Agilent 1100 HPLC system with a Vydac 218TP54 column (0.47 mm × 25 cm). For protein analysis, injected samples were eluted at 1 ml/min with a 5–35% (v/v) acetonitrile/water gradient containing 0.04% (v/v) trifluoroacetic acid over 15 min and with a 35–60% gradient over the next 15 min. Gradients of 5–10% over 10 min, 10–40% over 30 min, and 40–60% over 30 min were used for peptide analysis. A Finnigan MAT LCQ electrospray ion-trap mass spectrometer was used for analysis of Prx I proteins and sequence analysis of Cys51-containing peptides. Dried HPLC fractions dissolved in 75% acetonitrile and 0.1% acetic acid were introduced at 1 μl/min using a syringe pump. Data were collected for positive ions at 300–2000 m/z using the following settings: capillary temperature, 215 °C; maximum ion inject time, 100 ms; full scan target, 9 × 107; and three microscans/full scan. For proteins, ESI-MS spectra were obtained and used to calculate the masses of proteins by deconvolution using Finnigan MAT BioWorks software. For sequence analysis of peptides, once the parent ions were identified, the mass spectrometer was set up to obtain collision-induced dissociation MS/MS spectra of the parent ions. The instrument for MS/MS spectra was configured as follows: capillary temperature, 215 °C; maximum ion inject time, 100 ms; full scan target, 2 × 107; three microscans/full scan; mass window, 2.5; and collision energy, used within a range of 0–50%. Mass spectrometry analysis of the tryptic peptides was performed on a Voyager-STR MALDI-TOF instrument (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser. Samples were dissolved in 50% acetonitrile and 5% formic acid, mixed with 10 mg/ml 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; Aldrich) in 0.1% trifluoroacetic acid and 70% acetonitrile, and spotted on a sample plate. The spectra were obtained in the positive-ion reflector or linear mode with delayed extraction under standard conditions. Spectra were analyzed using DataExplorer software (PerSeptive Biosystems). Standard peptides were used for calibration of peptides, whereas apomyoglobin and carbonic anhydrase were used as internal standards for mass scale calibration of proteins. The peroxidase activity of human Prx I was monitored by following the decrease inA 340 attributable to the oxidation of NADPH in a reaction mixture containing NADPH, Trx, TrxR, and varying concentrations of H2O2. The initial rate of NADPH oxidation (the slope at t = 0) was independent of H2O2 at saturating concentrations (0.1–1 mm) (K m for H2O2< 20 μm (31Chae 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 (317) Google Scholar)) (Fig.1 A). However, the rate decreased with time, and the higher the H2O2concentration, the faster the rate of decrease. The H2O2 concentration dependence of this decrease is shown more quantitatively by plotting the rate of NADPH oxidation (the first derivative of the NADPH oxidation curve) versustime (Fig. 1 B). Replenishment of Prx I was shown to restore the enzyme rate, indicating that the markedly decreased NADPH oxidation rate was not attributable to exhaustion of substrate or to product inhibition (data not shown). Reactions containing 200 μmH2O2 were stopped at 0, 60, and 150 s, and the resulting samples were subjected to two-dimensional PAGE analysis. A single spot at a position corresponding to a pI value of 8.1 (the theoretical pI of Prx I is 8.2) was observed at 0 s. At 60 s, however, a new spot whose intensity was similar to that of the original spot appeared at a more acidic position (pI 7.6). At 150 s, the intensity of the acidic spot was enhanced at the expense of the original spot. The increasing proportion of the acidic species compared with the original spot is consistent with the notion that the 50 and 80% decreases in rate observed at 60 and 150 s, respectively, are related to the conversion of Prx I to a more acidic derivative. To identify the modification responsible for the acidic shift, Prx I was oxidized for 5 min in the presence of 1 mm H2O2 and 10 