Reduction of Cysteine Sulfinic Acid in Peroxiredoxin by Sulfiredoxin Proceeds Directly through a Sulfinic Phosphoryl Ester Intermediate

Abstract

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO2-. This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the γ-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8Å resolution crystal structure of Srx in complex with ATP and Mg2+. This structure reveals the role of the Mg2+ ion to position the γ-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO2- species, support a mechanism where phosphorylation of Prx-SO2- is the first chemical step. Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO2-. This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the γ-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8Å resolution crystal structure of Srx in complex with ATP and Mg2+. This structure reveals the role of the Mg2+ ion to position the γ-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO2- species, support a mechanism where phosphorylation of Prx-SO2- is the first chemical step. The ubiquitous peroxiredoxins (Prxs) 2The abbreviations used are:PrxperoxiredoxinCsdCys sulfinic acidCys-SPHperoxidatic Cys residueCys-SRHresolving Cys residueCys-SPOHCys sulfenic acidCys-SPO2-Cys sulfinic acidGSHglutathioneSrxsulfiredoxinTrxthioredoxin. 2The abbreviations used are:PrxperoxiredoxinCsdCys sulfinic acidCys-SPHperoxidatic Cys residueCys-SRHresolving Cys residueCys-SPOHCys sulfenic acidCys-SPO2-Cys sulfinic acidGSHglutathioneSrxsulfiredoxinTrxthioredoxin. are thiol-dependent peroxidases that reduce H2O2, peroxynitrite, and alkyl hydroperoxides to water, nitrite, and alcohols, respectively (1Hofmann B. Hecht H.J. Flohé L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (763) Google Scholar). Besides this detoxification role, Prxs can regulate hydrogen peroxide-mediated cell signaling in response to receptor stimulation and oxidative stress (2Rhee S.G. Science. 2006; 312: 1882-1883Crossref PubMed Scopus (1649) Google Scholar, 3Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (602) Google Scholar, 4Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar, 5Choi M.H. Lee I.K. Kim G.W. Kim B.U. Han Y.H. Yu D.Y. Park H.S. Kim K.Y. Lee J.S. Choi C. Bae Y.S. Lee B.I. Rhee S.G. Kang S.W. Nature. 2005; 435: 347-353Crossref PubMed Scopus (328) Google Scholar). Humans have six isoforms of Prxs that can be divided into three classes depending on the number and location of Cys residues: 2-Cys, atypical 2-Cys, and 1-Cys (6Rhee S.G. Kang S.W. Chang T.S. Jeong W. Kim K. IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (506) Google Scholar). Although all Prxs contribute to peroxide homeostasis, the 2-Cys Prxs (PrxI–IV) are unique in that they can be can be post-translationally inactivated by the peroxide substrate and subsequently repaired by an enzyme known as sulfiredoxin (Srx or Npn3) (7Woo H.A. Jeong W. Chang T.S. Park K.J. Park S.J. Yang J.S. Rhee S.G. J. Biol. Chem. 2005; 280: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 8Woo H.A. Chae H.Z. Hwang S.C. Yang K.S. Kang S.W. Kim K. Rhee S.G. Science. 2003; 300: 653-656Crossref PubMed Scopus (463) Google Scholar, 9Chevallet M. Wagner E. Luche S. van Dorsselaer A. Leize-Wagner E. Rabilloud T. J. Biol. Chem. 2003; 278: 37146-37153Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar, 11Jönsson T.J. Lowther W.T. Subcell. Biochem. 2007; 44: 115-141Crossref PubMed Scopus (62) Google Scholar). Inactivation of Prx is thought to lead to a local increase in peroxide levels and signal potentiation (2Rhee S.G. Science. 