Interactions of Sodium Selenite, Glutathione, Arsenic Species, and

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Interactions of Sodium Selenite, Glutathione, Arsenic Species, and Omega Class Human Glutathione Transferase Robert A. Zakharyan,†,‡ George Tsaprailis,† Uttam K. Chowdhury,† Alba Hernandez,†,§ and H. Vasken Aposhian*,†,‡ Department of Molecular and Cellular Biology and Center of Toxicology, The University of Arizona, Tucson, Arizona, 85721-0106 Received February 24, 2005

Human monomethylarsenate reductase [MMA(V) reductase] and human glutathione Stransferase omega 1-1 (hGSTO1-1) [because MMA(V) reductase and hGSTO1-1 are identical proteins, the authors will utilize the designation “hGSTO1-1”] are identical proteins that catalyze the reduction of arsenate, monomethylarsenate [MMA(V)], and dimethylarsenate [DMA(V)]. Sodium selenite (selenite) inhibited the reduction of each of these substrates by the enzyme in a concentration-dependent manner. The kinetics indicated a noncompetitive inhibition of the MMA(V), DMA(V), or arsenate reducing activity of hGSTO1-1. The inhibition of the MMA(V) reducting activity of hGSTO1-1 by selenite was reversed by 1 mM DLdithiothreitol (DTT) but not by reduced glutathione (GSH), which is a required substrate for the enzyme. Neither superoxide anion nor hydrogen peroxide was involved in the selenite inhibition of hGSTO1-1. MALDI-TOF and MS/MS analysis demonstrated that five molecules of GSH were bound to one monomer of hGSTO1-1. Four of the five cysteines of the monomer were glutathionylated. Cys-32 in the active center, however, exists mostly in the sulfhydryl form since it was alkylated consistently by iodoacetamide. MALDI-TOF mass spectra analysis of hGSTO1-1 after reaction with GSH and sodium selenite indicated that selenium was integrated into hGSTO1-1 molecules. Three selenium were found to be covalently bonded to the monomer of hGSTO1-1 with three molecules of GSH. It is proposed that the reaction products of the reduction of selenite inhibited the activity of hGSTO1-1 by reacting with disulfides of glutathionylated cysteines to form bis (S-cysteinyl)selenide and S-selanylcysteine and had little or no interaction with the sulfhydryl of Cys-32 in the active site of the enzyme.

Introduction Arsenite and the methylated trivalent arsenic species are metabolites of arsenate in humans (1-4). They are multisite carcinogens (5-8). The high affinity of trivalent arsenic species for dithiols, their uncoupling of mitochondrial oxidative phosphorylation, and the bursts of reactive oxygen species produced may result in chronic metabolic alterations (9-11). Selenium has been recognized as an arsenic antagonist for many years. It protects against genotoxic and teratogenic effects of inorganic arsenicals (12-14), decreases chromosome and chromatid breaks (15), alters in vivo retention and distribution of inorganic arsenic (16-18), and antagonizes induction of heme oxygenase by arsenite (19). The formation of seleno-bis (S-glutathionyl) arsinium ion has been suggested as a molecular basis for the antagonistic interaction between these metalloid compounds in vivo (20). Therapeutic dosages of selenium have been linked to induction of apoptosis and toxicity (21, 22). The chemopreventive or pharmacological effects * To whom correspondence should be addressed. E-mail: aposhian@ u.arizona.edu. † Department of Molecular and Cellular Biology. ‡ Center of Toxicology. § Present address: Grup de Mutage ` nesi, Departament de Gene`tica i de Microbiologia, Facultat de Cie`ncies, Universitat Auto´noma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Valle´s, Spain.