mm DTT. Reduced and oxidized enzymes were separated from the reaction mixture by reverse-phase HPLC (Fig.2 A). The molecular masses of the separated proteins were measured by ESI-MS (Fig. 2 B). The masses calculated fromm/z and total charge of multiply charged ions of the reduced and oxidized enzymes were 21,979 and 22,011 Da, respectively. The mass of the reduced enzyme was in good agreement with the theoretical mass of 21,979.2 Da. The difference of 32 mass units between the reduced and oxidized Prx I enzymes suggests the presence of two additional oxygen atoms in the oxidized species. To determine the site of oxidation, the reduced and oxidized Prx I enzymes separated in Fig. 2 A were digested with trypsin, and the resultant peptides were fractionated by reverse-phase HPLC (Fig.2 C). The HPLC elution profiles of the reduced and oxidized Prx I peptides were nearly identical except between 48 and 56 min (Fig.2 C). Major peptide peaks from the reduced and oxidized proteins were collected and analyzed by MALDI-TOF-MS, enabling the assignment of 12 such peaks to defined fragments of Prx I. In addition to the disulfide-forming Cys51 and Cys172residues, Prx I contains two additional cysteine residues at positions 70 and 82. In both the reduced and oxidized states, the Cys172-containing peptide encompassing residues 168–189 eluted from the HPLC column at 26.1 min. Peptides containing both Cys70 and Cys82 were also unchanged: in both states, the peptides encompassing residues 67–91 (representing one missed trypsin cleavage site at Lys67) and residues 68–91 eluted at 35.5 and 36.8 min, respectively. However, retention of Cys51-containing peptides was altered by oxidation. In the reduced state, the peptides encompassing residues 27–61 (representing two missed trypsin cleavage sites at Lys34 and Lys36), residues 35–61 (representing one missed cleavage site at Lys36), and residues 37–61 eluted at 50.8, 51.7, and 55.1 min, respectively (Fig. 2 D). In the oxidized protein, however, none of these peaks corresponding to the three Cys51-containing peptides were observed. Instead, several peaks eluted near the expected positions. When the peaks at 49.7, 50.4, and 53.0 min were analyzed by MALDI-TOF-MS, each of them displayed a dominant and a minor ion separated by 16 mass units (Fig.2 D). Furthermore, the dominant ions derived from the oxidized peptides eluting at 49.7, 50.4, and 53.0 min were each larger by 32 mass units than those derived from reduced peptides encompassing residues 27–61, 35–61, and 37–61, respectively (Fig. 2 Dand Table I). These results support a model wherein Cys51-SH, but not Cys70-SH, Cys82-SH, or Cys172-SH, is hyperoxidized by H2O2 to Cys-SO2H and cysteic acid (Cys-SO3H).Table ICharacterization of Cys51-containing peptidesResiduesRetention timeMassSequenceTheoretical1-aTheoretical averagem/z values of singly charged ions ([M + H]+) calculated using GPMAW software (Lighthouse Data, Odense, Denmark).m/zObserved1-bAverage of at least three separate experiments with S.D. <0.02%.m/zmin27–611-cCys51-SH-containing peptide.50.84145.84145.4DISLSDYKGKYVVFFFYPLDFTFVCPTEIIAFSDR27–61-O2 1-dCys51-SO2H-containing peptide.49.74177.84177.527–61-O3 1-eCys51-SO3H-containing peptide.4193.84193.135–611-cCys51-SH-containing peptide.51.73223.83222.9GKYVVFFFYPLDFTFVCPTEIIAFSDR35–61-O2 1-dCys51-SO2H-containing peptide.50.43255.83254.935–61-O3 1-eCys51-SO3H-containing peptide.3271.83270.837–611-cCys51-SH-containing peptide.55.13038.53038.3YVVFFFYPLDFTFVCPTEIIAFSDR37–61-O2 1-dCys51-SO2H-containing peptide.53.03070.53070.337–61-O3 1-eCys51-SO3H-containing peptide.3086.53086.11-a Theoretical averagem/z values of singly charged ions ([M + H]+) calculated using GPMAW software (Lighthouse Data, Odense, Denmark).1-b Average of at least three separate experiments with S.D. <0.02%.1-c Cys51-SH-containing peptide.1-d Cys51-SO2H-containing peptide.1-e Cys51-SO3H-containing peptide. Open table in a new tab To demonstrate directly the formation of Cys51-SO2H, the amino acid sequence of the dominant