2006; 312: 1882-1883Crossref PubMed Scopus (1649) Google Scholar, 3Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (602) Google Scholar, 4Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar). Moreover, inactivation appears to endow high molecular weight aggregates of Prx with chaperone activity (12Jang H.H. Lee K.O. Chi Y.H. Jung B.G. Park S.K. Park J.H. Lee J.R. Lee S.S. Moon J.C. Yun J.W. Choi Y.O. Kim W.Y. Kang J.S. Cheong G.W. Yun D.J. Rhee S.G. Cho M.J. Lee S.Y. Cell. 2004; 117: 625-635Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 13Moon J.C. Hah Y.S. Kim W.Y. Jung B.G. Jang H.H. Lee J.R. Kim S.Y. Lee Y.M. Jeon M.G. Kim C.W. Cho M.J. Lee S.Y. J. Biol. Chem. 2005; 280: 28775-28784Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The action of Srx restores peroxidase activity, thus turning off the peroxide signal and chaperone activity. This novel redox switch has been shown to regulate the transcription of antioxidant genes in Schizosaccharomyces pombe (14Bozonet S.M. Findlay V.J. Day A.M. Cameron J. Veal E.A. Morgan B.A. J. Biol. Chem. 2005; 280: 23319-23327Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 15Vivancos A.P. Castillo E.A. Biteau B. Nicot C. Ayte J. Toledano M.B. Hidalgo E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8875-8880Crossref PubMed Scopus (194) Google Scholar). Moreover, the return of human PrxII (hPrxII) to the reduced state can allow cells to renter the cell cycle after H2O2-dependent arrest (16Phalen T.J. Weirather K. Deming P.B. Anathy V. Howe A.K. van der Vliet A. Jönsson T.J. Poole L.B. Heintz N.H. J. Cell Biol. 2006; 175: 779-789Crossref PubMed Scopus (116) Google Scholar). Recent studies have also illustrated the importance of Srx in synaptic N-methyl-d-aspartic acid receptor activity and antioxidant defenses (17Papadia S. Soriano F.X. Leveille F. Martel M.A. Dakin K.A. Hansen H.H. Kaindl A. Sifringer M. Fowler J. Stefovska V. McKenzie G. Craigon M. Corriveau R. Ghazal P. Horsburgh K. Yankner B.A. Wyllie D.J. Ikonomidou C. Hardingham G.E. Nat. Neurosci. 2008; 11: 476-487Crossref PubMed Scopus (406) Google Scholar). peroxiredoxin Cys sulfinic acid peroxidatic Cys residue resolving Cys residue Cys sulfenic acid Cys sulfinic acid glutathione sulfiredoxin thioredoxin. peroxiredoxin Cys sulfinic acid peroxidatic Cys residue resolving Cys residue Cys sulfenic acid Cys sulfinic acid glutathione sulfiredoxin thioredoxin. Eukaryotic 2-Cys Prxs have evolved structural features that make them susceptible to elevated levels of peroxides (4Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar). In the catalytic cycle the peroxidatic cysteine (Cys-SPH) is oxidized by the peroxide to form a Cys sulfenic acid (Cys-SPOH). The interaction between the conserved YF and GGLG structural motifs is thought to impede the attack of the resolving Cys (Cys-SRH) from the adjacent monomer of the Prx homodimer on the Cys-SPOH to form a disulfide bond (Prx-Cys-SP–SR-Cys-Prx) (18Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar). The resulting catalytic “pause” permits excess peroxide to further oxidize Cys-SPOH to the catalytically inactive Cys sulfinic acid (Cys-SPO2-). Although a general thiol-disulfide oxidoreductase such as thioredoxin (Trx) readily returns the disulfide-bonded form of Prx to the active state, Cys sulfinic acid reduction requires Srx. The Srx repair reaction involves ATP, Mg2+, a conserved active site Cys (Cys-99 in hSrx), and an exogenous thiol reductant, such as glutathione (GSH) or Trx (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar, 19Chang T.S. Jeong W. Woo H.A. Lee S.M. Park S. Rhee S.G. J. Biol. Chem. 2004; 279: 50994-51001Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). Conflicting schemes for the reaction mechanism (Fig. 1) have been proposed, including which protein is phosphorylated in the first step (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar, 11Jönsson T.J. Lowther W.T. Subcell. Biochem. 2007; 44: 115-141Crossref PubMed Scopus (62) Google Scholar, 21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In the originally proposed reaction mechanism (Fig. 1, Path 1), Prx-SO2- is phosphorylated to generate a transient, novel sulfinic acid phosphoryl ester (Prx-SO2-PO32-) intermediate (6Rhee S.G. Kang S.W. Chang T.S. Jeong W. Kim K. IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (506) Google Scholar). In this mechanism Srx functions as a phosphotransferase (Fig. 1, Path 1, Step 1) and a thioltransferase (Path 1, Step 2). Another study proposed that Cys-99 of hSrx attacks the γ-phosphate of ATP resulting in the generation of a thiophosphate (Srx-S-PO32-) intermediate (Fig. 1, Path 2, Step 