have been attributed to the specific inhibition of enzymes having reactive thiol or selenol groups. When mice fed a selenium-supplemented diet were administered arsenate per os a significantly higher percentage of the dose of the administered arsenic was excreted in the urine. This was accompanied with a reduction of the ratio of methylated arsenicals to inorganic arsenic (17). Selenite lowered the tissue level of MMA(III) (18). The exposure of hepatocytes to selenite and arsenite in vitro resulted in the cellular accumulation of inorganic arsenic, decreased production of methylated metabolites of arsenite, and a significantly decreased DMA/MMA ratio (23) suggesting preferential inhibition of the conversion of MMA to DMA. It has been suggested that these effects were due to alterations in the enzymatic systems involved in the biotransformation of inorganic arsenic. This multistep pathway (Figure 1) is catalyzed by arsenate reductase (24-28), arsenite methyltransferase (29-31), hGSTO1-1 (32), and monomethylarsonous acid methyltransferase (29, 31, 33). hGSTO1-1 can reduce arsenate, MMA(V), and DMA(V) (24, 28). Although selenite has been reported to be an inhibitor of arsenite methyltransferases (23, 29, 34), no experimental observations have been reported regarding any effect of selenite on the modulation of hGSTO1-1 activity in reducing various chemical forms of arsenic.

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Figure 1. Biotransformation of inorganic arsenic.

hGSTO1-1 is a member of the glutathione-S-transferase superfamily. An excellent review of these important enzymes has appeared recently (35). There are seven major types of human cytosolic GSTs: alpha, mu, pi, sigma, theta, zeta, and omega. These enzymes detoxify xenobiotics usually by the catalysis of the nucleophilic attack by reduced glutathione on an electrophilic compound. hGSTO1-1 is a dimer of identical subunits. It has the characteristic GST fold of a N-terminal GSH-binding domain and a C-terminal domain made up of R-helices (36). Recently, Schmuck et al. solubilized hGSTO2-2 and found it had MMA(V) and DMA(V) reducing activity (37). The CLIC proteins, a family of intracellular chloride channels, have a structural similarity to GSTO (38). In addition, there has been the suggestion that hGSTO1-1 may have a role as a nuclear antioxidant system (39). GSTO1-1 has a wide distribution in human (39) and hamster tissue (40). hGSTO1-1 is a target of cytokine release inhibitory drugs (41). The predominant catalytic properties of hGSTO1-1 are as a reductase for arsenate, MMA(V), and DMA(V) converting them to their +3 oxidation state analogues (28, 42), a thiotransferase, and a dehydroascorbate reductase activity (36). The present study reports the interactions of selenite with hGSTO1-1 during the reduction of +5 arsenic species. MALDI-TOF and MS/MS analysis indicated the interactions of the five sulfhydryl groups of this protein with GSH and selenite during the selenite inhibition of the reduction of arsenic species.

Materials and Methods Caution: Inorganic arsenic is classified as a group 1 carcinogen for humans (43). Exposure to inorganic arsenic can lead to the development of skin cancer. Reagents. Sodium selenite pentahydrate was purchased from Fluka Chemical Corp. (Milwaukee, WI). 14C-MMA(V) (0.55 µCi/nmol) and 14C-DMA (0.67 µCi/nmol) were synthesized by Professor Eugene A. Mash, Jr., Department of Chemistry, The University of Arizona. Disodium methylarsenate was obtained from ChemService, Inc. (West Chester, PA). Sodium arsenate dibasic heptahydrate, dimethylarsinic acid sodium salt, TRIZMA (Reagent grade, minimum 99.9%), o-dainisidine dihydrochloride, bovine serum albumin, hydrogen peroxidase (HRP II) (EC1.11.1.7), luminol (HPLC grade), GSH, DL-dithiothreitol (DTT), 1880 U/mg bovine liver catalase (EC1.11.1.6), and sodium ascorbate were purchased from Sigma Chemical Co. (St. Louis, MO). Nanosep 10K Omega Centrifugal Devices were from Pall Gelman Lab (Ann Arbor, MI). Monoflow-3 scintillation cocktail was from National Diagnostics (Atlanta, GA). Recombinant hGSTO1-1. Recombinant human GSTO-1 (GST omega) with an N-terminal 6x His tag was prepared and purified as described previously (36).

