Portal de Periódicos da CAPES

A Novel Antioxidant Mechanism of Ebselen Involving Ebselen Diselenide, a Substrate of Mammalian Thioredoxin and Thioredoxin Reductase

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m206452200

1083-351X

Rong Zhao, Arne Holmgren,

Free Radicals and Antioxidants

The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m−1s−1. The diselenide also was a substrate of TrxR forming the selenol withK m of 40 μm andk cat of 79 min−1(k cat/K m of 3.3 × 104m−1s−1). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 × 103m−1s−1). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 μm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2was estimated to be faster than 350m−1s−1. We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target. The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m−1s−1. The diselenide also was a substrate of TrxR forming the selenol withK m of 40 μm andk cat of 79 min−1(k cat/K m of 3.3 × 104m−1s−1). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 × 103m−1s−1). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 μm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2was estimated to be faster than 350m−1s−1. We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target. 2-phenyl-1,2 benzisoselenazol-3(2H)-one (EbSe) thioredoxin reductase thioredoxin, Trx-(SH)2, reduced Trx oxidized Trx ebselen diselenide 5,5′-dithiobis-(2-nitrobenzoic acid) 5-thiol-2-nitrobenzoic acid glutathione peroxidase selenocysteine, DTT, dithiothreitol high performance liquid chromatography Tris/EDTA Ebselen1(2-phenyl-1,2-benzisoselenazol-3(2H)-one, also called PZ51) is a seleno-organic compound, originally synthesized while searching for compounds that mimic the activity of the endogenous antioxidant glutathione peroxidase (1Muller A. Cadenas E. Graf P. Sies H. Biochem. Pharmacol. 1984; 33: 3235-3239Crossref PubMed Scopus (754) Google Scholar). Numerous studies have provided a wealth of information that this compound has anti-inflammatory, antiatherosclerotic, and cytoprotective properties in both in vitro and in vivo models (2Sies H. Free Radic. Biol. Med. 1993; 14: 313-323Crossref PubMed Scopus (400) Google Scholar, 3Sies H. Methods Enzymol. 1994; 234: 476-482Crossref PubMed Scopus (61) Google Scholar, 4Schewe T. Gen. Pharmacol. 1995; 26: 1153-1169Crossref PubMed Scopus (388) Google Scholar, 5Nakamura Y. Feng Q. Kumagai T. Torikai K. Ohigashi H. Osawa T. Noguchi N. Niki E. Uchida K. J. Biol. Chem. 2002; 277: 2687-2694Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Zhang M. Nomura A. Uchida Y. Iijima H. Sakamoto T. Iishii Y. Morishima Y. Mochizuki M. Masuyama K. Hirano K. Sekizawa K. Free Radic. Biol. Med. 2002; 32: 454-464Crossref PubMed Scopus (62) Google Scholar). Unlike other selenium compounds, ebselen possesses a very low toxicity because of its unique stability in structure and because its selenium moiety is not liberated during biotransformation and therefore does not enter the selenium metabolism of the organism (7Fischer H. Terlinden R. Lohr J.P. Romer A. Xenobiotica. 1988; 18: 1347-1359Crossref PubMed Scopus (51) Google Scholar, 8Muller A. Gabriel H. Sies H. Terlinden R. Fischer H. Romer A. Biochem. Pharmacol. 1988; 37: 1103-1109Crossref PubMed Scopus (69) Google Scholar, 9Sies H. Wendel A. Selenium in Biology and Medicine. Springer-Verlag, Heidelberg1989: 153-162Crossref Google Scholar). In more recent animal model studies, ebselen was shown to reduce oxidative stress in ischemia-reperfusion in heart (10Maulik N. Yoshida T. Das D.K. Free Radic. Biol. Med. 1998; 24: 869-875Crossref PubMed Scopus (160) Google Scholar) and to have neuroprotective effects in brain (11Dawson D.A. Masayasu H. Graham D.I. Macrae I.M. Neurosci. Lett. 1995; 185: 65-69Crossref PubMed Scopus (140) Google Scholar, 12Takasago T. Peters E.E. Graham D.I. Masayasu H. Macrae I.M. Br. J. Pharmacol. 1997; 122: 1251-1256Crossref PubMed Scopus (147) Google Scholar, 13Imai H. Masayasu H. Dewar D. Graham D.I. Macrae I.M. Stroke. 2001; 32: 2149-2154Crossref PubMed Scopus (163) Google Scholar, 14Namura S. Nagata I. Takami S. Masayasu H. Kikuchi H. Stroke. 2001; 32: 1906-1911Crossref PubMed Scopus (93) Google Scholar). More importantly, ebselen has been demonstrated to have beneficial effects in clinical trials for the treatment of patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage (15Saito I. Asano T. Sano K. Takakura K. Abe H. Yoshimoto T. Kikuchi H. Ohta T. Ishibashi S. Neurosurgery. 