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Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E 0) of the Trx1 active site (–230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an α helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals. Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E 0) of the Trx1 active site (–230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an α helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals. Thioredoxin (Trx1) 1The abbreviations used are: Trx1, human thioredoxin-1; GSH, reduced glutathione; GSSG, oxidized glutathione; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; ESI-MS, electrospray ionization triple quadrupole mass spectrometry; MALDI-TOF MS, matrix assisted laser desorption ionization time-of-flight mass spectrometry; IAA, iodoacetic acid; mBBr, monobromobimane. is a ubiquitous 12-kDa protein that functions as a reductant for ribonucleotide reductase, peroxiredoxins, and transcription factors (e.g. Fos, Jun, NF-κB, p53), controlling key aspects of cell proliferation and survival (1Laurent T.C. Moore E.C. Reichard P. J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar, 2Matthews J.R. Wakasugi N. Virelizier J. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 3Tanaka T. Nakamura H. Nishiyama A. Hosoi F. Masutani H. Wada H. Yodoi J. Free Radic. Res. 2000; 33: 851-855Crossref PubMed Scopus (131) Google Scholar, 4Powis G. Montfort W.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 261-295Crossref PubMed Scopus (331) Google Scholar). The active site of Trx1, WCGPC, is conserved among species from cyanobacteria to humans (5Eklund H. Gleason F. Holmgren A. Proteins. 1991; 11: 13-28Crossref PubMed Scopus (329) Google Scholar). The active site cysteines are readily accessible on the surface of the protein and become oxidized to a disulfide upon reduction of a target protein. This disulfide is cycled back to the dithiol by Trx reductase (6Holmgren A. Antioxid. Redox Signal. 2000; 2: 811-820Crossref PubMed Scopus (417) Google Scholar). Unlike Trxs from lower species, mammalian Trx1 contains additional conserved cysteine residues (at positions 62, 69, and 73 of human Trx1; See Fig. 1). Whether these non-active site Cys residues have biologic function is unknown. Cys-73 was present as an intermolecular disulfide bond (Trx1 homodimer) in x-ray crystal studies (7Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure (Lond.). 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), suggesting a possible function for Cys-73. However, a mutant Trx1 bearing a serine at this position still appeared as a homodimer in the crystal structure, suggesting that Cys-73 was not essential for dimerization (7Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure (Lond.). 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). More recently, S-glutathionylation of Trx1 at Cys-73 has been found during oxidative stress (8Casagrande S. Bonetto V. Fratelli M. Gianazza E. Eberini I. Massignan T. Slamona M. Chang G. Holmgren A. Ghezzi P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9745-9749Crossref PubMed Scopus (305) Google Scholar). In addition, S-nitrosylation of Cys-69 has recently been described (9Haendeler J. Hoffmann J. Tischler V. Berk B.C. Zeiher A.M. Dimmeler S. Nat. Cell Biol. 2002; 4: 743-749Crossref PubMed Scopus (344) Google Scholar). The midpoint potential (E 0) for the active site dithiol of Trx is available for several lower species (10Krause G. Lundstrom J. Barea J.L. de la Cuesta C.P. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 11Moore E.C. Reichard P. Thelander L. J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google Scholar, 12Porque P.G. Baldesten A. Reichard P. J. Biol. Chem. 1970; 9: 2363-2370Abstract Full Text PDF Google Scholar, 13Joelson T. Sjoberg B-M. Eklund H. J. Biol. Chem. 1990; 265: 3183-3188Abstract Full Text PDF PubMed Google Scholar, 14Kanzok S.M. Schirmer R.H. Turbachova I. Iozef R. Becker K. J. Biol. Chem. 2000; 275: 40180-40186Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) but not for mammals. Equilibrium with NADPH in the presence of a catalytic amount of Trx reductase, where it is assumed that each mole of NADPH consumed translates into 1 mol of Trx reduced, indicated that Escherichia coli Trx had a midpoint potential of –270 mV (10Krause G. Lundstrom J. Barea J.L. de la Cuesta C.P. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 11Moore E.C. Reichard P. Thelander L. J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google Scholar). An alternate approach using the equilibrium between glutathione (GSH) redox buffers and E. coli Trx yielded a similar value for E 0 (15Lundstrom J. Holmgren A. Biochemistry. 