Zakharyan et al. Mass Spectrometry. MALDI-TOF mass spectra were acquired using a Micromass (Manchester, United Kingdom) MALDI-LR TOF mass spectrometer operating a 337 nm nitrogen laser. One hundred micrograms of hGSTO1-1 in 250 µL of 0.1 M Tris-HCl buffer (pH 8.0)-5 mM GSH was desalted and washed at 4 °C with ddH2O using a Nanosep 10 K Omega Centrifugal Devices and recovered in 50 µL of ddH2O for MALDI-TOF spectra mass analysis. An aliquot of hGSTO1-1 (50 µg) in 50 µL of ddH2O was reduced under a N2 atmosphere with 2 mM DTT in 0.1 M TrisHCL buffer, pH 7.4, and 1 mM EDTA. The reaction was allowed to proceed for 30 min at 37 °C (44). To remove the DTT and salts, the hGSTO1-1 was desalted and washed at 4 °C with ddH2O on a Nanosep 10 K Omega Centrifugal Devices and recovered in 50 µL of ddH2O for MALDI-TOF spectra mass analysis. hGSTO1-1 samples (0.5-1.0 mg/mL) in H2O were mixed with an equal volume of a saturated sinnapinic acid solution in 40% acetonitrile/60% H2O containing 0.1% trifluoroacetic acid. One microliter was spotted on the target plate and allowed to air dry prior to mass analysis. Mass spectra were collected in linear mode with a 15000 V source voltage using a 2.0 ns sample period. The detector was held at 1800 V, and five laser shots at 3 Hz were combined per mass spectrum recorded. hGSTO1-1 (0.5-4 µg) in the presence of 5 mM GSH was gel purified under nonreducing SDS-PAGE conditions using a 12.5% polyacrylamide mini gel from BioRad. Following staining with Biosafe Coomassie, hGSTO1-1 bands were cut out and subjected to in-gel chymotrypsin digestion using published procedures (45). LC-MS/MS analyses of in-gel chymotrypsindigested hGSTO1-1 were carried out using a quadrupole ion trap ThermoFinnigan LCQ Classic (San Jose, CA). The LCQ Classic was equipped with a Michrom (Auburn, GA) MAGIC 2002 HPLC and a nanoelectrospray ionization source (University of Washington, Seattle, WA). Peptides were eluted from a 10-20 cm pulled tip capillary column (100 µm i.d. × 360 µm o.d.; 3-5 µm tip opening) packed with 7 cm Vydac (Hesperia, CA) C18 material 5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% trifluoroacetic acid) over 60 min at a flow rate of 200-300 nL/ min. The LCQ ESI positive mode spray voltage was set at 2.2 kV, and the capillary temperature was set at 200 °C. Dependent data scanning was performed with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, an activation time of 30 ms, and a minimal signal of 10000 ion counts. Global-dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of 1 min, and exclusion duration of 5 min. Scan event series included one full scan with a mass range of 350-2000 Da followed by three dependent MS/MS scans of the of the most intense ion. The sequences of individual peptides were identified using the Turbo SEQUEST algorithm on a stand-alone web server or cluster computer to search and correlate the MS/MS spectra with amino acid sequences in the Swiss Prot protein database (46). Protein Assay. Protein concentrations were determined using bovine serum albumin as the standard (47).

Results Selenite Inhibited the Reduction of MMA(V), DMA(V), and Arsenate by hGSTO1-1. hGSTO1-1 can reduce MMA(V), DMA(V), or arsenate (24, 28). Selenite inhibited the enzymatic reduction of each of these substrates in a concentration-dependent manner (Figure 2). The kinetic parameters representing MMA(V) reducing activity in the presence of selenite were Km ) 52.7 × 10-3 M and Vmax ) 19.6 µmol/mg/h with a Ki ) 29.6 µM. In the absence of selenite, they were Km ) 53.6 × 10-3 M and Vmax ) 52.6 µmol/mg/h (Figure 3a,b). For arsenate

