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Copper,Zinc Superoxide Dismutase as a Univalent NO−Oxidoreductase and as a Dichlorofluorescin Peroxidase

2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês

10.1074/jbc.m104237200

ISSN

1083-351X

Autores

Stefan I. Liochev, Irwin Fridovich,

Tópico(s)

Vanadium and Halogenation Chemistry

Resumo

Nitroxyl (NO−) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO− to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO− and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O⨪2 was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were ∼100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO− caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O⨪2, SOD could catalyze the oxidation of NO− to NO. The reverse reaction, i.e. the reduction of NO to NO− by Cu(I),Zn-SOD, followed by the reaction of NO− with O2 would yield ONOO− and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H2O2 caused the HCO3−-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H2O2 production. Nitroxyl (NO−) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO− to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO− and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O⨪2 was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were ∼100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO− caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O⨪2, SOD could catalyze the oxidation of NO− to NO. The reverse reaction, i.e. the reduction of NO to NO− by Cu(I),Zn-SOD, followed by the reaction of NO− with O2 would yield ONOO− and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H2O2 caused the HCO3−-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H2O2 production. superoxide dismutase dichlorofluorescin Angeli's salt diethylene triamine pentaacetic acid dihydrorhodamine glutathione familial amyotrophic lateral sclerosis SODs1 protect against the deleterious actions of O⨪2, by catalyzing its dismutation (1Liochev S.I. Fridovich I. IUBMB Life. 1999; 48: 157-161Crossref PubMed Google Scholar). However, additional properties have been ascribed to these enzymes, including peroxidase (2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Crossref PubMed Scopus (217) Google Scholar, 3Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Crossref PubMed Scopus (54) Google Scholar, 4Liochev S.I. Chen L.L. Hallewell R.A. Fridovich L. Arch. Biochem. Biophys. 1997; 346: 263-268Crossref PubMed Scopus (42) Google Scholar), superoxide reductase (5Liochev S.I. Fridovich I. J. Biol. Chem. 2000; 275: 38482-38485Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), superoxide oxidase (5Liochev S.I. Fridovich I. J. Biol. Chem. 2000; 275: 38482-38485Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and reversible NO/NO−oxidoreductase (6Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar, 7Fukuto J.M. Hobbs A.J. Ignarro J. Biochem. Biophys. Res. Commun. 1993; 196: 707-713Crossref PubMed Scopus (135) Google Scholar, 8Fukuto J.M. Wink D.A. Sigel A. Sigel H. Metal Ions in Biological Systems. 36. Marcel Dekker Inc., New York1999: 547-595Google Scholar, 9Kim W.-K. Choi Y.-B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) activities. The biological significance of these latter activities remain to be solidified, but interest in them is increased by the finding that point mutations in Cu,Zn-SOD, most of which do not effect the SOD activity, have been associated with ∼20% of the cases of familial amyotrophic lateral sclerosis (FALS) (10Okado-Matsmoto A. Myint T. Fujii J. Taniguchi N. Free Radic. Res. 2000; 33: 65-73Crossref PubMed Scopus (23) Google Scholar, 11Ahmed M.S. Hung W.Y. Zu J.S. Hockberger P. Siddique T. J. Neurol. Sci. 2000; 176: 88-94Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We will now present data supporting the ability of SODs to catalyze the oxidation of NO−, utilizing O⨪2 as the oxidant; and of Cu,Zn-SOD to catalyze the HCO3−-dependent oxidation of DCF by H2O2. These findings could provide an alternative explanation for the observations of Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar). These findings will also shed ambiguity on the use of DCF oxidation as a measure of intracellular H2O2 and ONOO− production, in agreement with Rota et al. (13Rota C. Chignel C.F. Mason R.P. Free Radic Biol. Med. 1999; 27: 873-881Crossref PubMed Scopus (346) Google Scholar) who criticized this use of DCF. Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar) have reported that Zn-depleted Cu,Zn-SOD (Cu(II)-SOD) is reduced much more rapidly by ascorbate than is the Zn-replete enzyme and that other reductants, such as urate and GSH, act similarly but less effectively. Since DCF was oxidized by aerobic ascorbate + Cu(II)-SOD + NO, and is known to be oxidized by ONOO−, but not by NO, O⨪2, or H2O2, they proposed that the Cu(I) enzyme was reducing O2 to O⨪2, that then reacted with NO to give ONOO−. This mechanism had special significance in view of reports that the FALS-associated mutant Cu,Zn-SODs exhibit lowered affinity for Zn(II) (14Crow J.P. Sampson J.C. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Crossref PubMed Scopus (429) Google Scholar, 15Goto J.J. Zhu H. Sanchez R.J. Nersissian A. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 2000; 275: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and that Cu(II)-SODs, whether mutant or wild type, were toxic to motor neurons (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar). An alternative explanation for the results of Estevez et al.(12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar) would entail the reduction of NO to NO− by the Cu(I)-SOD, followed by production of ONOO− from the reaction of NO− with O2. The ONOO− thus produced would oxidize DCF. That reduced SODs can act as univalent reductants for NO was shown by Murphy and Sies (6Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar) and by Kim et al. (9Kim W.-K. Choi Y.-B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and might account for the observations of McBride et al. (16McBride A.G. Boritaite V. Brown G.C. Biochim. Biophys. Acta. 1999; 1454: 275-288Crossref PubMed Scopus (92) Google Scholar) in which case H2O2 was the reductant of Cu,Zn-SOD, in place of the ascorbate used by Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar). Angeli's Salt (Na2N2O3; AS) from the Cayman Chemical Co. was generously provided by J. S. Stamler. Stock solutions of ∼10 or 20 mm were prepared in 10 mm NaOH and were stored at −20 °C until used. The extinction coefficient of AS at 250 nm in the NaOH was taken to be 8,000 m−1 cm−1 (17Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hofman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (702) Google Scholar) and in sodium phosphate buffer, pH 7.4, to be 4,400 m−1cm−1. NADPH, diethylene triamine pentaacetic acid (DTPA), and bovine serum albumin were from Sigma; potassium ferricyanide was from J. T. Baker; cytochrome c was from Fluka; catalase was from Boehringer/Ingelheim; bovine Cu,Zn-SOD was from Grunenthal; and recombinant human Mn-SOD was from Biotechnology General. All reactions were performed at 23 °C in 100 mm sodium phosphate, 50 μm DTPA at pH 7.4. The decomposition of AS was followed at 250 nm (17Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hofman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (702) Google Scholar, 18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), the oxidation of NADPH at 340 nm (19Horecker B.L. Kornberg A. J. Biol. Chem. 1948; 175: 385-390Abstract Full Text PDF PubMed Google Scholar), the reduction of cytochrome c at 550 nm (20Massey V. Biochim. Biophys. Acta. 1959; 34: 255-256Crossref PubMed Scopus (530) Google Scholar), and the oxidation of DCF at 500 nm (21Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (559) Google Scholar). An alkaline stock solution of AS, when diluted into neutral buffer to 0.175 mm, decomposed at an initial rate of 9 μm/min as shown byline 1 in Fig. 1. When 100 μm NADPH was present it was oxidized by the AS at a rate of 5 μm/min (line 2). Thus the ratio of AS decomposed to NADPH oxidized was almost 2:1, in agreement with earlier reports (22Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Radic. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar, 23Wink D.A. Feelisch M. Fukuto J. Christodoulou D. Jourd'heuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S.M. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1998; 351: 66-74Crossref PubMed Scopus (185) Google Scholar). Since AS decomposition is known to be a source of NO− (7Fukuto J.M. Hobbs A.J. Ignarro J. Biochem. Biophys. Res. Commun. 1993; 196: 707-713Crossref PubMed Scopus (135) Google Scholar, 18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Wink D.A. Feelisch M. Fukuto J. Christodoulou D. Jourd'heuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S.M. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1998; 351: 66-74Crossref PubMed Scopus (185) Google Scholar, 24Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (245) Google Scholar), it appears that NO−, or a species derived therefrom, under aerobic conditions, probably ONOO−, oxidizes NADPH. Both Cu,Zn-SOD and Mn-SOD were able to inhibit the oxidation of NADPH by NO− as shown in Fig.2 and in agreement with Reif et al. (22Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Radic. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar). This was not due to an effect on the rate of decomposition of AS, which was not influenced by Cu,Zn-SOD (not shown). Moreover it was not due to a nonspecific protein effect, since comparable levels of serum albumin caused only a small and transient effect (Fig. 3) that was probably due to the consumption of NO− by oxidation of albumin cysteine and methionine residues. Thus GSH was also able to inhibit the oxidation of NADPH by NO− in a way that suggested consumption of the GSH (Fig. 4).Figure 2SODs prevent the oxidation of NADPH by AS. The conditions were the same as described in the legend to Fig. 1. At arrows Cu,Zn-SOD was added to 0.060 mg/ml and then to 0.36 mg/ml (line 1), or Mn-SOD was added to 0.36 mg/ml and then to 0.72 mg/ml (line 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of bovine serum albumin on the oxidation of NADPH by AS. Reaction conditions were the same as described in the legend of Fig. 1. Line 1, albumin added to 0.12 mg/ml at the arrow; line 2, albumin added to 0.6 mg/ml at the arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of GSH on the oxidation of NADPH by AS. Reaction conditions were the same as described in Fig. 1. GSH was added to 5 μm at the arrow (line 1) or to 20 μm (line 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The inhibitory effect of the SODs did not appear to be due to the dismutation of O⨪2, since the levels of SOD needed to cause the inhibitions seen in Fig. 2 are ∼100-fold greater than would be required to cause comparable inhibition of the reduction of cytochromec by O⨪2. An interesting possibility is that NO− can reduce the redox-active metals (Cu(II) or Mn(III)) of the SODs and that O⨪2 then reoxidizes these metal centers, thus providing for catalytic consumption of NO−. The reactions envisioned are as follows.Me(n)+NO−⇄Me(n−1)+NOREACTION 1 Me(n−1)+O⨪2+2H+⇄Me(n)+H2O2REACTION 2 Exploration of this possibility required demonstration that NO− could reduce the redox-active metals at the active sites of the SODs and that O⨪2 was produced during the oxidation of NADPH by NO−. Addition of AS to 0.2 mm to a buffered solution of 4 mg/ml of Cu,Zn-SOD resulted in bleaching of the enzyme (Fig. 5). That this bleaching was due to reduction of the Cu(II) to Cu(I) was shown by the ability of ferricyanide to restore the lost absorbance. Catalase did not prevent the reduction of Cu,Zn-SOD by NO− (not shown). Thus H2O2 was not involved. This attests to the reality of Reaction 1. The less than complete bleaching shown in Fig. 5 probably was due to our stopping the process after 30 min or to limitation in the amount of AS. Production during the Oxidation of NADPH by NO−—When AS (0.175 mm) was oxidizing aerobic 0.1 mm NADPH, in the presence of 10 or 40 μmcytochrome c, reduction of the cytochrome c was seen (Fig. 6), and this could be inhibited by very low levels of Cu,Zn-SOD. It should be noted that the low levels of Cu,Zn-SOD, that were sufficient to compete with cytochrome c for O⨪2, were unable to detectably inhibit NADPH oxidation by NO−. Thus O⨪2 was produced during the oxidation of NADPH and that O⨪2 could drive Reaction 2. A likely source of that O⨪2 would be the oxidation of NADPH to NADP⋅ by NO−, followed by the autoxidation of NADP⋅ to NADP+. There was no reduction of cytochrome c by aerobic AS in the absence of NADPH. The oxidation of DCF to its fluorescent product has been widely used as a measure of intracellular production of H2O2 (11Ahmed M.S. Hung W.Y. Zu J.S. Hockberger P. Siddique T. J. Neurol. Sci. 2000; 176: 88-94Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Rota C. Chignel C.F. Mason R.P. Free Radic Biol. Med. 1999; 27: 873-881Crossref PubMed Scopus (346) Google Scholar, 25Ying W. Anderson C.M. Chen Y. Stein B.A. Fahlman C.S. Copin J.C. Chan P.H. Swanson R.A. J. Cereb. Blood Flow Metab. 2000; 20: 359-368Crossref PubMed Scopus (39) Google Scholar), and such use of DCF has been criticized (13Rota C. Chignel C.F. Mason R.P. Free Radic Biol. Med. 1999; 27: 873-881Crossref PubMed Scopus (346) Google Scholar). Nevertheless increased DCF oxidation as a consequence of the overproduction of normal Cu,Zn-SOD, or of the FALS-associated mutants, thereof, have been so interpreted (11Ahmed M.S. Hung W.Y. Zu J.S. Hockberger P. Siddique T. J. Neurol. Sci. 2000; 176: 88-94Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,25Ying W. Anderson C.M. Chen Y. Stein B.A. Fahlman C.S. Copin J.C. Chan P.H. Swanson R.A. J. Cereb. Blood Flow Metab. 