1993; 32: 6649-6655Crossref PubMed Scopus (160) Google Scholar). In the latter approach, the relative amounts of reduced and oxidized Trx were determined by quantification of the number of thiols with Ellman's reagent (dithionitrobenzoic acid) because E. coli Trx contains only one redox-active pair of cysteines. Oxidized and reduced forms of bovine Trx1 have been separated by carboxymethylation of thiols, native gel electrophoresis, and immunoblotting (16Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (201) Google Scholar). The fully reduced (fully carboxymethylated) and fully oxidized forms of bovine Trx1 were identified, but intermediate bands on the immunoblot were only identified as “partially carboxymethylated” (16Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (201) Google Scholar). In the present report we have used mass spectrometry to positively identify the forms of human Trx1 that are resolved by this native gel electrophoresis/Western blot (Redox Western blot) method. The results show that the resolved bands include fully reduced Trx1, a band with the active site in the disulfide form, and a band in which the protein has two disulfides, one at the active site and the other involving Cys-62 and Cys-69. The midpoint potential (E 0) for the active site was found to be –230 mV. Using this value and redox Western blot analysis of THP1 cells, the steady state redox (Eh) of Trx1 was –280 mV, a value that is comparable with the Eh for GSH/GSSG in these cells (–259 mV). During oxidative stress induced by diamide, both the Trx1 active site and non-active site disulfides were observed. In vitro, the non-active site disulfide was found to inhibit the regeneration of the active form of Trx1 by thioredoxin reductase, supporting the interpretation that oxidation of this non-active site dithiol could provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. Cell Culture—THP1 human monocytes were obtained from American Tissue Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Cells were grown in a humidified atmosphere of 5% CO2 at 37 °C. Eh calculations for cells were done using the Nernst equation with E 0 values for pH 7.4 and assuming a 5-μl cell volume per mg of cell protein. Glutathione Redox buffers were in a pH containing 1 were by with for at The GSH/GSSG redox potential was to the Nernst equation E 0 where E 0 mV at pH J. S. 1964; PubMed Scopus Google Scholar), and for the oxidation of to The concentrations were used to the and mV, and mV, and mV, and mV, and –270 mV, and mV, and with Trx1 at for were for the of and and for of the redox state of studies that from to yielded similar that equilibrium had been and were by as relative to as an J.L. P. 275: PubMed Scopus Google Scholar). Redox Western of the redox forms of Trx1 was upon the of Holmgren and A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and (16Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (201) Google Scholar). Trx1 was in pH containing iodoacetic at 37 for was by were in pH and separated on a native gel were to and for Trx1 using and by with x-ray were determined by analysis of with of bands containing Trx1 were to with and the were as described G. M. E. A. PubMed Scopus Google Scholar). The were using analysis by mass analysis of the was on a triple quadrupole mass with electrospray and in the of of the were to MALDI-TOF MALDI-TOF analysis was using MALDI-TOF mass with a was used as the and of were with of was in the for at 37 and was with The were with in pH at and 10% was The were separated by on an using a with a of in The of the was at an of with an of were and the of the in each was by for Trx1 with a disulfide bond between Cys-62 and Cys-69 was with the The were from the reduced human crystal structure, The was using the disulfide The disulfide was and the was of the were using J. 1991; Google Scholar), J. PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar). human Trx1 was a of of were using the in of The and were by of the with of the and containing the were by and of Trx1 in E. Trx1 were into of the of the an site and a a by a and a site were the and the were with and and at °C. cells were with of the mutant was induced by 1 for and were under native conditions using of Oxidized Thioredoxin by Thioredoxin and and were oxidized with 1 for at two of pH In a volume of of Trx1 were reduced in the presence of NADPH and bovine thioredoxin reductase of NADPH was by the in at A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of and Oxidized Trx1 was reduced with or oxidized with diamide, with IAA, separated by native and by Trx1 as a band as in Fig. Trx1 as a band 1 in Fig. but more Fig. the MALDI-TOF mass of of reduced and oxidized The and of the are in I. cysteines were in Trx1 The mass of Trx1 the presence of two disulfides, one between and and one between Cys-62 and Cys-69 were that to carboxymethylation at of these cysteines. and Cys-73 was present in the redox state of the of Cys-73 was a of redox The of the the as as the positions of the these were by mass spectrometry of of oxidized and reduced of MALDI-TOF mass of for Trx1 and Trx1 of Trx1 are is is and of and of from mass in in in identified in a Fig. of from the of and oxidized The and were with and the were and by on a triple the reduced species, the at to the addition of two was for The a mass of for each in an in mass of for the and and an in mass of for the as with the In addition, the by and the and by to the the oxidized species, the at to the was for Redox of Trx1 with Redox Trx1 was with GSH/GSSG redox buffers a of to mV) to be found cells J. Free Radic. Biol. PubMed Scopus (305) Google Scholar, 2002; PubMed Scopus Google Scholar), and separated on a native gel as The redox of the redox buffers were by analysis of the and of the redox buffers at the and the of the these were found to be the of the were with GSH/GSSG redox buffers at of and results were the equilibrium of 1991; PubMed Scopus Google Scholar), were to results obtained with and The results were not the conditions used not