Selenium, Arsenic, and Human GSTO1-1

Figure 2. Inhibition of the MMA(V), arsenate, or DMA(V) reducing activity of hGSTO1-1 by Se(IV). (a) The reaction mixture (250 µL) contained 0.1 M Tris-HCL, pH 8.0, 0.55 µCi 14C-MMA(V), 16 mM sodium MMA(V), 5 mM GSH, 12-96 µM sodium selenite, and 4 µg of hGSTO1-1 and was incubated at 37 °C for 60 min. The product formed was measured as previously described (32). (b) The reaction mixture, 250 µL, was the same as in panel a with 3 µg of hGSTO1-1 except nonradioactive arsenate, 15 mM, replaced MMA(V) and the product, arsenite, was measured by HPLC/ICP/MS (68). (c) The reaction mixture (250 µL) was the same as in panel a except that 0.67 µCi 14C-DMA and 10 mM sodium DMA(V) replaced MMA(V). The product of the reaction that was formed was measured as previously described (32). Note different units of coordinates. Each value represented the mean ( SE of three independent experiments. The SEs of some data points are so small that they are not visible in this figure.

reducing activity in the presence of selenite, they were Km ) 31.8 × 10-3 M, Vmax ) 8.1 µmol/mg/h, and Ki ) 34 µM. In the absence of selenite, they were Km ) 34.8 × 10-3 M and Vmax ) 12.8 µmol/mg/h (Figure 4a,b). For DMA(V) reducing activity in the presence of selenite, they were Km ) 32 × 10-3 M, Vmax ) 10.4 µmol/mg/h, and Ki ) 13.6 µM. In the absence of selenite, they were Km ) 30.6 × 10-3 M and Vmax ) 18.0 µmol/mg/h (Figure 5a,b). The kinetics indicated a noncompetitive inhibition by selenite of the MMA(V), DMA(V), or arsenate reducing activity of hGSTO1-1. To obtain 50% inhibition of MMA(V), DMA(V), or arsenate reducing activity under our experimental conditions, approximately 40, 12.8, or 12 µM selenite, respectively, was required. Inhibition of hGSTO1-1 by Selenite Was Reversed by Dithiothreitol. The inhibition of hGSTO1-1 by 30 µM selenite was substantially reversed (90%) by incubation with 1 mM DTT for an additional 30 min (Figure 6). Inhibition by selenite was not prevented by 5 mM GSH (data not shown). Reactive Oxygen Species Were Not Involved in Selenite Inhibition of hGSTO1-1. One of the products of the reaction of selenite with GSH is hydrogen selenide, which can be converted to Se° with the production of superoxide anion. By measuring chemiluminescence, the superoxide anion can be used as an indicator of selenite reduction and hydrogen selenide formation (52) (Scheme 1). In the presence of 1.6-20.8 µM selenite (Figure 7), a dose-dependent formation of superoxide anion occurred. Because one of the products of superoxide anion metabolism is hydrogen peroxide, it was pertinent to determine the possible involvement of superoxide anion (O2•-) and H2O2 in the selenite inhibition of hGSTO1-1. L-Ascorbic acid is a scavenger of reactive oxygen species such as O2•-, HO•, and singlet dioxygen. Sodium ascorbate, 5 or 10 mM, did not prevent the selenite (30 µM) inhibition of MMA(V) reducing activity (Figure 8), neither did catalase (Figure 9). Selenite (30 µM) inhibited the

