Thiol Oxidase Activity of Copper,Zinc Superoxide Dismutase

The ability of copper,zinc superoxide dismutase (Cu,Zn-SOD) to catalyze autoxidation of cysteine and other thiols was investigated by measuring thiol loss and oxygen consumption. The reaction occurred equally well with the bovine and human enzymes and produced hydrogen peroxide and the corresponding disulfide. It did not occur with manganese SOD and is not, therefore, due to the dismutase activity of the enzyme. Cysteine and cysteamine were highly reactive: the Km for cysteine was 1.4 mm andVmax (with 40 μg/ml SOD) 35 μm/min; the equivalent values for cysteamine (with 20 μg/ml SOD) were 1.4 mm and 36 μm/min. With 1 mm thiol and 40 μg/ml SOD, rates of oxidation of other thiols (μm/min) were as follows: GSH, 1.0; dithiothreitol, 2.1; dihydrolipoic acid, 1.7; homocysteine, 1.6; cys-gly, 1.4; penicillamine, 0.6; and N-acetylcysteine, 0.1. SOD-mediated oxidation of cysteine, in the absence of chelating agents, proceeded only after a variable lag phase. The lag was decreased but not eliminated with Chelex-treated reagents and is attributed to interference by submicromolar concentrations of iron and possibly other transition metal ions. SOD-catalyzed oxidation of the other thiols was variably affected by adventitious metal ions and chelating agents. Reactions were all performed in the presence of desferrioxamine to obviate these effects. SOD-catalyzed oxidation of GSH and homocysteine was enhanced by cysteine through a thiol-disulfide exchange mechanism. This study characterizes a novel pro-oxidant thiol oxidase activity of Cu,Zn-SOD. It is a potential source of reactive oxidants and may contribute to the cytotoxicity of reactive thiols such as cysteine and cysteamine. The ability of copper,zinc superoxide dismutase (Cu,Zn-SOD) to catalyze autoxidation of cysteine and other thiols was investigated by measuring thiol loss and oxygen consumption. The reaction occurred equally well with the bovine and human enzymes and produced hydrogen peroxide and the corresponding disulfide. It did not occur with manganese SOD and is not, therefore, due to the dismutase activity of the enzyme. Cysteine and cysteamine were highly reactive: the Km for cysteine was 1.4 mm andVmax (with 40 μg/ml SOD) 35 μm/min; the equivalent values for cysteamine (with 20 μg/ml SOD) were 1.4 mm and 36 μm/min. With 1 mm thiol and 40 μg/ml SOD, rates of oxidation of other thiols (μm/min) were as follows: GSH, 1.0; dithiothreitol, 2.1; dihydrolipoic acid, 1.7; homocysteine, 1.6; cys-gly, 1.4; penicillamine, 0.6; and N-acetylcysteine, 0.1. SOD-mediated oxidation of cysteine, in the absence of chelating agents, proceeded only after a variable lag phase. The lag was decreased but not eliminated with Chelex-treated reagents and is attributed to interference by submicromolar concentrations of iron and possibly other transition metal ions. SOD-catalyzed oxidation of the other thiols was variably affected by adventitious metal ions and chelating agents. Reactions were all performed in the presence of desferrioxamine to obviate these effects. SOD-catalyzed oxidation of GSH and homocysteine was enhanced by cysteine through a thiol-disulfide exchange mechanism. This study characterizes a novel pro-oxidant thiol oxidase activity of Cu,Zn-SOD. It is a potential source of reactive oxidants and may contribute to the cytotoxicity of reactive thiols such as cysteine and cysteamine. superoxide dismutase Zn-SOD, copper,zinc superoxide dismutase dithiothreitol manganese superoxide dismutase diethylenetriaminepentaacetic acid Superoxide dismutase (SOD)1 is an essential component of the antioxidant defense of aerobic organisms. Three forms of the enzyme, namely Cu,Zn-SOD, Mn-SOD, and extracellular Cu,Zn-SOD, are found in mammals (1Lyons T.J. Gralla E.B. Valentine J.S. Met. Ions Biol. Syst. 1999; 36: 125-177PubMed Google Scholar). Their prime function is to catalyze dismutation of superoxide (Reaction 1).O⨪2+SOD−Cu2+→O2+SOD−Cu+O⨪2+2H++SOD−Cu+→H2O2+SOD−Cu2+REACTION1A key feature of the enzyme is the maintenance of high dismutase activity while shielding the active site copper from other redox transformations. However, side reactions have been documented (2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 3Yim M.B. Boon Chock P. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar, 4Goss S.P. Singh R.J. Kalyanaraman B. J. Biol. Chem. 1999; 274: 28233-28239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 5Crow J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Crossref PubMed Scopus (429) Google Scholar), and superoxide oxidase and reductase activities, involving in each case only one-half of the copper redox cycle, have recently been reported (6Liochev S.I. Fridovich I. J. Biol. Chem. 2000; 275: 38482-38485Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 7Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 2001; 388: 281-284Crossref PubMed Scopus (8) Google Scholar). If SOD does have superoxide reductase activity, then thiols are potential biological substrates. Thiols are generally viewed as antioxidants. GSH is present intracellularly in millimolar concentrations and acts with glutathione peroxidase and glutathione reductase to remove peroxides. GSH and other thiols can also scavenge radicals and in combination with SOD constitute an efficient radical removal system (8Munday R. Winterbourn C.C. Biochem. Pharmacol. 1989; 38: 4349-4352Crossref PubMed Scopus (128) Google Scholar, 9Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (323) Google Scholar, 10Winterbourn C.C. Metodiewa D. Free Radic. Biol. Med. 1999; 27: 322-328Crossref PubMed Scopus (655) Google Scholar, 11Winterbourn C.C. Metodiewa D. Arch. Biochem. Biophys. 1994; 314: 284-290Crossref PubMed Scopus (237) Google Scholar, 12Zhang N. Schuchmann H.-P. von Sonntag C. J. Phys. Chem. 1991; 95: 4718-4722Crossref Scopus (52) Google Scholar). However, thiols can act as pro-oxidants by undergoing autoxidation to produce hydrogen peroxide (13Misra H.P. J. Biol. Chem. 1974; 249: 2151-2155Abstract Full Text PDF PubMed Google Scholar, 14Biaglow J.E. Varnes M.E. Radiat. Res. 1983; 95: 437-455Crossref PubMed Scopus (212) Google Scholar, 15Kachur A.V. Koch C.J. Biaglow J.E. Free Radic. Res. 1999; 31: 23-34Crossref PubMed Scopus (96) Google Scholar, 16Kachur A.V. Koch C.J. Biaglow J.E. Free Radic. Res. 1998; 28: 259-269Crossref PubMed Scopus (134) Google Scholar, 17Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (59) Google Scholar). The reaction is metal-catalyzed, with copper being a particularly effective catalyst. Autoxidation is considered to be important in the well established cytotoxicity of low molecular weight thiols (18Munday R. Adv. Pharmacol. 1994; 27: 237-270Crossref PubMed Scopus (32) Google Scholar, 19Held K.D. Sylvester F.C. Hopcia K.L. Biaglow J.E. Radiat. Res. 1996; 145: 542-553Crossref PubMed Scopus (106) Google Scholar). Cu,Zn-SOD, if it were a catalyst of this reaction, could be a promoter of thiol toxicity. We have investigated the ability of Cu,Zn-SOD to catalyze autoxidation of cysteine, GSH, and a range of physiological relevant thiols. In previous studies of superoxide-mediated thiol oxidation, we noted that addition of SOD to some thiols alone appeared to promote oxidation (10Winterbourn C.C. Metodiewa D. Free Radic. Biol. Med. 1999; 27: 322-328Crossref PubMed Scopus (655) Google Scholar). Others have made similar observations with cysteine (20Biaglow J.E. Issels R.W. Gerweck L.E. Varnes M.E. Jacobson B. Mitchell J.B. Russo A. Radiat. Res. 1984; 100: 298-312Crossref PubMed Scopus (53) Google Scholar, 21Saez G. Thornalley P.J. Hill H.A. Hems R. Bannister J.V. Biochim. Biophys. Acta. 1982; 719: 24-31Crossref PubMed Scopus (245) Google Scholar). The explanation given, that superoxide somehow protects cysteine against oxidation (13Misra H.P. J. Biol. Chem. 1974; 249: 2151-2155Abstract Full Text PDF PubMed Google Scholar, 21Saez G. Thornalley P.J. Hill H.A. Hems R. Bannister J.V. Biochim. Biophys. Acta. 1982; 719: 24-31Crossref PubMed Scopus (245) Google Scholar), is not consistent with current knowledge of how superoxide interacts with thiols (11Winterbourn C.C. Metodiewa D. Arch. Biochem. Biophys. 1994; 314: 284-290Crossref PubMed Scopus (237) Google Scholar, 12Zhang N. Schuchmann H.