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• W2024994588 abstract "Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O⨪2 gateway in Mn-SOD. Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O⨪2 gateway in Mn-SOD. Peroxynitrite anion (ONOO−) 1The term peroxynitrite is used to refer to both peroxynitrite anion (ONOO−) and peroxynitrous acid (ONOOH). IUPAC recommended names are oxoperoxonitrate(1−) and hydrogen oxoperoxonitrate, respectively. 1The term peroxynitrite is used to refer to both peroxynitrite anion (ONOO−) and peroxynitrous acid (ONOOH). IUPAC recommended names are oxoperoxonitrate(1−) and hydrogen oxoperoxonitrate, respectively. is a potent biological oxidant that has been implicated in diverse forms of free radical-induced tissue injury (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1961) Google Scholar). Peroxynitrite has been demonstrated to readily oxidize or nitrate various enzymes such as metalloproteinase-1 inhibitor (2Frears E.R. Zhang Z. Blake D.R. O'Connell J.P. Winyard P.G. FEBS Lett. 1996; 381: 21-24Crossref PubMed Scopus (148) Google Scholar), alcohol dehydrogenase (3Crow J.P. Beckman J.S. McCord J.M. Biochemistry. 1995; 34: 3544-3552Crossref PubMed Scopus (239) Google Scholar), aconitase (4Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar), glutamine synthetase (5Berlett B.S. Friguet B. Yim M.B. Chock P.B. Stadman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1776-1780Crossref PubMed Scopus (165) Google Scholar), and superoxide dismutase (6Ischiropoulos H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1423) Google Scholar). However, little is known about the molecular basis of the nitration or oxidation of these enzymes. Peroxynitrite is produced by the reaction of superoxide (O⨪2) and NO with a very rapid reaction rate (4 ∼ 6.7 × 109m−1s−1) (7Huie R.E. Podmain S. Free Radical Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2015) Google Scholar, 8Kobayashi K. Miki M. Tagawa S. J. Chem. Soc. Dalton Trans. 1995; : 2885-2889Crossref Google Scholar). Therefore, various cell types, such as macrophages (9Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Crossref PubMed Scopus (1092) Google Scholar), Kuppfer cells (10Wang J.F. Komorov P. Sies H. deGroot H. Biochem. J. 1991; 279: 311-314Crossref PubMed Scopus (106) Google Scholar), and endothelial cells (11Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Crossref PubMed Scopus (242) Google Scholar), that simultaneously produce and release O⨪2 and NO could produce peroxynitrite. Mammalian mitochondria are one of the most important targets of the cytotoxicity of peroxynitrite. Recently, a mechanism for peroxynitrite-mediated dysfunction of mitochondria has been proposed as follows. When mitochondria are exposed to NO, NO diffuses easily through the membranes and reversibly inhibits cytochrome oxidase. This inhibition causes inhibition of the mitochondrial respiratory chain and as a consequence increases mitochondrial O⨪2release, leading to peroxynitrite formation (12Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (674) Google Scholar, 13Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (608) Google Scholar). Peroxynitrite then irreversibly inhibits complexes I and II in the mitochondrial respiratory chain (13Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (608) Google Scholar). In this mechanism, accumulation of O⨪2 results in production of a large amount of peroxynitrite. Mitochondrial Mn-SOD 2The abbreviations used are; SOD, superoxide dismutase; Mn-SOD, manganese-SOD; Cu,Zn-SOD, copper,zinc-SOD; Fe-SOD, iron-SOD; PTH, phenylthiohydantion; HPLC, high performance liquid chromatography. 2The abbreviations used are; SOD, superoxide dismutase; Mn-SOD, manganese-SOD; Cu,Zn-SOD, copper,zinc-SOD; Fe-SOD, iron-SOD; PTH, phenylthiohydantion; HPLC, high performance liquid chromatography. has a function to eliminate O⨪2 from the mitochondrial matrix space. However, O⨪2 can react with NO more than three times faster than with mitochondrial Mn-SOD (14Hsu J.-L. Hsieh Y. Tu C. O'Connor D. Nick H.S. Silverman D.N. J. Biol. Chem. 1996; 271: 17687-17691Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Therefore, if mitochondria are exposed to a relatively large amount of NO, a sufficient amount of peroxynitrite could be formed even in the presence of Mn-SOD. In this context, a clear understanding of the reactivity of mitochondrial Mn-SOD with peroxynitrite is necessary to clarify the mechanism of mitochondrial respiratory damage caused by NO. Ischiropoulos et al. (6Ischiropoulos H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1423) Google Scholar) and Smith et al. (15Smith C.D. Carson M. van der Woerd M. Chen J. Ischiropoulos H. Beckman J.S. Arch. Biochem. Biophys. 