1) (7Woo H.A. Jeong W. Chang T.S. Park K.J. Park S.J. Yang J.S. Rhee S.G. J. Biol. Chem. 2005; 280: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). This intermediate would then be attacked by the Prx sulfinic moiety to generate the same Prx-SO2PO32- intermediate. It was further proposed that this latter intermediate is resolved by an external thiol (i.e. GSH, Fig. 1, Path 2, Step 2) and not Srx-Cys-99 (Path 1, Step 2). In Path 2 Srx functions exclusively to transfer the phosphate to Prx. Neither of these studies were able to obtain direct experimental evidence for formation of the sulfinic acid phosphoryl ester (Prx-SO2PO32-) nor was Jeong et al. (21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) able to demonstrate the existence of the proposed thiophosphate (Srx-S-PO32-) intermediate. In an effort to resolve the uncertainty surrounding the first step of the repair mechanism, we solved the crystal structure of Srx in complex with ATP·Mg2+. This allowed us to generate a model of the quaternary enzyme·substrate complex, ATP·Mg2+·Srx·Prx-SO2-. Phosphorylation studies on mutant proteins were performed to test this model. Overall, our results support that Prx-SO2- repair proceeds through a sulfinic acid phosphoryl ester intermediate formed by the direct transfer of the γ-phosphate from ATP to Prx. Protein Preparation and Modification—An engineered truncation of hSrx (residues 32–137) was expressed from a pET19 (Novagen) vector derivative containing a PreScission protease (GE Healthcare) cleavage site between Srx and the N-terminal His tag. The protein was expressed in C41(DE3) Escherichia coli cells and purified using nickel affinity and size-exclusion chromatography after the removal of the His tag (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). His-tagged hPrxII was purified in a similar manner as described previously (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar, 22Jönsson T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (113) Google Scholar). Mutant forms of hSrx (C99S and C99A) and hPrxII (C70S, C172S, and C51D) were generated using the QuikChange site-directed mutagenesis kit from Stratagene, and the proteins were purified as described for the wild-type proteins. Oxidation of PrxII to the sulfinic acid form was performed with excess dithiothreitol and H2O2 as described previously (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). A PD10 desalting column (GE Healthcare) was used to remove excess dithiothreitol and H2O2. Crystallization—Human SrxET-C99S was crystallized by the vapor diffusion method. Equal volumes of protein (16.6 mg/ml in 20 mm HEPES, pH 7.5, 100 mm NaCl) and well solutions (1 m sodium-potassium phosphate, pH 8.8, 8–13% (±)-2-methyl-2,4-pentanediol (MPD)) were mixed and incubated at 20 °C for a week as sitting drops. The crystal drop solution was slowly changed over the course of a day to a phosphate-free solution containing 1 m sodium malonate, pH 7.5, and 10% MPD. The ligands, 90 mm ATP and 5 mm Mg2+ in the same solution, were slowly to the crystal drop over an and allowed to A of the ATP solution with was to the crystal drop over 2 the crystal in at 100 K. and was on at were using and to was performed with of the that were The of the crystal was with cell a for the Srx complex were by molecular using and the structure as the model (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). The molecular solution was with using of and and P. J.S. J. M. T. Biol. PubMed Scopus Google Scholar). was performed with P. K. Biol. 2004; PubMed Scopus Google Scholar). were an at and with The of were performed with Biol. PubMed Scopus Google Scholar). The structure was using the which of the in the Murray J.S. 2004; PubMed Scopus Google Scholar). The and for the structure are in structural were with the and in are for the resolution Cell in are for the resolution to to to of in are for the resolution in a (Srx at 2 and PrxII at 1 and 1 ATP with were incubated at °C in a reaction containing mm HEPES, pH 7.5, 100 mm 1 mm The were by the of containing in mm pH 5 mm The were on that were also on The were in and