1998; 42: 269-278Crossref PubMed Scopus (222) Google Scholar) and acute ischemic stroke (16Yamaguchi T. Sano K. Takakura K. Saito I. Shinohara Y. Asano T. Yasuhara H. Stroke. 1998; 29: 12-17Crossref PubMed Scopus (547) Google Scholar, 17Ogawa A. Yoshimoto T. Kikuchi H. Sano K. Saito I. Yamaguchi T. Yasuhara H. Cerebrovasc. Dis. 1999; 9: 112-118Crossref PubMed Scopus (181) Google Scholar). The thioredoxin system, a collective name for thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is the most powerful protein disulfide reductase system in cells and is present in all living organisms (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 21Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar). TrxR is a dimeric FAD-containing enzyme, which catalyzes the NADPH-dependent reduction of the active site disulfide in oxidized Trx (Trx-S2) to give a dithiol in reduced Trx (Trx-(SH)2) (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). The reduced Trx is a hydrogen donor for ribonucleotide reductase, the essential enzyme for DNA synthesis and a powerful general protein disulfide reductase with a large number of functions in growth and redox regulations (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 21Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar). As an endogenous antioxidant, the thioredoxin system serves to keep the redox state of a cell balanced (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 21Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar). Mammalian TrxRs are large selenoproteins (M r 114,000 or larger) with structures showing a close homology to glutathione reductase but with an elongation containing a unique catalytically active selenolthiol/selenenylsulfide in the conserved C-terminal sequence Gly-Cys-Sec-Gly (22Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar, 23Tamura T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1006-1011Crossref PubMed Scopus (483) Google Scholar, 24Zhong L. Arnér E.S. Ljung J. Åslund F. Holmgren A. J. Biol. Chem. 1998; 273: 8581-8591Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 25Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (414) Google Scholar, 26Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 27Sandalova T. Zhong L. Lindqvist Y. Holmgren A. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9533-9538Crossref PubMed Scopus (296) Google Scholar). Mammalian TrxRs have a remarkably wide substrate specificity (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 21Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar, 22Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar), reducing not only different thioredoxins but also e.g.selenite (28Kumar S. Björnstedt M. Holmgren A. Eur. J. Biochem. 1992; 207: 435-439Crossref PubMed Scopus (208) Google Scholar), selenodiglutathione (29Björnstedt M. Kumar S. Holmgren A. J. Biol. Chem. 1992; 267: 8030-8034Abstract Full Text PDF PubMed Google Scholar), and selenocystine (30Björnstedt M. Kumar S. Bjorkhem L. Spyrou G. Holmgren A. Biomed. Environ. Sci. 1997; 10: 271-279PubMed Google Scholar). The mammalian enzymes also are NADPH-dependent lipid hydroperoxide reductases (31Björnstedt M. Hamberg M. Kumar S. Xue J. Holmgren A. J. Biol. Chem. 1995; 270: 11761-11764Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) and may serve directly as an electron donor of human plasma glutathione peroxidase (32Björnstedt M. Xue J. Huang W. Akesson B. Holmgren A. J. Biol. Chem. 1994; 269: 29382-29384Abstract Full Text PDF PubMed Google Scholar). In a previous study, we reported that ebselen is a substrate of mammalian TrxR, which is reduced by NADPH forming ebselen selenol with a K m value of 2.5 μm and a k cat of 588 min−1 (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar). We also showed that ebselen strongly enhanced the hydrogen peroxide reductase activity of mammalian TrxR, acting as a TrxR peroxidase. In the presence of Trx, ebselen acted as a mimic of a peroxiredoxin (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar). In reduction of ebselen by mammalian TrxR, we observed that under certain conditions, ebselen reacted with its selenol forming ebselen diselenide (2,2-diselenobis-(N-phenyl)-benzamid in Reaction FR1), which absorbs strongly at 340 nm and has a low solubility giving rise to a precipitate and increase in A 340(33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar). Here we report a study on the interaction of ebselen diselenide with the mammalian thioredoxin system to clarify the role of this compound in the H2O2 reductase activity of ebselen. We found that ebselen diselenide also is a direct substrate of mammalian TrxR and that Trx increases the reduction rate. The kinetics of the formation of ebselen diselenide (Reaction FR1) from ebselen reacting with its selenol was measured. Ebselen has been widely used as an antioxidant in experimental models assuming that it is acting via a GSH peroxidase-like mechanism (1Muller A. Cadenas E. Graf P. Sies H. Biochem. Pharmacol. 1984; 33: 3235-3239Crossref PubMed Scopus (754) Google Scholar, 2Sies H. Free Radic. Biol. Med. 1993; 14: 313-323Crossref PubMed Scopus (400) Google Scholar, 3Sies H. Methods Enzymol. 1994; 234: 476-482Crossref PubMed Scopus (61) Google Scholar, 4Schewe T. Gen. Pharmacol. 1995; 26: 1153-1169Crossref PubMed Scopus (388) Google Scholar, 5Nakamura Y. Feng Q. Kumagai T. Torikai K. Ohigashi H. Osawa T. Noguchi N. Niki E. Uchida K. J. Biol. Chem. 2002; 277: 2687-2694Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Zhang M. Nomura A. Uchida Y. Iijima H. Sakamoto T. Iishii Y. Morishima Y. Mochizuki M. Masuyama K. Hirano K. Sekizawa K. Free Radic. Biol. Med. 2002; 32: 454-464Crossref PubMed Scopus (62) Google Scholar). The discovery of its reactivity with thioredoxin reductase and thioredoxin (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar) changed this picture. Using glutathione as the reductant, the H2O2 reductase activity of ebselen was compared with that in the presence of the mammalian thioredoxin system. Our results demonstrate that ebselen uses the thioredoxin system far more efficiently than glutathione. Formation of ebselen diselenide may serve as a dose-dependent storage form of ebselen, which can be relatively slowly activated to the catalytically active selenol by the mammalian thioredoxin system. NADPH, DTT, and DTNB were from Sigma. Hydrogen peroxide (perhydrol) (30%) and dimethyl sulfoxide (Me2SO) were from Merck. TrxR from calf thymus or human placenta was purified to homogeneity (25 μmol of NADPH oxidized per min per mg) essentially as described for the rat liver enzyme (20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 22Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar). Trx from Escherichia coli was a homogeneous preparation, and recombinant human Trx and the mutant C61S/C72S prepared as described by Ren et al. (34Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar) were from IMCO Ltd, Stockholm, Sweden (www.imcocorp.se). The sources of other materials have been described in previous publications (25Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (414) Google Scholar, 28Kumar S. Björnstedt M. Holmgren A. Eur. J. Biochem. 1992; 207: 435-439Crossref PubMed Scopus (208) Google Scholar, 29Björnstedt M. Kumar S. Holmgren A. J. Biol. Chem. 1992; 267: 8030-8034Abstract Full Text PDF PubMed Google Scholar). Ebselen and ebselen diselenide were products of Daiichi and were dissolved in fresh Me2SO before addition into the aqueous solvents. Concentrations of Me2SO were less than 5% of the solvent buffer, effective in dissolving the drug. The activity of enzymes was determined at room temperature using an Ultrospec 3000 uv/visible spectrophotometer (Amersham Biosciences). Measurements of TrxR activity were performed in a buffer containing 50 mm Tris-Cl, 1 mm EDTA, pH 7.5, generally with 100 μm NADPH and the indicated amounts of ebselen. Reactions were started with addition of 5 or 10 μl of a stock solution of TrxR in a final total volume of 0.50 ml. Cuvettes containing reference mixtures contained the same amount of Me2SO in the samples and TrxR. The glutathione peroxidase activity of ebselen was measured according to Wilson's method (35Wilson S.R. Zucker P.A. Huang R.R.C. Spector A. J. Am. Chem. Soc. 1989; 111: 5936-5939Crossref Scopus (413) Google Scholar). The reactions were carried out at 37 °C in 0.5 ml of solution containing 50 mm potassium phosphate buffer, pH 7.5, 1 mm EDTA, 1 mm sodium azide, 1 mm GSH, 1 unit of GSH