1992; 299: 350-355Crossref PubMed Scopus (70) Google Scholar) reported that bovine Cu,Zn-SOD reacts with peroxynitrite to form nitrotyrosine at Tyr-108 without inactivation of enzymatic activity. In contrast, Mn- and Fe-SOD from Escherichia coli are inactivated by peroxynitrite (6Ischiropoulos H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1423) Google Scholar). Nitration of tyrosine residues was also observed. MacMillan-Crow et al. (16MacMillan-Crow L.A. Crow J.P. Kerby J.D. Beckman J.S. Thompson J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11853-11858Crossref PubMed Scopus (717) Google Scholar) reported inactivation of Mn-SOD activity and concomitant increase of 3-nitrotyrosine in a tissue homogenate of transplanted allogripha during chronic rejection. They also reported that recombinant human Mn-SOD was inactivated by peroxynitrite with an IC50 of 10 μm and concomitant increase of nitration of tyrosine residues. However, the number of nitrated residues and an exact correlation between the nitration of tyrosine residue(s) and inactivation of human Mn-SOD has not been confirmed. Recently, we showed that nitration of one tyrosine residue of human Mn-SOD by peroxynitrite was linearly related to inactivation of human Mn-SOD by monitoring the absorption increase at 428 nm (17Yamakura F. Moncad S. Toda N. Maeda H. Higgs E.A. The Biology of Nitric Oxide. Portland Press, London1998: 34Google Scholar). In this study, we report that exclusive nitration of Tyr-34 is responsible for the inactivation of human Mn-SOD by peroxynitrite. This is the first example of peroxynitrite-mediated enzyme inactivation that is caused by nitration of a single specific amino acid. Peroxynitrite, synthesized by the reaction of sodium nitrite and hydrogen peroxide, was purchased from Dojindo Laboratory Co., Japan. Staphylococcal serine protease (EC 3.4.21.19) and lysylendopeptidase (EC 3.4.21.50) were purchased from Pierce and Wako Chemical Co., Japan, respectively. Human Mn-SOD was generously provided by Bio-Technology General (Israel) Ltd. The SOD preparation showed a specific activity of 2,684 ± 296 units/mg/mol of manganese/mol of subunit and metal contents of 0.81 ± 0.028 and 0.036 ± 0.02 g atom/mol of subunit for manganese and iron, respectively. This preparation lacked two amino acids from the N terminus (Lys-His-) and therefore had a calculated molecular weight of 21,939 (18Borgstahl G.E. Parge H.E. Hickey M.J. Beyer Jr., W.F. Hallewell Z.A. Tainer J.A. Cell. 1992; 71: 107-118Abstract Full Text PDF PubMed Scopus (422) Google Scholar). The reaction of peroxynitrite with SOD for structural studies was carried out as follows. 0.1-ml reaction mixtures containing 1 mg/ml of SODs in 150 mm potassium phosphate buffer, pH 7.4, with 0.1 mm diethylenetriamine pentaacetic acid and peroxynitrite at a concentration of 1 mm were incubated at 25 °C. For control experiments, peroxynitrite was added 1 min before the addition of SOD. The reaction mixtures were analyzed after a 2-min incubation. Superoxide dismutase activity was measured by the inhibition of cytochrome c reduction by xanthine oxidase (19McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar) with minor modifications (20Yamakura F. Rardin R.L. Petsko G.A. Ringe D. Hiraoka B.Y. Nakayama K. Fujimura T. Taka H. Kimie M. Eur. J. Biochem. 1998; (in press)PubMed Google Scholar). Metal contents were determined by using atomic absorption spectrometry with a Hitachi Z-9000 atomic absorption spectrophotometer. Amino acid analyses were performed with a Hitachi L-8500 amino acid analyzer, and the samples were hydrolyzed with 6m hydrochloric acid containing 2% thioglycolic acid for 10, 15, and 20 min at 165 °C. Native and peroxynitrite-inactivated Mn-SOD (5.8 nmol) were denatured in 150 μl of 67 mm ammonium acetate buffer, pH 4.0, at 95 °C for 10 min and then digested with staphylococcal serine protease (substrate:enzyme = 1:50, mol/mol) at 37 °C for 18 h. After freeze-drying the digested sample, 100 μl of 0.1% trifluoroacetic acid solution was added. The peptides were separated on a Jasco HPLC system with a ultraviolet/visible monitor (Tokyo, Japan). An octadecyl silica gel reverse-phase column, 4.6 × 250 mm (120T, TOHSO Co. Ltd., Tokyo, Japan), was used. Solvent A was 0.1% trifluoroacetic acid in ultra pure water (Milli-Q), and solvent B was 0.1% trifluoroacetic acid in 