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reduction of MMA(V) by 50%. In the presence of selenite and catalase, the activity of hGSTO1-1 was inhibited up to 40%. In independent experiments, 50 µM selenite, in the absence of hGSTO1-1, did not inhibit catalase in reducing hydrogen peroxide (100-500 µM). Thus, the experimental results indicated that reactive oxygen species resulting from the reaction of selenite with GSH were not involved in the selenite inhibition of hGSTO1-1. Properties of the Cysteine Residues of hGSTO11. Glutathionylation. Because hGSTO1-1 has an absolute requirement for GSH and because the monomer of this dimeric enzyme has five cysteine residues, it was of interest to determine the extent, if any, of glutathionylation of the monomer of the enzymes. Each monomer of recombinant hGSTO1-1 with an N-terminal 6 × His tag has a molecular mass of 28389. The monomer contains Cys-32, Cys-90, Cys-112, Cys-192, and Cys-237 (Figure 10). MALDI-TOF and mass spectra analysis of hGSTO1-1 under our assay conditions, which include 5 mM GSH, demonstrated that 95% of the molecules of hGSTO1-1 were glutathionylated and contained 3-5 GSH molecules per protein monomer. The molecular mass of the subunit was determined to be 29301 ( 2.8 (calculated, 29304), 29609 ( 4.1 (calculated, 29609), or 29912 ( 2.5 (calculated, 29914) correspondingly (Figure 11). After reduction by 2 mM DTT of the glutathionylated hGSTO1-1, only one glutathione molecule was attached per hGSTO1-1 monomer with a molecular mass of 28691 ( 3.41 (calculated, 28694) (Figure 11). Active Center Cysteine-32 Was in the Reduced Form. LC-MS/MS of in-gel chymotrypsin digested hGSTO1-1 revealed that, in the absence of DTT, the alkylating agent iodoacetamide reacted primarily with the sulfhydryl of Cys-32 (Figure 10). This suggested that the Cys-32 was reduced and was not in disulfide linkage with the essential GSH. This observation was consistent with the MALDI-TOF mass analysis, suggesting that the sulfhydryl of Cys-32 was predominantly free of disulfide bonds with GSH. In the presence of excess GSH, Cys-32 was maintained in its sulfhydryl form in the active center. Cys-237, however, could be alkylated by iodoacetamide, or it could form a disulfide with GSH. Cys-237 containing chymotryptic peptides were found alkylated or bound to GSH. The remaining three cysteine residues of the monomer were found to be glutathionylated. The resulting disulfide bonds can be reduced by DTT but not by a GSH excess. On LC-MS/MS analysis of hGSTO1-1 molecules that had been treated with DTT followed by iodoacetamide, all five cysteines were found in the alkylated form. Se(IV) Was Integrated into hGSTO1-1. To understand how Se(IV) interacted with hGSTO1-1, an incubation was performed, which included, under our assay conditions, the required 5 mM GSH and the inhibitory 50 µM Se(IV). MALDI-TOF mass spectra analysis of hGSTO1-1 demonstrated the molecular masses of 29074.7 ( 3.3 (calculated, 29079), 29381.52 ( 3.2 (calculated, 29384), and 29541.09 ( 1.73 (calculated, 29544). The data indicated that selenium was integrated in the hGSTO1-1 monomer with the GSH:Se ratios of 2:1, 3:1, and 3:3 (Figure 11).

Discussion The biotransformation of inorganic arsenic in mammals and especially the human involves a series of

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Figure 3. Substrate saturation for MMA(V) reductase activity of hGSTO1-1 in the presence or absence of Se(IV). The reaction mixture (250 µL) contained 0.1 M Tris-HCl, pH 8.0, 0.55 µCi 14C-MMA(V), 5 mM GSH, 4 µg of hGSTO1-1, and an increased concentration of sodium MMA(V) in the absence or presence of 50 µM selenite and was incubated at 37 °C for 60 min. The product formed was measured as previously described (32). (a) Substrate saturation; (b) Lineweaver-Burk plots. Each experiment was performed three times.

Figure 4. Substrate saturation for As(V) reductase activity of hGSTO1-1 in the presence and absence of Se(IV). The reaction mixture (250 µL) contained 0.1 M Tris-HCl, pH 8.0, 5 mM GSH, 3 µg of hGSTO1-1, and an increased concentration of sodium arsenate in the absence and presence of 20 µM selenite and was incubated at 37 °C for 60 min. The product formed was measured as previously described (68). (a) Substrate saturation; (b) Lineweaver-Burk plots. Each experiment was performed three times.

Figure 5. Substrate saturation for DMA(V) reductase activity of hGSTO1-1 in the presence and absence of Se(IV). The reaction mixture (250 µL) contained 0.1 M Tris-HCl, pH 8.0, 0.55 µCi 14C-DMA(V), 5 mM GSH, 4 µg of hGSTO1-1, and an increased concentration of sodium DMA(V) in the absence and presence of 10 µM selenite at 37 °C for 60 min. The product formed was measured as previously described (32). (a) Substrate saturation; (b) Lineweaver-Burk plots. Each experiment was performed three times.

reductions and oxidative methylations (Figure 1). Various reports have implicated different steps in this pathway as being susceptible to inhibition by selenite (29, 34), but none of these concerned GSTO1.