reductase, and 2 μmebselen. The mixtures were preincubated for 10 min, and NADPH was added to a final concentration of 250 μm. The reactions were then initiated by addition of 0.5 mmH2O2, and the activities were followed by the decrease of NADPH at 340 nm against blanks without ebselen. Protein fluorescence was measured with a thermostatic SPEX-FluoroMax Spectrofluorometer. Trx-(SH)2 was prepared from E. coliTrx-S2, which was incubated at room temperature for 20 min with 10 mm DTT. DTT was subsequently removed by gel chromatography on a NAP-5 column (Amersham Biosciences) using N2-equilibrated buffer. Trx-(SH)2 was mixed with ebselen in a total volume of 3 ml of 0.1 m potassium phosphate, 1 mm EDTA, pH 7.5 directly at 22 °C. Excitation of fluorescence was at 290 nm and emission spectra from 300 to 500 nm were recorded. Emission at 340 nm was followed to record the rate of oxidation of Trx-(SH)2 by ebselen diselenide (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 26Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Using the authentic compound, the absorption spectrum of 30 μm ebselen diselenide in TE buffer, pH 7.5, containing 2% Me2SO was measured (Fig. 1). The ebselen diselenide has two strong and broad absorption bands (from 250 to 300 nm and from 300 to 420 nm) overlapping each other. The band from 250 to 300 nm can be ascribed to the phenyl moieties, and a tailed broad band from 300 to 420 nm is caused by the Se-Se moiety (36Alam M.M. Ito O. Koga Y. Ouchi A. Int. J. Chem. Kinet. 1998; 30: 193-200Crossref Scopus (6) Google Scholar). The extinction coefficient of (EbSe)2 at 340 nm (ε340) was calculated to be 21,000 m−1cm−1 using several known concentrations of (EbSe)2. It should be noted that most assays of ebselen and its reactivity toward hydroperoxides were measured at 340 nm, where the kinetic oxidation of NADPH to NADP+ was followed. The high and broad absorption of (EbSe)2 has to be taken into consideration when a decrease of A 340 is interpreted. Another useful wavelength for (EbSe)2 measurement is 390 nm with ε390 of 8000m−1cm−1, where most of the other substances used in the assays have no or very weak absorbance. Reduction of ebselen by NADPH catalyzed by mammalian TrxR produces ebselen selenol (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar). However, when lower concentrations of the enzyme and higher concentrations of ebselen were used, the A 340 showed a complex change. Fig.2 shows the change ofA 340 upon the reduction of 10–100 μm ebselen by NADPH catalyzed by 7.5 nm TrxR. With higher concentrations of ebselen (>20 μm), theA 340 first increased, followed by a decrease to give the final reduction product ebselen selenol. The highest concentrations (50 and 100 μm) gave visible precipitates. This is due to a fast reaction of excess ebselen with its reduction product ebselen selenol through Reaction FR1. The rate constant of Reaction FR1 was measured in a way where 40 μm ebselen selenol is produced by reduction of 40 μm ebselen with 100 μm NADPH catalyzed by 50 nm calf liver TrxR (Fig.3 A). After 20 min, where the reaction was complete, the enzyme in the solution was removed by filtering though an Ultrafree®-MC Millipore 10,000 cutoff filter. To this solution containing 40 μm ebselen selenol, another 40 μm ebselen was added, and the kinetics of formation of (EbSe)2 was followed at 390 nm (Fig. 3 B). The formation of ebselen diselenide was fitted to second-order rate Equation 1, where x is the micromolar concentration of (EbSe)2 using ε390 of 8000m−1s−1, and a is the initial concentration of ebselen and ebselen selenol, in this case, 40 μm. x/a(a−x)=k1·tEquation 1 As seen from the inset of Fig. 3 B, a plot of the left side of Equation 1 against time gave a straight line confirming second-order kinetics with a slope corresponding to the second-order rate constant k 1 of 0.0223 μm−1min−1 (372m−1s−1). To TE buffer solutions (pH 7.5) containing 10 and 20 μm ebselen diselenide, 100 μm NADPH, 50 nm calf thymus TrxR were added. The decreases of (EbSe)2 were followed against an identical blank without TrxR (Fig. 4). Ebselen diselenide was a direct substrate of mammalian TrxR, and addition of human Trx increased the reaction rate. Reduction of ebselen diselenide by NADPH catalyzed by TrxR produced ebselen selenol (Reaction FR2), as evidenced by the final