a and at The were using a (GE The are of at three of Srx in with the structural the first step of the repair mechanism, we to the crystal structure of hSrx (residues 32–137) in complex with the substrate ATP and Mg2+. ATP was by the active site Cys-99 to to Srx in the of ATP were of the with the high of phosphate (1 in the solution (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). that removal of phosphate was for ATP into the the were with a solution containing sodium ATP and Mg2+ were then and The structure was determined to resolution and solved by molecular using wild-type as the model. The protein structure of the solved complex was to our previously wild-type engineered truncation of the hSrx crystal structure The Srx of a mixed in the of the and in and the The the ATP molecule and the Mg2+ ion was (Fig. The of ATP hydrogen with and on The phosphate of of and in the conserved active site with the and γ-phosphate from these by hydrogen with the with our structure of the of has that the with the γ-phosphate the of ATP (20Jönsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (46) Google Scholar). The Mg2+ ion is by the three phosphate of ATP and and (Fig. The in the is repair of 2-Cys Prxs has been to (Fig. 1) (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar, 21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In the originally proposed reaction mechanism (Fig. 1, Path 1), Prx-SO2- is phosphorylated to generate a sulfinic acid phosphoryl ester (Prx-SO2PO32-) intermediate (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar). this mechanism, Jeong et al. (21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) that the hSrx mutant was This the to that Cys-99 attacks the γ-phosphate of ATP resulting in the generation of a thiophosphate (Srx-S-PO32-) intermediate. In the complex (Fig. was of phosphorylation the crystal was with an excess of ATP The of this mutant is from the of the γ-phosphate of Moreover, as a of interaction with the Mg2+ the γ-phosphate into to This that Cys-99 of the wild-type enzyme would not be able to an in-line attack on the ATP the of attack for and A. Full Text Full Text PDF PubMed Scopus Google and The importance of Mg2+ for the of the γ-phosphate is further by the of activity the of (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar). of to the shown in Fig. of the hPrxII active site in a to and is by the GGLG and YF structural to and the for structural to Srx can and repair (4Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar, 22Jönsson T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (113) Google Scholar, E. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the of an in-line attack on the ATP molecule with the Srx active further the structural for the first step of the repair mechanism, we the complex the structure of the complex a disulfide bond between the active site Cys Fig. T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (113) Google Scholar). The of the γ-phosphate of ATP is from the of and from the of this we generated an model (Fig. by the disulfide bond and the peroxidatic Cys to the sulfinic acid a the active site was to the In this model of is from γ-phosphate The between the sulfinic acid moiety and ATP that the of a direct in-line attack on the In Cys-99 of Srx from the phosphate transfer to this residue in Srx and Prx test the enzyme substrate model and to between the proposed repair we the phosphorylation of Srx and Prx. to phosphorylated wild-type proteins were that the were (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar, 21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). the reaction is thiol-dependent and the of the first reaction the were performed the of or Moreover, all the active site cysteine of PrxII were to and C172S, to as Srx Cys Furthermore, we that a reduced would increase the of the to studies with wild-type these were (Fig. and phosphorylated Prx were In to the we generated reduction inactive of the the thiol attack by Srx to form the intermediate (Fig. 1, Path 1, Step 2) would be to phosphorylation of with this Srx mutant (Fig. we