2000; 20: 359-368Crossref PubMed Scopus (39) Google Scholar). We now present data that should decrease enthusiasm for this use of DCF. Thus Fig. 7 demonstrates that DCF is not oxidized by H2O2 per se but is oxidized by Cu,Zn-SOD plus H2O2 and that HCO3− markedly stimulates this oxidation. HCO3−-dependent peroxidations catalyzed by Cu,Zn-SOD have been reported previously (3Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Crossref PubMed Scopus (54) Google Scholar, 26Yim M.B. Chock P.B. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar, 27Sankarapandi S. Zweier J.L. J. Biol. Chem. 1999; 274: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 28Zhang H. Joseph J. Felix C. Kalyanaraman B. J. Biol. Chem. 2000; 275: 14038-14045Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and have been explained on the basis of the oxidation of HCO3− to CO⨪3, at the active site, followed by diffusion of the carbonate radical into the bulk solvent where it can cause diverse oxidations (3Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Crossref PubMed Scopus (54) Google Scholar, 28Zhang H. Joseph J. Felix C. Kalyanaraman B. J. Biol. Chem. 2000; 275: 14038-14045Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Thus the data in Fig. 7 indicates that increased oxidation of DCF within cells could signal increased oxidation of HCO3− by Cu,Zn-SOD plus H2O2due to increased Cu,Zn-SOD, or due to increased H2O2, or to increased peroxidase activities of the FALS-associated, or of the Zn-depleted, enzymes. DCF can also be oxidized by ascorbate + SOD + a source of NO under aerobic conditions (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar). Fig. 8(line 1) demonstrates that 10 μm DCF is oxidized when exposed to 20 μm AS and that 30 μg/ml Cu,Zn-SOD inhibited. Catalase at 260 units/ml had no significant effect, in the presence or absence of SOD. When 10 μm DCF was exposed to 50 μm AS in an argon-purged buffer, a slow and rapidly decreasing rate of DCF oxidation was seen. Subsequent aeration increased this rate dramatically (line 2). Dihydrorhodamine has similarly been seen to be oxidized by AS in an oxygen-dependent manner (18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). It appears that NO− + O2 yields an oxidant capable of rapid oxidation of DCF or DHR. We assume that this oxidant is ONOO− or something derived from it. It has been shown that NO− produced by reduction of NO by Cu(I),Zn-SOD is in the triplet ground state (9Kim W.-K. Choi Y.-B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), which is known to most readily react with triplet ground state O2 yielding ONOO− (24Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (245) Google Scholar,29Jay-Gerin J.-P. Ferradini C. Biochimie (Paris). 2000; 82: 161-166Crossref PubMed Scopus (28) Google Scholar). It should be noted that Miranda et al. (18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) proposed that the product of the AS-generated NO− + O2reaction is somehow different from ONOO−. That could be an isomeric form of ONOO− produced by the reaction of singlet NO− with O2. However, when NO− is produced biologically, as in the measurements of Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar), it would be triplet NO−, and this would yield ONOO-, per se, when reacting with O2. In any case ONOO−, or the similar product proposed by Miranda et al. (18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), would rapidly oxidize DCF and DHR. The sum of Reactions 1 plus 2 is as follows.NO−+O⨪2+2H+⇄NO+H2O2REACTION 3 In essence this is an expression of the superoxide reductase activity (5Liochev S.I. Fridovich I. J. Biol. Chem. 2000; 275: 38482-38485Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) of Cu,Zn-SOD and is in accord with Murphy and Sies (6Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar). It should be noted that Cu,Zn-SOD was not inactivated by incubation with 0.25 mm AS for 50 min (not shown). NO−/HNO can oxidize NADPH anaerobically as well as aerobically (22Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Radic. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar, 23Wink D.A. Feelisch M. Fukuto J. Christodoulou D. Jourd'heuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S.M. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1998; 351: 66-74Crossref PubMed Scopus (185) Google Scholar). The anaerobic reaction presumably involves the divalent oxidation of NADPH and the concomitant reduction of HNO to NH2OH, while the aerobic reaction could additionally be due to univalent oxidation of NADPH by ONOO− formed from the rapid reaction of NO− with O2 (24Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (245) Google Scholar, 29Jay-Gerin J.