The inhibition of enzyme activity by selenium compounds usually has been stated to be due to their affinity for sulfhydryl groups in the active site of the enzyme. In our experiments, this was not so.


Figure 6. Inhibition of hGSTO1-1 by Se(IV) was reversed by dithiothreitol. (A) Four tubes of reaction mixtures, 250 µL per tube, as in Figure 2, were incubated for 30 min at which time 125 µL was removed from each tube to determine hGSTO1-1 activity. (B) As in panel A but in the presence of 30 µM selenite. (C) A 125 µL amount of A was incubated for an additional 30 min. (D) A 125 µL amount of B was incubated for an additional 30 min. (E) DTT to 1 mM was added to 125 µL of A and incubated for an additional 30 min. (F) DTT to 1 mM was added to 125 µL of B and incubated for an additional 30 min. Each value represented the mean ( SE of four independent experiments.

Scheme 1

Selenite inhibited the reduction of MMA(V), DMA(V), and arsenate by hGSTO1-1 in a noncompetitive manner indicating that the active site was not involved. Selenite requires bioreductive activation prior to eliciting its therapeutic or pro-oxidant activity. Six electrons are involved in the bioactivation of one selenium(IV) atom to selenodiglutathione (RS-Se-SR), with the subsequent formation of selenopersulfide (RS-SeH) and selenide (H2Se) (48-51). The latter is a critical metabolite in the utilization of selenium (22, 51) (Scheme 1). We hypothesized that selenite in the presence of GSH might modify the structure of hGSTO1-1 due to the production of reduced forms of selenite, namely, hydrogen selenide, O2•-, and hydrogen peroxide. Our experiments were performed in the presence of GSH (5 mM) and the reduced form of hGSTO1-1. GSH is absolutely required for the catalytic activity of this enzyme. The generation of superoxide that occurred (Figure 7) is evidence for the formation of hydrogen selenide. The pro-oxidant catalytic activity of selenite is


Figure 7. Selenite and GSH, in the presence or absence of hGSTO1-1, generated superoxide anion. The reaction mixture (250 µL) contained 0.1 M Tris-HCL, pH 8.0, 1.6 mM sodium MMA(V), 0.5 mM GSH, 1.6-20.8 µM sodium selenite, and 4 µg of hGSTO1-1. Luminol was injected (final concentration 0.1%) (53), and chemiluminescence was recorded every 30 s for 180 s at 37 °C: +, in the presence of hGSTO1-1; -, in the absence of hGSTO1-1. Chemoluminescence was measured by a Microtiter Plate Luminometer ML 3000 (Dynatech Laboratories). Each value represented the mean ( SE of three independent experiments.

Figure 8. L-Ascorbic acid did not prevent selenite inhibition of the MMA(V) reducing activity of hGSTO1-1. The reaction mixture described in Figure 2 contained 16 mM sodium MMA(V), 30 µM sodium selenite, and 5 or 10 mM sodium ascorbate. (A) No Se(IV), no ascorbate; (B) plus Se(IV); (C) plus Se(IV), plus ascorbate, 5 mM; (D) plus Se(IV) plus ascorbate, 10 mM. Each value represented the mean ( SE of three independent experiments.

thought to be due to the reaction of hydrogen selenide with oxygen to produce superoxide (O2•-), hydrogen

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Figure 9. hGSTO1-1 activity in the presence of selenite(IV) and catalase. (A) The reaction mixture as described in Figure 2 contained 16 mM sodium MMA(V); (B) plus 30 µM sodium selenite; (C) plus 30 µM sodium selenite and 10 or 20 µg of catalase. Reaction mixtures were incubated at 37 °C for 60 min. Each value represented the mean ( SE of three independent experiments.