ion ([M + 2H]2+, m/z1535.90) derived from peptide 37–61 was determined from the masses of the fragments arising from collision-induced dissociation of the peptide. The collision-induced dissociation MS/MS spectrum of the ion is shown in Fig. 2 E, along with the interpretation. The major daughter ions are y10 (m/z1148.5) and b15 (m/z 1922.5) ions, which derive from cleavage of the peptide bond between Cys51 and Pro52. The mass differences between b14 and b15 (135.2 Da), between y10and y11 (135.0 Da), and between z10 and z11 (135.0 Da) clearly indicate the presence of Cys-SO2H at Cys51. Mass spectral analysis of the tryptic peptides suggested that peroxide oxidation of Prx I Cys51 yielded mainly Cys51-SO2H, but also Cys51-SO3H to a lesser extent. In Fig. 2, Prx I was digested with trypsin for only 3 h before HPLC analysis. However, when oxidized Prx I was digested overnight with trypsin, Cys51-SO3H-containing peptides became the major products (data not shown). Furthermore, purified Cys51-SO2H-containing peptides were slowly autoxidized to Cys51-SO3H-containing peptides upon exposure to air (data not shown). However, longer exposure of intact Prx I to either H2O2 or air did not increase cysteic acid content (data not shown). Therefore, it appears that the formation of cysteic acid derivatives is the result of the air oxidation of Cys-SO2H-containing peptides. The sensitivity of Cys-SO2H to air has been reported (32Poole L.B. Claiborne A. J. Biol. Chem. 1989; 264: 12330-12338Abstract Full Text PDF PubMed Google Scholar). Cysteines 51 and 172 were individually replaced by serine residues to generate the C51S and C172S mutant enzymes, respectively. The wild-type and mutant enzymes were subjected to two-dimensional PAGE analysis before and after incubation with a peroxidase reaction mixture containing H2O2, NADPH, Trx, and TrxR (Fig.3). As observed in Fig. 1 C, the wild-type enzyme migrated predominantly to the more acidic position upon incubation with H2O2, suggesting that Cys51-SH was hyperoxidized to Cys-SO2H. As expected, no such shift was observed for C51S. C172S, which is catalytically inactive because Cys172-SH is required for disulfide formation by Cys51-SOH, did not undergo hyperoxidation. In contrast, when reducing equivalents were provided by DTT rather than by the Trx system (Trx, TrxR, and NADPH), C172S became hyperoxidized just as the wild-type enzyme did, whereas C51S was still resistant to hyperoxidation. As previously shown with a yeast Prx mutant in which the C-terminal conserved Cys was changed to Ser (1Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar), C172S is fully active when supported by DTT even though it lacks peroxidase activity in the presence of the Trx system (data not shown). Such catalysis is possible because a small diffusible thiol molecule like DTT can replace Cys172-SH in the formation of a disulfide with Cys51-SOH. The disulfide is subsequently reduced by DTT. These results indicate that oxidation of Cys51-SH to Cys51-SO2H occurs only when Prx is engaged in the catalytic cycle. To further examine whether the accumulation of the hyperoxidized protein requires continuous passage through the catalytic cycle, Prx I was inactivated by incubation with H2O2, NADPH, TrxR, and varying concentrations of Trx. An aliquot of the reaction mixture was removed at various times and assayed for peroxidase activity by measuring NADPH oxidation coupled to H2O2 reduction. For each Trx concentration, the initial rate of NADPH oxidation was plotted against duration of inactivation to yield rates of enzyme inactivation (Fig.4 A). Prx I inactivation proceeded slowly in the absence of Trx and increased gradually with increasing Trx concentrations, reaching saturation at 4 μm Trx. The Trx dependence of inactivation is displayed by plotting the residual activities measured after a 3-min incubation with the inactivation reaction mixture against Trx concentration. Half-maximal inactivation occurred at <0.5 μm Trx. When either TrxR or NADPH was omitted from the i