spectrum and HPLC analysis (data not shown). Addition of one volume of 6 mguanidine hydrochloride containing 10 mm DTNB at the end of the reaction also gave formation of TNB, showing the reduction of DTNB by the ebselen selenol in Reaction FR2(33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar).Figure FR2View Large Image Figure ViewerDownload (PPT) The steady state kinetics of ebselen diselenide as a substrate of TrxR was also measured directly using 100 nmmammalian TrxR. As shown in Fig. 5, theK m value of 40 μm andk cat of 79 min−1 can be derived from a Lineweaver-Burk Plot of an assay, and thek cat/K m was calculated to be 3.3 × 104m−1s−1. From Fig. 4, it was obvious that addition of Trx to TrxR increased reduction rate of NADPH toward ebselen diselenide. This indicated that Trx-(SH)2 is a fast reductant of (EbSe)2 according to Reactions FR3 and FR4 in the following reaction scheme.Figure FR4View Large Image Figure ViewerDownload (PPT) Mammalian and E. coli Trx have the same active site (WCGPC) and reactivity with disulfides (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 26Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Since E. coli Trx-(SH)2 has a 3-fold higher tryptophan fluorescence than TrxS2 (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 26Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), this was used to follow the reaction with ebselen diselenide. The fluorescence decay of 5 μm reduced E. coli Trx-(SH)2 was recorded after adding 5 μm (EbSe)2 (Fig.6). The inverse of the Trx-(SH)2 fluorescence intensity was found to be linear against time indicating a typical second-order kinetics (Fig. 6,inset). Thus the slope of the linear fit gave a second-order rate constant k 5 of 1.7 × 103m−1s−1. This rate constant is lower than the one for the reduction of insulin by reduced Trx (105m−1s−1) (37Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar,38Holmgren A. Structure. 1995; 3: 239-243Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Ebselen diselenide also was reduced by DTT to form two equivalents of ebselen selenol (Reaction FR5). The rate constant of this reaction was directly measured by following the decrease of 20 μm (EbSe)2 at 390 nm after adding 2 mm reduced DTT. The ebselen diselenide has an ε390 of 8000m−1cm−1, and all other species involved have little or no absorbance at this wavelength. Since the concentration of DTTred is 100-fold higher than that of (EbSe)2, the decrease of (EbSe)2 absorption at 390 nm should follow a pseudo first order kinetics. The decrease of (EbSe)2 was found to be first order with a half-life of 5 s. Thus k 6 was calculated to be ∼70 ± 2 m−1s−1. This rate constant can be compared with the one for the reduction of insulin by DTT as 5 m−1s−1 (39Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Reduction of Trx by DTT is much faster with a rate constant of 1647m−1s−1 (39Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Because reduction of ebselen diselenide by NADPH produces two equivalents of ebselen selenol, which will rapidly reduce H2O2 and generate ebselen, a much more efficient substrate of mammalian TrxR and thus a TrxR peroxidase (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar), ebselen diselenide should also stimulate the H2O2 reductase activity of mammalian TrxR. Fig.7 showed that the changes inA 340 as recorded after addition of 1–10 μm (EbSe)2 to TE buffers, pH 7.5, containing 250 μm NADPH, 18 nm mammalian TrxR, and 0.5 mm H2O2. These H2O2 reductase activities of (EbSe)2 were compared with that of 2 μmebselen (dark line). Obviously (EbSe)2stimulated the H2O2 reductase activity of TrxR. However, we also noticed that this activity was higher than expected, since ebselen diselenide is about a 100-fold less efficient substrate of the TrxR than ebselen, as we have described above. Although we cannot account for the high reactivity of ebselen diselenide, which may have to do with a direct interaction of the oxidized form of ebselen diselenide with TrxR, it will be active as an antioxidant in the presence of the thioredoxin system. At higher (EbSe)2concentrations, the H2O2 reductase activity reached saturation, probably because (EbSe)2 was regenerated through Reaction FR1. The molecular mechanism of ebselen acting as an antioxidant has long been attributed to its activity as a glutathione peroxidase mimic, catalyzing the glutathione detoxification of hydroperoxides (1Muller A. Cadenas E. Graf P. Sies H. Biochem. Pharmacol. 