Figure 10. Primary sequence of hGSTO1-1. The cysteine residues that were found by LC-MS/MS to be alkylated by iodoacetamide in the absence of DTT (bold and underlined); alkylated by iodoacetamide only in the presence of DTT (bold); Cys-237 (bold and italics) was alkylated or nonalkylated in the absence of DTT but always alkylated in the presence of DTT.

peroxide, other cascading oxyradicals, and elemental selenium (52-55). Thus, reactions by which selenite might noncompetitively inhibit or modify hGSTO1-1 might involve selenodiglutathione, glutathione selenopersulfide, hydrogen selenide, O2•-, and/or hydrogen peroxide, but neither sodium ascorbate, a scavenger of superoxide, nor catalase blocked the selenite inhibition of hGSTO1-1 (Figures 8 and 9). Thus, it was unlikely that hydrogen peroxide or superoxide was involved in the selenite modification of hGSTO1-1. Other studies have suggested four different types of reactions by which selenite might modify proteins: (i) formation of bis (S-cysteinyl) selenide (selenotrisulfide) bonds (-S-Se-S-); (ii) formation of S-selanylcysteine, selenoylsulfide bonds (-S-Se-); (iii) catalysis of disulfide bonds formation (-S-S-); or (iv) the reversal of the latter (51). Hydrogen selenide was one of the products formed in our reaction mixture. Ganther proposed that selenium toxicity was due to its interaction with proteins to form bis (S-cysteinyl) selenide, (RS-Se-SR) (49). Recently, it was suggested that a hydrogen selenide bonded to a disulfide not to a thiol, i.e., to one of the 17 disulfide bonds in an isoform of albumin forming GSSeH (56, 57). Selenotrisulfides, bis (S-cysteinyl) selenide have been proposed by others as a part of the inhibitory action of

Figure 11. MALDI-TOF mass spectra analysis of hGSTO1-1. (A) The sample (0.5-1.0 mg hGSTO1-1/mL) from a mixture of 0.1 M Tris-HCL, pH 8.0, with 5 mM GSH was treated as described in the Materials and Methods; (B) MALDI-TOF mass spectra analysis of hGSTO1-1 sample (0.5-1.0 mg/mL) reduced by the presence of 2 mM DTT, as described in the Materials and Methods; (C) MALDI-TOF mass spectra analysis of hGSTO11. The sample (0.5-1.0 mg hGSTO1-1/mL) was from a mixture of 0.1 M Tris-HCl, pH 8.0, with 5 mM GSH and 50 µM selenite after incubation at 37 °C for 15 min.

selenite. A selenotrisulfide type of adduct was suggested as the mechanism by which selenite inhibited rat brain prostaglandin-D synthetase. This inhibition was reversed by an excess of DTT (58). The inhibitory effect of selenite on the DNA-binding activity of AP-1 (59) or NF-kb (60) also was suggested to be via formation of RS-Se-SR adducts of protein thiols. DTT, but not GSH, affected the inhibitory action of selenite on Caspase-3, suggesting formation of a selenotrisulfide or disulfide (61). In our experiments, MALDI-TOF and MS/MS analysis of samples of hGSTO1-1 treated first by alkylation and then without or with DTT showed that four cysteines of the hGSTO1-1 monomer formed disulfide bonds with GSH and thus were glutathionylated. Also, Cys-32 in the active center exists mostly in the reduced state since it consistently was alkylated with iodoacetamide. Hydrogen selenide, formed in our reaction mixture, can catalyze sulfhydryl-disulfide exchange reactions that involve the nucleophilic attack of thiolate along the S-S bond axis of the disulfide with production of bis (S-cysteinyl) selenide and S-selanylcysteine (56, 62-64). The noncompetitive inhibition by selenite of the MMA(V), DMA(V), or arsenate reducing activity of hGSTO1-1 suggested formation of bis (S-cysteinyl) selenide. This could be the result of the incorporation of reduced selenide between