60% acetonitrile. The gradient program of the solvent was linear from 0–30% B in 7 min, 30–80% B in 60 min, and 80–100% B in 6 min with a flow rate of 1.0 ml/min. The ultraviolet-visible detector was used at 230 and 428 nm, respectively. The nine major peptides were collected and subjected to amino acid sequence analysis and molecular mass determination on a mass spectrometer. The amount of these peptides was estimated by the absorption at 210 nm (21Webster G.C. Biochim. Biophys. Acta. 1970; 207: 371-373Crossref PubMed Scopus (75) Google Scholar). The single peptides from each sample that had different elution positions were further digested with lysylendopeptidase (S:E = 150:1, mol/mol) in 10 mm Tris-HCl buffer at pH 9.5 at 35 °C for 14 h. The peptides resulting from this digestion were separated by the same method as above except that the gradient program of the solvent was linear from 0 to 100% B in 60 min. The single peptides from each sample that had different elution positions were collected and subjected to amino acid sequence and molecular mass analysis. The molecular weights of the native and peroxynitrite-inactivated Mn-SOD and protease-digested peptides were determined with a TSQ 700 electrospray ionization mass spectrometer (Thermo-Quest Finnigan Mat Co., Ltd., San Jose, CA). The analytical conditions were as follows; spray voltage, 4.5 kV; electron multiplier, 1500 V; manifold vacuum, 7.0 × 10−6; manifold temperature, 70 °C; capillary temperature, 150 °C; scan range, m/z500–3000; scan time, 5 s. Mn-SOD and the peptides were dissolved in a mixture of methanol and 0.5% acetic acid (1:1 v/v) to a final concentration of 10 pmol/μl and infused into the ion source of the TSQ 700 with a pump. Amino acid sequences of the peptides derived from Mn-SOD were determined by using an Applied Biosystems 477A gas liquid phase protein sequencer equipped with an on-line 120A PTH amino acid analyzer. PTH-3-nitrotyrosine was obtained by application of 3-nitrotyrosine into the PTH analyzer. Inactivation of recombinant human Mn-SOD by peroxynitrite shows different IC50 values dependent on the enzyme concentration, that is 0.43, 2.28, and 11.8 μmMn-SOD exhibits IC50 values of 6, 30, and 170 μm peroxynitrite, respectively, at pH 7.4 (17Yamakura F. Moncad S. Toda N. Maeda H. Higgs E.A. The Biology of Nitric Oxide. Portland Press, London1998: 34Google Scholar). For this structural analysis study, we used 11.8 μm Mn-SOD and 1 mm peroxynitrite at pH 7.4. Under these conditions, more than 90% of the activity was lost after a 2-min incubation. The inactivated enzyme still contained over 75% of the original content of manganese in the native enzyme and showed two bands that migrated a little faster than those of the native enzyme in acrylamide gel electrophoresis (data not shown). Fig. 1shows the results of mass spectrometry of the native and peroxynitrite-inactivated Mn-SOD. Deconvolution of the protein mass spectra (Fig. 1, A and B, insets) revealed that the molecular mass of the enzyme had increased by 46 Da (21,939 Da to 21,985 Da) after inactivation, consistent with the addition of a single nitro group into the inactivated enzyme. Deconvolution of the spectra also showed subpeaks corresponding to a molecular mass of 21,977 Da for the native enzyme and 22,023 Da for the inactivated enzyme. Each subpeak had an increase in molecular mass (38 Da) compared with the respective main peaks that corresponds to binding of potassium ion to each enzyme preparation. Amino acid analyses of the native and the inactivated enzymes showed a decrease of 0.81 ± 0.15 mol of tyrosine residue/mol of subunit in the inactivated Mn-SOD (average of three measurements). No significant differences in the composition of the other amino acids was observed between the two samples. From this evidence, we conclude that a single nitro group was introduced onto a tyrosine residue specifically by the reaction of peroxynitrite with Mn-SOD. After digestion of the native and peroxynitrite-inactivated Mn-SOD by staphylococcal serine protease, we separated nine major peptides by reverse-phase HPLC. Fig.2 shows a chromatogram of the peptides from the native (A) and peroxynitrite-inactivated (B) Mn-SOD. Among the nine major peptides separated, peptides 5 and 5′ had different elution positions between the two samples, which suggested that peptide 5′ was nitrated by peroxynitrite. A minor peak that has the same elution position as peak 5 in Fig.2 A also appears in Fig. 2 B. The ratio of the area between this peak and peak 5′ in Fig. 2 B is 17:83, which