-P. von Sonntag C. J. Phys. Chem. 1991; 95: 4718-4722Crossref Scopus (52) Google Scholar). After further investigation, we report here that the enzyme does not act in these systems simply by dismutating superoxide. Instead, we demonstrate a novel activity of Cu,Zn-SOD, namely the catalysis of thiol oxidation by oxygen. This thiol oxidase activity is high for cysteine and cysteamine but much lower with GSH and a range of other biological thiols. Unless stated otherwise, all biochemicals, including thiol compounds, were purchased from Sigma. Catalase, bovine erythrocyte, and liver Cu,Zn-SOD, human Cu,Zn-SOD, and Mn-SOD were used without further purification. Human erythrocyte Cu,Zn-SOD was isolated in our laboratory from red cell lysate by successive chromatography on DEAE-Sepaharose and phenyl-Sepharose and ran as single band on SDS-PAGE in the same position as Sigma human Cu,Zn-SOD. SOD concentrations were determined by weight or using the Bio-Rad (Lowry-based) DC protein assay standardized with bovine serum albumin. Micromolar concentrations of the bovine enzyme were determined spectrophotometrically, using ε258 = 10.3 × 103m−1 cm−1 (22Rotilio G. Calabrese L. Bossa F. Barra D. Finazzi-Agro A. Mondovi B. Biochemistry. 1972; 11: 2182-2187Crossref PubMed Scopus (100) Google Scholar), and on this basis, 40 μg/ml corresponded to 1.2 μm. Where the term SOD is used in the text, it refers to the bovine erythrocyte Cu,Zn enzyme. The apoenzyme was prepared by initial dialysis against 50 mm acetate buffer, pH 3.8, + 10 mm EDTA followed by exhaustive dialysis with 0.1m NaCl to remove the bound chelator (23Valentine J.S. Pantoliano M.W. McDonnell P.J. Burger A.R. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4245-4249Crossref PubMed Scopus (110) Google Scholar). Dismutase activities were measured by a microtiter plate assay using WST-1 (water-soluble tetrazolium salt; Dojindo Laboratories (Kumamoto, Japan)) and a xanthine oxidase/hypoxanthine superoxide generating system (24Peskin A.V. Winterbourn C.C. Clin. Chim. Acta. 2000; 293: 157-166Crossref PubMed Scopus (395) Google Scholar). The amount of protein required per well (200 μl volume) to give 50% inhibition of WST-1 reduction was 2.5 ± 0.4, 3.3 ± 0.4, and 2.4 ± 0.9 ng for the bovine erythrocyte, bovine liver, and human erythrocyte enzymes, respectively. Except where indicated otherwise, buffers and thiol solutions were treated with washed Chelex 100 resin (Bio-Rad) for and then by were in and with was concentrations indicated in the were with SOD in in a of of buffer, pH and with present indicated were and with a acid to thiol were measured on a and thiol concentrations using = and Scholar). With GSH, was by a glutathione system of and Scholar). was measured with a thiol oxidase activity was due to bound metal bovine Cu,Zn-SOD was with (200 in mm buffer, pH then after 10 μl was through a in the same without Cysteine oxidation was then measured with 40 μg/ml SOD and 1 mm cysteine in the presence and absence of and to dismutase activity and protein oxidation studies were performed pH in the presence of for 1 these cysteine alone oxidation, but in the presence of bovine erythrocyte Cu,Zn-SOD was thiol loss Human erythrocyte Cu,Zn-SOD and bovine liver Cu,Zn-SOD also cysteine oxidation a to with the bovine erythrocyte enzyme. The reaction was to Cu,Zn-SOD, as with Mn-SOD loss of This that the of thiol oxidation by Cu,Zn-SOD is not simply a of ability to catalyze superoxide was required to promote cysteine oxidation, as the apoenzyme was not SOD a with a T.J. A. J.A. J.A. C. Gralla E.B. Valentine J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). addition of cysteine, was a that cysteine the active site GSH, only with SOD loss is an catalyst of thiol oxidation, it was important to the that cysteine oxidation was simply due to adventitious or bound copper in the enzyme With we oxidation of cysteine a of 50 10 on a with Others have Sigma bovine Cu,Zn-SOD and found of the copper to be bound S. F. D. S. J. Biochem. 