1984; 33: 3235-3239Crossref PubMed Scopus (754) Google Scholar, 2Sies H. Free Radic. Biol. Med. 1993; 14: 313-323Crossref PubMed Scopus (400) Google Scholar, 3Sies H. Methods Enzymol. 1994; 234: 476-482Crossref PubMed Scopus (61) Google Scholar, 4Schewe T. Gen. Pharmacol. 1995; 26: 1153-1169Crossref PubMed Scopus (388) Google Scholar). A number of other thiols, e.g. N-acetyl-l-cysteine (40Cotgreave I.A. Sandy M.S. Berggren M. Moldeus P.W. Smith M.T. Biochem. Pharmacol. 1987; 36: 2899-2904Crossref PubMed Scopus (68) Google Scholar), dithiothreitol (41Muller A. Gabriel H. Sies H. Biochem. Pharmacol. 1985; 34: 1185-1189Crossref PubMed Scopus (139) Google Scholar), and dihydrolipoate (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar) were also used. The endogenous dihydrolipoate was found to be a better cofactor than glutathione for the peroxidase activity of ebselen (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar). We compared the H2O2 reductase activities of ebselen using a lower than normal physiological concentrations of TrxR (50 nm), with or without the presence of 5 μmTrx (43Holmgren A. Luthman M. Biochemistry. 1978; 17: 4071-4077Crossref PubMed Scopus (123) Google Scholar, 44Rozell B. Hansson H.A. Luthman M. Holmgren A. Eur. J. Cell Biol. 1985; 38: 79-86PubMed Google Scholar, 45Hansson H.A. Holmgren A. Rozell B. Täljedal I.B. Cell Tissue Res. 1986; 245: 189-195Crossref PubMed Scopus (18) Google Scholar), with the activity in the presence of 1 mmglutathione. As shown in Fig. 8, the rate of NADPH turnover was 1.66 μm/min for 2 μmebselen in the presence of 1 mm GSH, excess glutathione reductase and 0.5 mm H2O2. When 50 nm calf thymus TrxR was used, the rate was found to be 13 μm/min, thus an increase of H2O2reductase activity of 8-fold. With the further addition of 5 μm Trx, the rate was increased to 21 μm/min, i.e. a relative increase of the H2O2 reductase activity of ebselen to 13-fold. This experiment clearly demonstrated that ebselen reacted with TrxR and Trx far more efficiently than with glutathione. Direct measurement of the rate constant for ebselen selenol reducing H2O2(Reaction FR6) was complicated by the reaction between ebselen and ebselen selenol to form the (EbSe)2 (Reaction FR1). However, an estimate of the lowest limit of this rate constant can be made from the enzyme and H2O2 coupled assays. As seen from Fig. 8, the rate of NADPH oxidation was ∼21 μm/min in a solution containing 250 μmNADPH, 2 μm ebselen, 50 nm TrxR, 5 μm Trx, and 0.5 mmH2O2. If we assume a saturation of this reaction by the turnover of ebselen selenol to ebselen, i.e.the rate of ebselen selenol with H2O2 becomes rate-limiting step, we get Equation 2. v=k1·[H2O2]·[EbSeH]=21μM/minEquation 2 Because the highest concentration for ebselen selenol was 2 μm, one can estimate a rate constantk 1 to be at least larger than 21 mm−1min−1, i.e. 350m−1s−1. The actual rate constant should be even larger since with 50 nm TrxR and 5 μm Trx, the oxidation of NADPH has not reached saturation and then the concentration of the ebselen selenol should be much lower than 2 μm. Nevertheless, this rate constant is ∼7.5 times higher than the previously reported 2.8 mm−1min−1 (46Morgenstern R. Cotgreave I.A. Engman L. Chem. Biol. Interact. 1992; 84: 77-84Crossref PubMed Scopus (72) Google Scholar), most probably because the interference of the diselenide formation was not considered. The high rate constant k 7 also demonstrates that ebselen selenol is the most active form of ebselen for its H2O2 reductase activity. The selenium-containing glutathione peroxidase catalyzes the reduction of hydroperoxides by reduced glutathione with a ter uni ping pong mechanism (Scheme FS1 A) (47Ursini F. Maiorino R. Brigelius-Flohe R. Aumann K.D. Roveri A. Schomburg D. Flohe L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (671) Google Scholar). Thus, a similar reaction pathway can be drawn for ebselen acting as a Trx peroxidase mimic (Scheme FS1 B). It is well known that the active site of glutathione peroxidase is its selenocysteine residue (CysSeH), which because of its much lower pKavalue and higher nucleophilicity of the corresponding selenolate, will easily