the disulfides of GSH and the cysteines of hGSTO1-1. The noncompetitive nature of the inhibition of hGSTO1-1 suggested that selenite has little or no interaction with Cys-32 of the active site. In addition, selenodiglutathione (GS-Se-SG) and glutathione selenol (GSSeH) may catalyze the formation of bis (S-cysteinyl) selenide of glutathionylated hGSTO1-1 directly via exchange reaction. MALDI-TOF mass spectra analysis of hGSTO1-1 after reaction with GSH and selenite (Figure 11) indicated that selenium was integrated into hGSTO1-1 molecules. Three Se were found to be covalently bonded to the monomer of hGSTO1-1 with three molecules of GSH (molecular mass, 29541.09 ( 1.73). The small 3:1 or 2:1, GSH:Se ratio probably is the result of some instability of bis (S-cysteinyl) selenide and possible formation of S-selanylcysteine. While many selenotrisulfides tend to be unstable at neutral pH, the instability is not universal and moderately stable selenotrisulfide derivatives of protein and lipoic acid have been described (49, 56, 65, 66). Consistent with the above conclusion are data that indicate the inhibition of hGSTO1-1 by 30 µM selenite was substantially reversed when treated with 1 mM DTT but not with 5 mM GSH (Figure 6). From the crystal structure of hGSTO1-1, Board has pointed out that one of the distinguishing features of hGSTO1-1 is that Cys-32 can form a mixed disulfide with GSH and that there is no interaction between GSH bound to one monomer and groups of the other monomer (36). This may be so in the crystal but may not represent the resting state in a cell. Another distinguishing feature of hGSTO1-1 found in our experiments was that Cys-32 in the presence of millimolar concentrations of GSH was predominantly free of disulfide bonds. However, four other cysteines remained in a glutathionylated form. In the bis (S-cysteinyl) selenide formed in reactions with protein or thiols, selenium may be further reduced by reducing equivalents of GSH, liberating selenium as hydrogen selenide. In the presence of oxygen, the hydrogen selenide would be converted to elemental selenium, Se°. Elemental Se, formed in the redox system of selenite and GSH, is unstable and further aggregates into insoluble gray and black Se°. Such a reaction involving albumin in the formation of hydrogen selenide and its liberation resulting in the aggregation of elemental selenium has been described (50, 67). However, in the presence of albumin, bright red, highly stable, soluble, nano-defined size, and physiologically active elemental Se° was formed (67). In the presence of hGSTO1-1, GSH, and selenite, we detected aggregation as bright red elemental Se°. In the absence of hGSTO1-1, however, aggregated black elemental Se° was noted suggesting the involvement of bis (S-cysteinyl) selenide of hGSTO1-1 in controlling the release of hydrogen selenide and affecting the formation of nano red elemental Se°. In summary, the inhibition of hGSTO1-1 by selenite(IV) has a broad influence on the multistep biotransformation of inorganic arsenic since it decreases the conversion of arsenate to arsenite, of MMA(V) to the very toxic MMA(III), and DMA(V) to DMA(III). Along with the previously reported selenite inhibition of arsenic methyltransferases (29, 34), the inhibition of hGSTO1-1 may be the critical mechanism by which selenium compounds protect against arsenic toxicity. MALDI-TOF and MS/MS analysis demonstrated that, under our experimental conditions, five molecules of GSH are bound to one subunit monomer of hGSTO1-1. Four


of the five cysteines were glutathionylated when GSH was present. Cys-32 in the active center, however, exists mostly in the sulfhydryl form since it consistently was alkylated by iodoacetamide. It is proposed that selenite inhibits the activity of hGSTO1-1 by reacting with four of the cysteines to form bis (S-cysteinyl) selenide and has little or no interaction with the sulfhydryl of Cys-32 in the active site of the enzyme.

Acknowledgment. This work was supported in part by the Superfund Basic Research Program NIEHS Grant ES-04940 from the National Institute of Environmental Health Sciences and the Southwest Environmental Health Sciences Center P30-ES-06694. We thank Dr. H. E. Ganther of the University of Wisconsin and Dr. P. G. Board of the John Curtin School of Medical Research, Australian National University (Camberra, Australia), for reading and constructive criticism of the manuscript. We also thank Andrea Hunt for performing the MALDITOF measurements.

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