1989; PubMed Scopus Google Scholar). that we by the of J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Crossref PubMed Scopus (429) Google Scholar), consistent with this if cysteine oxidation could have been by adventitious we the SOD with a of then the on a Dismutase activities and protein concentrations were measured and after through the to for to rates of thiol oxidation by the treated and enzyme were with successive and through the dialysis of the SOD against buffer, with or without with also did not ability to promote cysteine it is that this activity was by bound Cysteine loss in the presence of Cu,Zn-SOD was by oxygen consumption. The of thiol loss to oxygen measured 20 in the same was ± = The oxygen was to by the addition of that hydrogen peroxide was produced in the hydrogen peroxide be to cysteine oxidation (10Winterbourn C.C. Metodiewa D. Free Radic. Biol. Med. 1999; 27: 322-328Crossref PubMed Scopus (655) Google Scholar, L. I. F. Chem. 1989; Google Scholar). We measuring thiol oxidation to due to this In the absence of (with 1 mm cysteine and 40 μg/ml cysteine oxidation was ± = with The of was the same μg/ml and of the enzyme was are consistent with hydrogen peroxide being produced in and further cysteine as in SOD also GSH autoxidation but a much lower was measured in this reaction and for of the GSH This is also consistent with the hydrogen peroxide could the SOD (2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar), of the SOD to cysteine the absence of then loss of dismutase activity or ability to promote further cysteine oxidation not The of relevant thiol with bovine Cu,Zn-SOD were determined as as With all the other addition of SOD a in oxidation but were reactive that It is that the GSH, was activity was not simply a of or with low thiol as was dihydrolipoic acid, homocysteine, and the of GSH, but was oxidase activity of bovine ± ± 1.2 ± ± ± ± = ± = ± ± 1 thiol were with SOD in mm buffer, pH desferrioxamine and The was and all reagents were in the same rates of thiol autoxidation in the absence of SOD were μm/min. The were dihydrolipoic acid and dithiothreitol, rates μm/min. Thiols with oxidation rates cysteine were for to to that was in oxidation with The of is in In all was a thiol oxidation in the presence and absence of = in a 1 thiol were with SOD in mm buffer, pH desferrioxamine and The was and all reagents were in the same rates of thiol autoxidation in the absence of SOD were μm/min. The were dihydrolipoic acid and dithiothreitol, rates μm/min. Thiols with oxidation rates cysteine were for to to that was in oxidation with The of is in In all was a thiol oxidation in the presence and absence of Cu,Zn-SOD. SOD oxidase activity with GSH and homocysteine, addition of cysteine the oxidation of as by thiol loss in of the cysteine is high to cell we measured cysteine oxidation rates of oxidation were and with to with mm mm not The rates of loss of cysteine and cysteamine with thiol In each case was of With 40 μg/ml SOD, cysteine a Km of 1.4 mm andVmax of 35 μm/min. (with 20 μg/ml also a Km of 1.4 mm and of 36 μm/min. The of cysteine oxidation was on SOD but with the 20 μg/ml enzyme the for cysteine and cysteamine were and respectively. The of cysteine oxidation the SOD was by for 40 This is not as the enzyme the active site a or of Mn-SOD Cu,Zn-SOD did not the of cysteine oxidation with Mn-SOD = ± of that without = cysteine, the same as for initial rates of oxidation pH and were ± 0.4, ± and ± pH in this With desferrioxamine initial rates of SOD-mediated thiol oxidation were for cysteine in In the absence of chelating agents, the were cysteine was a lag and the oxidation was rates on the and were with if the reagents were treated with Chelex the of the reaction, the did not the lag eliminated the lag but a with desferrioxamine The of cysteine oxidation was the same in the presence of 1 or 10 with 1.2 SOD or with SOD This that it was not to have as much chelator as active site in the presence of desferrioxamine were the same in and explanation for the in is that the by and bound copper from the enzyme and that in the absence of cysteine alone the for the This can be on the that did not the activity of the enzyme the lag was with SOD that been with and it further was This that the chelator