in Trx1 redox state during –270 mV, the band with the MALDI-TOF analysis that this band only fully Trx1 mV, Trx1 was between and and at mV, MALDI-TOF analysis that both dithiol and disulfide forms of and that is a of two form a disulfide between and with Cys-62 and Cys-69 present as thiols, and the other form, which had a a disulfide between Cys-62 and with and present as of the of the of immunoblotting yielded results comparable with obtained with of the redox of Trx1 (e.g. and redox state were with the redox Western blot Trx1 was with redox buffers from to mV, and and were by E 0 was by the to the Nernst equation using the of for the reduced form of the active site and the of 1 and as the disulfide The the to the Nernst a midpoint potential of –230 mV for provide an of the E 0 of the active site, Trx1 was at redox with the mBBr, with separated by liquid and by to of were and the of the were by thiols, but not disulfides, the in of the to and was of the oxidation state of the thiols in each this analysis of the of the active site was at a potential of mV. results are in with the value of –230 mV from the redox Western blot approach and show that the E 0 for the active site of human Trx1 is more that for E. coli in the of –230 mV –270 mV (10Krause G. Lundstrom J. Barea J.L. de la Cuesta C.P. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 11Moore E.C. Reichard P. Thelander L. J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google analysis of of are for or The of the of Cys could not be determined to of a to of could be positively in the are for or The of the of Cys could not be determined to of a to of could be positively in the in a Redox of the mass spectrometry that the oxidized form of Trx1 that as 1 on the redox Western blot two disulfide 1 was at as low as –270 mV, and the of 1 at mV and mV. However, was an of and the of a band at redox more oxidizing of the and was at mV the E 0 for the non-active site is more mV. The of the for the of in the redox Western blot at more mV are Redox of Trx1 in the redox state of Trx1, from and THP1 human monocytes were by redox Western blot and with the redox state of Trx1 at redox of the Trx1 from cells was in the fully reduced with and fully oxidized Trx1 1). This to an Eh value of –280 mV for the of pH the redox state of glutathione in these cells was mV of three of the cells to diamide, was a oxidation of the The amount of the form of Trx1 from to a of of the Trx1 of to diamide, the this was a oxidation of the active site relative to the from to mV). the of diamide a oxidation of the active site of the in the Trx1 crystal of fully reduced human Trx1 is but not that of the In the reduced structure, the for Cys-62 and Cys-69 at of a In this the disulfide bond form. However, this helix is only to the of the protein two and this helix in could from the protein and to disulfide bond this with the crystal for the fully reduced protein Cys-62 and Cys-69 and The the that the disulfide to form and the in this proximal to the active of Trx1 by Thioredoxin studies that fully oxidized mammalian Trx1 was more reduced by thioredoxin reductase was Trx1 that was oxidized only at the active site A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Holmgren A. Biochemistry. PubMed Scopus Google Scholar, J.R. Montfort M. M. Powis G. Biochem. Pharmacol. 1996; PubMed Scopus Google Scholar). results that oxidation of the non-active site thiols could the of reduction by thioredoxin reductase and provide a redox for of Trx1 the disulfide was for the with thioredoxin reductase, a mutant of Trx1 bearing Cys at positions and was and Fig. the reduction of and mutant Trx1 by thioredoxin reductase and NADPH to the NADPH oxidized Trx1 reduced A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Holmgren A. Biochemistry. PubMed Scopus Google Scholar, J.R. Montfort M. M. Powis G. Biochem. Pharmacol. 1996; PubMed Scopus Google Scholar), Trx1 an during which it was reduced by a of more In not the the of reduction was and only as the amount of substrate The amount of NADPH oxidized was with the interpretation that the mutant form one disulfide the An oxidized mutant of Trx1 in which the active site was was not a substrate for thioredoxin reductase However, upon the addition of a amount of Trx1 of the amount of mutant Trx1 on a 1 mol of NADPH was reduced per mol of the active site results show that was one disulfide in this mutant active site thiols and that this non-active site disulfide was reduced by the non-active site disulfide is a substrate for reduction by the active site of Trx1, and the active site in reduced by thioredoxin reductase. The of the are similar among species are of (5Eklund H. Gleason F. Holmgren A. Proteins. 1991; 11: 13-28Crossref PubMed Scopus (329) Google Scholar, A. Gasdaska J.R. Powis G. Montfort W.R. Structure (Lond.). 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The of the containing the active site cysteines is is at the of the and is in the between the and Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The E 0 of the active site dithiol of Trx1 is –270 mV in E. coli (10Krause G. Lundstrom J. Barea J.L. de la Cuesta C.P. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 11Moore E.C. Reichard P. Thelander L. J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google Scholar), mV in P.G. Baldesten A. Reichard P. J. Biol. Chem. 1970; 9: 2363-2370Abstract Full Text PDF Google Scholar), and –230 mV in T. Sjoberg B-M. Eklund H. J. Biol. Chem. 1990; 265: 3183-3188Abstract Full Text PDF PubMed Google Scholar). The present show that the active site of human Trx1 has an E 0 of –230 mV, at the higher of the of The redox state of E. coli Trx has been to be reduced A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a more report has that E. coli Trx1 is to reduced J. 2002; PubMed Scopus Google Scholar). In mammalian bovine Trx1 was reduced in cells (16Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (201) Google Scholar), and human Trx1 was reduced in cells J.R. 2002; PubMed Scopus Google Scholar) and in E. R. U. R. Biochem. Res. 2002; PubMed Scopus Google Scholar). results show that human Trx1, bovine Trx1, is in a more reduced state cells is E. coli Trx1 and that the redox Western blot an of the redox state of Trx1 that with In addition to the active site mammalian Trx1 contains three additional cysteines that are not found in from other species or in mammalian Cys-62 and Cys-69 are the and Cys-73 is on a on the surface of the protein Fig. The show that a disulfide bond form between Cys-62 and Cys-69. In analysis of that oxidized forms of human M. Holmgren A. Biochemistry. 1993; 32: PubMed Scopus Google Scholar), bovine A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and M. Holmgren A. Biochemistry. PubMed Scopus Google Scholar) Trx1 thiols as by reduction of A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Holmgren A. Biochemistry. 1993; 32: PubMed Scopus Google Scholar), supporting the interpretation that the non-active site thiols of mammalian Trx1 are to However, oxidation of Cys-62 and Cys-69 was not in the crystal of oxidized human Trx1 (7Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure (Lond.). 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). This could have resulted from the of because the of oxidized Trx1 were obtained by oxidation of the reduced form. The the reduced protein could have the of the second disulfide upon oxidation (7Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure (Lond.). 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). of oxidized and reduced human Trx1 are available J. A.M. Structure (Lond.). 2: Full Text Full Text PDF PubMed Scopus Google Scholar) but not provide on the non-active site disulfide because a mutant of Trx1 was used in which three non-active site cysteines were with the present the and that non-active site cysteines form a disulfide upon oxidizing conditions. for the of the for this disulfide bond The helix is to the of the protein two with the of helix The factors for the helix in the x-ray structure, which is a of in the are for the for the However, of the helix is in the found in the crystal and not The the helix are not in the crystal and have higher the of disulfide bond between the Cys residues of the helix is by the crystal The of a disulfide between Cys-62 and Cys-69 is to have a by the of the helix of Trx1 Fig. In this helix were to in with and J. J.R. A.M. Structure (Lond.). Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. A.M. Structure (Lond.). 1996; 4: Full Text Full Text PDF PubMed Scopus Google Scholar), two for Thioredoxin reductase most Trx1 in this as Trx1 was in the homodimer form, by an intermolecular disulfide bond in to the the active site was to thioredoxin reductase J.R. Montfort M. M. Powis G. Biochem. Pharmacol. 1996; PubMed Scopus Google Scholar). The reduction of the form of Trx1 Fig. for the interpretation that the between Trx1 and thioredoxin reductase is to the presence of a non-active site disulfide in the mutant of Trx1 that was to form the disulfide was not as a substrate for thioredoxin reductase by the of these two be that this mutant still Cys-73 and still of J.R. Montfort M. M. Powis G. Biochem. Pharmacol. 1996; PubMed Scopus Google Scholar, M. Holmgren A. Biochemistry. 1993; 32: PubMed Scopus Google Scholar). dimerization was not for the in the of reduction by thioredoxin reductase under the conditions used The presence of the non-active site disulfide the at which the active site of Trx1 was by thioredoxin reductase. the midpoint potential of the non-active site dithiol is more that of the active site, of this non-active site disulfide is more to under conditions in which the active site is oxidized the non-active site disulfide provide a by which Trx1 be and to regeneration by thioredoxin reductase. This time for signaling to that from the of thioredoxin include of the transcription from in the K. N. M. P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar) and of of the 1 M. H. M. K. K. M. K. H. J. PubMed Scopus Google Scholar). In E 0 for the active site of human Trx1 is –230 mV, among the of Trx to In addition to oxidation at the active site, human Trx1 forms a second disulfide under oxidizing conditions. the redox state of the active site is by thioredoxin reductase, both Trx1 disulfides are at redox the of the GSH/GSSG redox The of the non-active site disulfide the protein a number of possible as substrate the of other to Trx1 and of activity with thioredoxin reductase, as in the oxidation of the conserved non-active site Cys residues of mammalian Trx1 an in the function of for Fig. and for the
Watson et al. (Fri,) studied this question.
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