-P. Ferradini C. Biochimie (Paris). 2000; 82: 161-166Crossref PubMed Scopus (28) Google Scholar). There are reasons for believing that nitric-oxide synthase can produce both NO− and O⨪2, particularly when tetrahydrobiopterin is limiting (30Stuehr D. Pou S. Rosen G.M. J. Biol. Chem. 2001; 276: 14533-14536Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 31Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 32Vasquez-Vivar J. Hogg N. Martasek P. Karoui H. Pritchard Jr., K.A. Kalyanaraman B. J. Biol. Chem. 1999; 274: 26736-26742Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), and the observation that Cu,Zn-SOD increases the yield of NO produced per NADPH consumed could be partially explained by the superoxide reductase activity of Cu,Zn-SOD as expressed in Reaction 3. Nitric-oxide synthase plus NADPH and l-arginine produces more O⨪2 in the absence of tetrahydrobiopterin (32Vasquez-Vivar J. Hogg N. Martasek P. Karoui H. Pritchard Jr., K.A. Kalyanaraman B. J. Biol. Chem. 1999; 274: 26736-26742Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). That can partially be explained on the basis of NO− production. Thus the NO− plus O2 would yield ONOO−, which would oxidize NADPH to NADP⋅, and that in turn would reduce O2 to O⨪2. Hence the stoichiometry of NADPH consumed per NO produced, by nitric-oxide synthase, could be influenced by SOD both on the basis of the oxidation of NO− to NO and by the prevention of the nonenzymic oxidation of NADPH by NO−/ONOO−, in agreement with Reif et al. (22Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Radic. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar). It has been suggested by Stoyanovsky et al. (33Stoyanovsky D. Clancy R. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (30) Google Scholar) that HNO can dimerize and then decompose to N2 + 2HO⋅. If this were the case under our conditions, then NADPH oxidation should have been appreciably due to HO⋅ and should have been noticeably inhibited by an HO⋅ scavenger such as ethanol. However ethanol, added to 2% of the reaction volume, did not at all inhibit the oxidation of NADPH by NO− (not shown). Having previously noted that the oxyethyl radical does not oxidize NADPH (34Liochev S. Fridovich I. Arch. Biochem. Biophys. 1991; 291: 379-382Crossref PubMed Scopus (34) Google Scholar) we can now exclude HO⋅ from having a role in the oxidation NADPH by NO− under our conditions. It should be noted that Stoyanovsky et al. (33Stoyanovsky D. Clancy R. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (30) Google Scholar), using spin trapping, observed maximal HO⋅ production from HNO at pH 5, but very little at neutrality, while we worked at pH 7.4. The reduction of the Cu(II) at the active site of Cu,Zn-SOD by NO− is viewed as a reversible reaction, in accord with Murphy and Sies (6Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar). That being said, the observation of Estevezet al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar) might now be explained in terms of the reduction of NO to NO− by the ascorbate-reduced Cu-SOD, rather than by the reduction of O2 to O⨪2. The NO−, thus produced, would lead to formation of ONOO− that would account for the oxygen-dependent DCF oxidation that they observed. Another possible explanation is based on the observations of McBride et al. (16McBride A.G. Boritaite V. Brown G.C. Biochim. Biophys. Acta. 1999; 1454: 275-288Crossref PubMed Scopus (92) Google Scholar) who suggested that O⨪2, derived from the oxidation of H2O2 by the Cu(II) of Cu,Zn-SOD, might then react with NO, yielding ONOO− that could oxidize DHR. In the work of Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar) the autoxidation of ascorbyl radical is another possible source of O⨪2, and that, in the presence of NO, would yield ONOO−. The differences in reactivity between singlet and triplet NO− adds complexity to the data presented herein and to that in the literature (8Fukuto J.M. Wink D.A. Sigel A. Sigel H. Metal Ions in Biological Systems. 36. Marcel Dekker Inc., New York1999: 547-595Google Scholar, 9Kim W.-K. Choi Y.-B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Wink D.A. Feelisch M. Fukuto J. Christodoulou D. Jourd'heuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S.M. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1998; 351: 66-74Crossref PubMed Scopus (185) Google Scholar, 24Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (245) Google Scholar). Thus GSH may be oxidized by1NO− and not by 3NO−(9Kim W.-K. Choi Y.