was with a of the reaction and not with the enzyme. This of observations could be by the reagents of adventitious transition metal that the of cysteine oxidation by SOD and give to a lag phase. The with desferrioxamine to iron being L. I. F. Chem. 1989; Google have noted that iron can the autoxidation of cysteine by copper ions. We have found this inhibition to be such that with an of 50 and C. submicromolar concentrations of ions to SOD and cysteine a in the of cysteine for iron in the of the metal was by for the that of iron other transition in the reagents with SOD-mediated cysteine oxidation and that to study the system without this it is to EDTA and were not as effective as desferrioxamine the of adventitious and in the presence or absence of of these rates of cysteine oxidation in the pH range not SOD-mediated oxidation of the other thiols was also by adventitious but not in the same as with This with metal ions similar to on thiol oxidation by copper study of the of and on the thiol of copper and SOD be We have that Cu,Zn-SOD the autoxidation of low molecular weight thiols. The reaction reduction of the active site and hydrogen peroxide and disulfide. It is not a of the dismutase activity of the enzyme, as thiol oxidation was with it is a of the Cu,Zn enzyme, and as with cysteine, the human and bovine enzymes The oxidase activity was particularly high with the cysteine, and cysteamine. including GSH, were much It is well that copper ions are efficient of thiol autoxidation and that are or GSH to oxidation A.V. Koch C.J. Biaglow J.E. Free Radic. Res. 1998; 28: 259-269Crossref PubMed Scopus (134) Google Scholar, 17Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (59) Google Scholar, J.E. Issels R.W. Gerweck L.E. Varnes M.E. Jacobson B. Mitchell J.B. Russo A. Radiat. Res. 1984; 100: 298-312Crossref PubMed Scopus (53) Google Scholar, L. I. F. Chem. 1989; Google Scholar). a equivalent basis, the cysteine oxidase activity we with SOD was a that of This activity was not due to adventitious or bound copper that could be by of the enzyme with SOD from and liver, and human erythrocyte SOD prepared by all It is therefore, that it was due to a or enzyme, as these We that the active site copper was and that Cu,Zn-SOD thiol oxidase active from a physiological this activity of Cu,Zn-SOD is a cell SOD could mm with hydrogen peroxide with GSH, 10 this reaction could be a source of hydrogen peroxide in the the of thiol oxidation, our of with cysteine and cysteamine that an with the enzyme is The of inhibition by Mn-SOD that superoxide is not required to the It also a from oxygen to the to the of the enzyme and a acid of radicals and superoxide is the in Reactions in the acid with thiol to give the and the SOD be to This is similar to the for is for some superoxide (13Misra H.P. J. Biol. Chem. 1974; 249: 2151-2155Abstract Full Text PDF PubMed Google Scholar, 15Kachur A.V. Koch C.J. Biaglow J.E. Free Radic. Res. 1999; 31: 23-34Crossref PubMed Scopus (96) Google Scholar, 16Kachur A.V. Koch C.J. Biaglow J.E. Free Radic. Res. 1998; 28: 259-269Crossref PubMed Scopus (134) Google Scholar, 17Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (59) Google N. J. Biochem. 1999; PubMed Scopus Google Scholar). forms a with cysteine D. C. S. G. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). With SOD, where is a J.A. J.S. 1983; 284-290Crossref PubMed Scopus Google Scholar), it is that only cysteine could the active site copper or that is range generating hydrogen the reaction Cu,Zn-SOD and thiols is a potential source of reactive oxidants in biological The of thiols to is well with the cysteine and being (18Munday R. Adv. Pharmacol. 