reduce H2O2 to form H2O and the cysteine selenenic acid (CysSeOH). The latter consumes two glutathione molecules as hydrogen donors to finish a catalytic cycle and regenerating the selenocysteine. A corresponding reaction mechanism for ebselen (Scheme FS1 B) would imply that ebselen needs an efficient two-electron donor to form the active selenol to carry out its H2O2 reductase activity. As the most effective dithiol reductant of the cell (18Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 20Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar, 21Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar), the mammalian thioredoxin and particularly the selenocysteine-containing thioredoxin reductase are therefore electron donors for ebselen and its hydroperoxide reductase activity. The ebselen selenol was found to be much less oxygen sensitive than the cysteine selenol (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar). This is expected for an aryl selenol in ebselen, because correspondingly, an aryl thiol has much lower oxygen sensitivity than an alkyl thiol,e.g. cysteine (48Zhao R. Lind J. Gábor M. Eriksen T.E. J. Am. Chem. Soc. 1998; 120: 2811-2816Crossref Scopus (20) Google Scholar). Though ebselen is known for being a glutathione peroxidase mimic, the activity is rather weak (1Muller A. Cadenas E. Graf P. Sies H. Biochem. Pharmacol. 1984; 33: 3235-3239Crossref PubMed Scopus (754) Google Scholar, 49Wendel A. Fausel M. Safayhi H. Tiegs G. Otter R. Biochem. Pharmacol. 1984; 33: 3241-3245Crossref PubMed Scopus (370) Google Scholar, 50Maiorino M. Roveri A. Coassin M. Ursini F. Biochem. Pharmacol. 1988; 37: 2267-2271Crossref PubMed Scopus (146) Google Scholar). Ebselen was shown to react with glutathione forming an ebselen selenenylsulfide, which in turn is only slowly converted, in the presence of an excess of glutathione, to ebselen selenol and (EbSe)2 (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar, 51Cotgreave I.A. Morgenstern R. Engman L. Ahokas J. Chem. Biol. Interact. 1992; 84: 69-76Crossref PubMed Scopus (67) Google Scholar). The occurrence of the selenol as an intermediate in the reduction of the selenenylsulfide to (EbSe)2 by thiols was clearly demonstrated by Cotgreaveet al. (51Cotgreave I.A. Morgenstern R. Engman L. Ahokas J. Chem. Biol. Interact. 1992; 84: 69-76Crossref PubMed Scopus (67) Google Scholar) by trapping the selenol with 1-chloro-2,4-dinitrobenzene. Formation of diselenide of ebselen was evident from both HPLC and 77Se NMR analysis (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar). However, the mechanistic explanations of the chemical conversions of ebselen reacting with thiols were controversial (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar, 52Fischer H. N. D. Bull. Soc. Chim. Belg. 1987; 96: 757-768Crossref Scopus (136) Google Scholar), and Reaction FR1 was not recognized. The argument also included whether the active form of ebselen toward H2O2 is ebselen diselenide (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar,52Fischer H. N. D. Bull. Soc. Chim. Belg. 1987; 96: 757-768Crossref Scopus (136) Google Scholar) or the selenol (46Morgenstern R. Cotgreave I.A. Engman L. Chem. Biol. Interact. 1992; 84: 77-84Crossref PubMed Scopus (72) Google Scholar, 50Maiorino M. Roveri A. Coassin M. Ursini F. Biochem. Pharmacol. 1988; 37: 2267-2271Crossref PubMed Scopus (146) Google Scholar). Formation of a diselenide was suggested to be a key step for catalytic activity as the slowest one, and its reaction with the H2O2 would yield the parent compound and water (42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar, 52Fischer H. N. D. Bull. Soc. Chim. Belg. 1987; 96: 757-768Crossref Scopus (136) Google Scholar). However, Maiorino et al. (50Maiorino M. Roveri A. Coassin M. Ursini F. Biochem. Pharmacol. 1988; 37: 2267-2271Crossref PubMed Scopus (146) Google Scholar) concluded that the ebselen mechanism appeared kinetically identical to the enzyme reaction and showed that carboxymethylation of intermediates by iodoacetamide formed inactive derivatives suggested that an ebselen selenol is involved. Morgenstern et al. (46Morgenstern R. Cotgreave I.A. Engman L. Chem. Biol. Interact. 