-B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Hence if the biologically generated NO− is in the triplet state then GSH might not be as effective a scavenger of NO− as proposed by Miranda et al. (18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The oxidations of NADH and of DCF could have been due to NO−or to ONOO− derived therefrom. NO− is a small anion, akin to O⨪2, and should readily gain access to the active site of SOD, but the possibility of SOD reacting with ONOO−cannot be excluded. If the effect of SOD was mainly due to reaction with NO−, we can now attempt to estimate the rate constant for that interaction. The NO− produced by the decomposition of AS is said to be 1NO−(18Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S.M. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), which can relax to 3NO− and that, in turn, reacts with 3O2 to yield ONOO−. We have seen that ∼2 × 10−6m SOD 50% inhibited the oxidation of NADPH, or of DCF, by AS. Suppose that SOD only reacts with 1NO− and not with 3NO−. Then 2 × 10−6 SOD must halve the [1NO−] in competition with its conversion to 3NO−, plus its reactions with all targets for 1NO−attack. If there are no scavengers for 1NO−, or if we disregard them, we will get a minimal estimate for the rate constant and we can write: k 1 [SOD] [1NO−] = k 2[1NO−], where k 1 is the rate constant for the oxidation of 1NO− by SOD, and k 2 is the rate of relaxation of1NO− to 3NO−, thenk 1 [SOD] = k 2. The rate constant for the relaxation of 1NO− is not known, but we can assume that it is similar to that for the relaxation of 1O2, which is of the order of 2 × 105 s−1 (35Wilkinson F. Helman W.P. Ross A.B. J. Phys. Chem. Ref. Data. 1995; 24: 663-1021Crossref Scopus (1181) Google Scholar). Then k 1 = 1011m−1 s−1. This is unrealistically fast and might suggest that the relaxation of1NO− is slower than that of1O2. But even if k 2 was 2 × 103 s−1 the value of k1would be 109m−1 s−1and that is comparable with the rate constant for the reaction of O⨪2 with SOD. Now suppose that SOD reacts with 3NO− and not with 1NO−. In that case the SOD would be competing with the reaction of 3NO− with O2 and any other scavengers for3NO−. If we again ignore reactions with other targets we can write: k 1[SOD][3NO−] = k 2[3NO−][3O2] ork 1 [SOD] = k 2[3O2]. [SOD] is 2 × 10−6m and [3O2] in aqueous solutions equilibrated with air is ∼2 × 10−4m. Two values have been reported for k 2 and these are 4 × 107m−1 s−1 (29Jay-Gerin J.-P. Ferradini C. Biochimie (Paris). 2000; 82: 161-166Crossref PubMed Scopus (28) Google Scholar) and 7 × 107m−1s−1 (24Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (245) Google Scholar). If we take the lower value we getk 1 = 4 × 109m−1 s−1, and this is again a minimal estimate. Thus whether SOD reacts with1NO− or 3NO−, or both, its rate constant must be very high to account for its observed ability to inhibit the oxidations caused by NO−. Whether this has relevance in the biological milieu is not yet known. There is uncertainty in the literature concerning the pK a, redox potential, and even the spin state of NO− in aqueous solution. That being the case we must explain repeatable observations as best we can and hope that, in time, the physical chemistry and theoretical treatments of this fascinating molecule will yield certain results, which in turn, will lead to refinements of those currently reasonable explanations. It should be noted that we were not necessarily dealing with free NO/NO− but rather with these species at the active site of SOD, and binding may substantially change their physicochemical character. Thus the interaction of NO with reduced SOD could give rise to a bound nitroxyl, which can be represented by the following equilibrium: Enz-Cu(I)NO ⇄ Enz-Cu(II)NO− (where Enz indicates enzyme) and which could react with O2yielding ONOO− that could diffuse into the bulk solvent and there oxidize DCF or DHR. A similar scenario applies to the results of Sharpe and Cooper (36Sharpe M.A. Cooper C.E. Biochem. J. 1998; 332: 9-19Crossref PubMed Scopus (189) Google Scholar) who reported that aerobic ferrocytochromec plus NO oxidized DHR. This recalls the proposal made by Estevez et al. (12Estevez A.G. Crow J.P. Sampson J.B. Reiter L. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (515) Google Scholar) with respect to the putative bound O⨪2 made by the autoxidation of Cu(I)-SOD. The oxidation of NAD(P)H by ONOO−, with subsequent production of O2− by autoxidation of NAD(P)⋅, has been described by Kirsch and de Groot (37Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).

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