phosphorylation of the mutant as previously described by Jeong et al. (21Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In studies Srx not Srx was which the to that Srx was phosphorylated at to form a this we also phosphorylation of Srx and to wild-type Srx. Moreover, in the of the Srx were phosphorylated that the phosphorylation of Srx be to (Fig. In the mechanism proposed for Srx (Fig. 1), on the sulfinic moiety functions as the in the attack on the γ-phosphate of ATP, resulting in the formation of a sulfinic acid phosphoryl ester intermediate (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar). This mechanism is of the of a by a of including and 2002; PubMed Scopus Google Scholar). In the phosphorylation of of the of a phosphate which in is attacked by an ion in with similar to generate phosphate and we that the of the sulfinic acid moiety in with a of to would a phosphorylated Prx Although this the and of the the is the of the sulfinic acid the for a sulfinic acid we that the would be a F. V. PubMed Scopus Google Scholar, M.J. of and Scholar). phosphorylation of the is with Srx to the residue of the inactive then phosphorylation of Prx first would be the for the our is in the for a thiol attack on a that the step of the Srx reaction be in this Prx this of the mutant with wild-type Srx in a PrxII intermediate after of that hPrxII can be phosphorylated in the active site and that this mutant a mimic of the sulfinic acid substrate (Fig. test Srx be phosphorylated in the reaction, we incubated the mutant with the and Srx (Fig. In phosphorylation of PrxII as well as and In all the of phosphorylation in Prx was the Srx further test the of the reaction we the of phosphorylation in Prx Srx. In the of phosphorylation of was after and with (Fig. In formation of in the mutant was 1 of with all Srx (Fig. to Phosphorylation of Srx in the of was for support that is the substrate for The same was with the Srx are in with phosphorylation of Prx the first step in the reaction Moreover, the phosphorylation of Prx appears to be of Cys-99 Srx. of the have the enzymatic mechanism of sulfinic acid reduction by Srx. studies support for the first step of the reaction mechanism proposed by Biteau et al. (10Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (783) Google Scholar). This is by phosphorylation studies of Srx and Prx (Fig. which that the acid form of the active site residue of Prx be phosphorylated the phosphorylation of Srx be Furthermore, the crystal structure of Srx in complex with ATP and Mg2+ (Fig. 2) that Prx-SO2- is to attack ATP over Cys-99 in Srx. This with the for where the is attacked by in an in-line to a intermediate J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). crystal structure of Srx in complex with Prx T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (113) Google Scholar) and the complex a structural model of the repair Srx the active site of Prx by the YF of Prx (Fig. The of the conserved of Prx into a of Srx is also thought to in the of the Prx active site a of PrxII to the γ-phosphate of ATP for an in-line attack (Fig. these are on the and of the of Prx the of Srx from the Prx and Srx active T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (113) Google Scholar). This latter interaction is for repair to The step of the reaction, the thiol-dependent attack on the sulfinic phosphoryl ester is (Fig. 1, 1 and 2, Step 2). external thiol such as the phosphoryl ester from the from the site and attack the sulfinic phosphoryl ester moiety from the A is the direct attack by Cys-99 of Srx on the Prx intermediate. In our quaternary model is from of the Prx-SO2PO32- has been Cys-99 is in and in a position to attack the of Prx-SO2PO32- resulting in the formation of a intermediate (Fig. This intermediate be resolved by the external GSH, resulting in the formation of a mixed (Fig. 1, Path 1, Step or glutathione disulfide (Path 2, Step are to clarify the reaction Step The and of the and would further to resolve the unique reaction mechanism of Srx and the between signaling Prxs and the of and for and the is by the of and of with