1992; 84: 77-84Crossref PubMed Scopus (72) Google Scholar) also concluded that the selenol is the predominant molecular species responsible for the GSH-dependent peroxidase activity of ebselen. Because we have a reaction system where ebselen directly undergoes a fast two-electron reduction by NADPH catalyzed by mammalian TrxR, forming the ebselen selenol, the occurrence of Reaction FR1 became evident. The formation of the ebselen diselenide, measured to have a second order rate constant of 372m−1s−1, was apparent when both ebselen and ebselen selenol are present, e.g. in reduction reactions at high concentrations of ebselen and low concentrations of enzymes. Formation of the diselenide was not a unique feature of the enzyme since it could be mimicked by using a low non-stoichiometric concentration of DTT (10 μm) and 100 μmebselen, whereas excess DTT only gave the selenol as is also shown by HPLC (data not shown). It is therefore not surprising that dithiols like DTT or dihydrolipoate are better cofactors of ebselen than glutathione as observed (41Muller A. Gabriel H. Sies H. Biochem. Pharmacol. 1985; 34: 1185-1189Crossref PubMed Scopus (139) Google Scholar, 42Haenen G.R., De Rooij B.M. Vermeulen N.P. Bast A. Mol. Pharmacol. 1990; 37: 412-422PubMed Google Scholar). When the mammalian thioredoxin system was used, ebselen was highly efficiently reduced to its selenol, which in the presence of H2O2, rapidly forms H2O and a selenenic acid, and then spontaneously eliminates H2O and regenerates the ebselen for another catalytic cycle (Reaction FR6). We have clearly shown that ebselen diselenide is also a substrate of mammalian TrxR with a K m value of 40 μm and k cat of 79 min−1; thus thek cat/K m is calculated to be 3.3 × 104m−1s−1. Compared with the reduction of ebselen by NADPH catalyzed by mammalian TrxR having aK m value of 2.5 μm, ak cat of 588 min −1 and ak cat/K m of 3.9 × 106m−1s−1 (33Zhao R. Masayasu H. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8579-8584Crossref PubMed Scopus (216) Google Scholar), the efficiency of ebselen diselenide reduction is 100 times slower. The mechanism of mammalian TrxR acting on ebselen diselenide can be illustrated in Scheme FS2. The C-terminal active site selenenylsulfide in TrxR is reduced by NADPH via the active site disulfide in the second subunit forming a thiol and a selenolate (25–27,53). The latter will reduce (EbSe)2 via a short-lived intermediate, TrxRSe-SeEbSe, and will form two equivalents of ebselen selenol. Ebselen diselenide also was shown to have an H2O2 reductase activity (Fig. 7), which was surprisingly high compared with that of ebselen, taking into account the difference as the substrates for the enzyme as described above. A simple mechanism for (EbSe)2 acting as an H2O2 reductase is given in SchemeFS3. Reduction of (EbSe)2 by the mammalian TrxR slowly forms the ebselen selenol, which will rapidly reduce H2O2 and generate ebselen, a 100-fold more efficient substrate of mammalian Trx system. At the same time, ebselen will also react with the ebselen selenol to reform the ebselen diselenide through Reaction FR1. Thus the H2O2reductase activity of (EbSe)2 is actually achieved through a bridge connecting the active form of the catalytic cycle,i.e. the ebselen selenol and ebselen, with H2O2. The pathways described in Scheme FS3 also stand for a complete mechanism of ebselen for its H2O2 reductase activity. A more simplified form of Scheme FS3 is drawn in Scheme FS4, including a previously unrecognized part of the antioxidant mechanism of ebselen, i.e. the ebselen itself will compete with H2O2 for the ebselen selenol to form the ebselen diselenide, which in fact limits the level of its H2O2 reductase activity. Since the reduction of (EbSe)2 by NADPH catalyzed by the mammalian Trx system is much slower than that of ebselen, (EbSe)2 may then serve as a storage form of ebselen, which is slowly reactivated by the mammalian Trx system. Formation of the diselenide is definitely a part of ebselen antioxidant action, which will affect its efficiency as a peroxidase but will not change the character of ebselen as a thioredoxin reductase and thioredoxin peroxidase mimic.Figure FS3View Large Image Figure ViewerDownload (PPT)Figure FS4View Large Image Figure ViewerDownload (PPT) Dr Hiroyuki Masayasu from Daiichi Pharmaceutical Company, Ltd, Tokyo, Japan, is gratefully thanked for his gifts of the ebselen and ebselen diselenide compounds and helpful discussions.