1994; 27: 237-270Crossref PubMed Scopus (32) Google Scholar, 19Held K.D. Sylvester F.C. Hopcia K.L. Biaglow J.E. Radiat. Res. 1996; 145: 542-553Crossref PubMed Scopus (106) Google F. 1983; PubMed Scopus Google Scholar, J. 1993; Google Scholar, Biaglow J.E. L. Gerweck L.E. Res. 1984; Google Scholar, G. J. Clin. PubMed Scopus Google Scholar, K.D. Biaglow J.E. Radiat. Res. 1993; PubMed Scopus Google Scholar, G. J. Google Scholar). In a of these hydrogen peroxide by autoxidation been in the and a that Cu,Zn-SOD is a catalyst of this are other where Cu,Zn-SOD been to the of cysteine J. M.B. 1979; PubMed Google Scholar, J. 1979; 38: PubMed Google Scholar). The was attributed to copper from the enzyme by but our that SOD may have been GSH was much reactive cysteine and was in our This low millimolar concentrations of GSH to with Cu,Zn-SOD in the cell without generating of This not be for of low concentrations and of GSH as the thiol have the pro-oxidant of SOD and thiols. by GSH that for effective radical the of GSH and SOD is required (8Munday R. Winterbourn C.C. Biochem. Pharmacol. 1989; 38: 4349-4352Crossref PubMed Scopus (128) Google Scholar, 9Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (323) Google Scholar). The low the not this antioxidant GSH contribute to the of reactive thiols such as We that of cysteine autoxidation of GSH, SOD-catalyzed oxidation of cysteine and a exchange to the in therefore, the potential to all the GSH to With of the this could of hydrogen of homocysteine oxidation by cysteine and SOD was also homocysteine is a in J.S. A. 1996; PubMed Scopus Google Scholar), and of superoxide and hydrogen peroxide from homocysteine autoxidation could be a to the J. Clin. 1997; 100: PubMed Scopus Google Scholar). Autoxidation of homocysteine is in the presence of copper but it can be by of N. Free Radic. Med. 1999; 27: PubMed Scopus Google Scholar). that the same with Cu,Zn-SOD that it may be relevant copper as a catalyst. the other thiols we and dihydrolipoic acid to GSH with Cu,Zn-SOD, were the concentrations to be we is to cell or biological in high millimolar reaction with Cu,Zn-SOD this reaction be to give a of hydrogen peroxide and may in of We that thiol oxidation was by adventitious metal ions. this is relevant in a physiological is but it we highly variable and of cysteine oxidation, we to interference by of iron and possibly other metal ions that we were to remove from the of other thiols was also in that we in was eliminated by also affected the oxidation but was not to that of and it is that iron to the be that thiol oxidation is to interference by adventitious metal ions and that this on the of and a chelator is can be to this We have explanation for how metal ions cysteine oxidation by Cu,Zn-SOD. to the active site copper were that the interference with the and oxygen a We and L. I. F. Chem. 1989; Google have that low iron concentrations cysteine oxidation by and it is that a similar is with SOD generally been considered as an enzyme that is in other activities, particularly in the of with (1Lyons T.J. Gralla E.B. Valentine J.S. Met. Ions Biol. Syst. 1999; 36: 125-177PubMed Google Scholar). activity that the oxidation of to a radical been documented for Cu,Zn-SOD (2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 3Yim M.B. Boon Chock P. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar, 4Goss S.P. Singh R.J. Kalyanaraman B. J. Biol. Chem. 1999; 274: 28233-28239Abstract Full Text Full Text PDF PubMed Scopus (87) Google as catalysis of J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Crossref PubMed Scopus (429) Google Scholar). Cu,Zn-SOD been reported to of to D. M.B. Arch. Biochem. Biophys. 1999; PubMed Scopus Google Scholar), that this hydrogen of copper R.J. N. S.P. Kalyanaraman B. Arch. Biochem. Biophys. 1999; PubMed Scopus Google Scholar). This activity to be with with J.B. B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and Fridovich have the of the superoxide oxidase and reductase activities of SOD can be (6Liochev S.I. Fridovich I. J. Biol. Chem. 2000; 275: 38482-38485Abstract Full Text Full Text PDF PubMed Scopus (84) Google and a superoxide reductase activity of Cu,Zn-SOD in the catalysis of acid oxidation S.I. Fridovich I. Arch. Biochem. Biophys. 2001; 388: 281-284Crossref PubMed Scopus (8) Google Scholar). With we that the enzyme does not as a superoxide reductase but acts as a thiol This novel pro-oxidant function of Cu,Zn-SOD the potential to of hydrogen peroxide and may contribute to the of thiols such as cysteine and cysteamine and the of thiol We are to for on thiol oxidation and to and for