suggests that 83% of the intact peptide was converted to the modified peptide by the peroxynitrite treatment. Fig. 2 C shows the elution pattern of the peptides from the peroxynitrite-inactivated enzyme monitoring the absorption at 428 nm, which arises from 3-nitrotyrosine. Although a few very small peaks were observed other than the major peak in Fig. 2 C, the elution position of the major peak coincided with the position of peak 5′ in Fig.2 B. Molecular masses of 4,681 and 4,727 Da were obtained for the peptides from the native and inactivated enzymes, respectively. The difference in molecular mass (45 Da) corresponds to the molecular mass of a nitro group substituted onto a tyrosine residue. The same sequence of 23 amino acids from the N terminus was obtained for both peptides as Ser-Leu-Pro-Ala-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Ile-Asn-Ala-Gln-Ile-Met-Gln-Leu-, which are the same as the reported sequence from Ser-3 to Leu-25. These results suggest that the fifth peptide in both HPLC elutants corresponds to a peptide of 41 amino acids at Ser-3 to Glu-43 (S3LPALPYD10YGALEPHINA20QIMQLHHSKH30HAAYVNN L NV40TEE). The calculated molecular mass of the peptide from the native Mn-SOD (4,681 Da) was exactly the same as that obtained from the mass spectrometry of the peptide. Because each peak in Fig.2 B had the same molecular mass as corresponding peaks in Fig. 2 A (except for peaks 5 and 5′) and the whole amino acid sequence of the enzyme was covered by the peptides of the peak 1 (Lys-44–Glu-47, 567 Da), peak 6 (Ala-48–Glu-95, 4,969 Da), peak 7 (Ala-96–Glu-131, 3,963 Da), peak 9 (dimer of Arg-132–Glu-191, 13,897 Da), and peak 8 (heterodimer between Arg-132–Glu-191 and Arg-192–Lys-198, 7,847 Da), we conclude that no other amino acid was modified by peroxynitrite. The peak 3 in Fig. 2 (A andB) corresponded to the peptide Ala-96–Gln-109, which might be caused by the conversion of glutamine109 to glutamic acid during the course of the protease digestion or purification of the enzyme. To clarify the position of the modified tyrosine and identify the product of the modification, we further digested the peptide with lysylendopeptidase. The two peptides obtained from digestion of native and peroxynitrite-inactivated Mn-SOD were separated by reverse-phase HPLC (data not shown). The second peptides eluted at the same elution position, whereas the first peptides eluted at different elution positions. When the elution was monitored at 428 nm for the peroxynitrite-inactivated Mn-SOD sample, a single peak that had the same elution position as the first peptide of the same sample was obtained. The molecular masses of the first peptides of the samples from the native Mn-SOD and the peroxynitrite-inactivated Mn-SOD were 1,610 and 1,655 Da, respectively. The difference in molecular mass (45 Da) again corresponds to the mass of a nitro group substituted onto tyrosine. The same sequence from the N terminus was obtained for each peptide except for the fifth amino acid, that is His-His-Ala-Ala-(Tyr/Xaa)-Val-Asn-Asn-Leu-Asn-Val-Thr-Glu-Glu. This sequence corresponds to the peptide at His-30 to Glu-43, which has a calculated molecular mass of 1,610 Da. A tyrosine was obtained as the fifth amino acid for the peptide from the native Mn-SOD (Fig.3 A); however, a new peak that eluted at the same position as authentic 3-nitrotyrosine was found as the fifth amino acid from the N terminus for the peptide from the peroxynitrite-inactivated Mn-SOD as shown in Fig. 3 (B andC). From this evidence, we conclude that the single nitrated tyrosine in peroxynitrite-inactivated human Mn-SOD is Tyr-34. Peroxynitrite is known to attack many amino acids including cysteine (22Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), methionine (23Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Crossref PubMed Scopus (361) Google Scholar), tryptophan (24Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 93: 390-396Crossref Scopus (233) Google Scholar), phenylalanine (25Ramezanian M.S. Padmaja S. Koppenol W.H. Chem. Res. Toxicol. 1996; 93: 232-240Crossref Scopus (155) Google Scholar,26Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar), and tyrosine (25Ramezanian M.S. Padmaja S. Koppenol W.H. Chem. Res. Toxicol. 1996; 93: 232-240Crossref Scopus (155) Google Scholar, 26Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar). Although human Mn-SOD contains one methionine residue, two cysteine residues, six tryptophan residues, six phenylalanine residues, and nine tyrosine residues (18Borgstahl G.E. Parge H.E. Hickey M.J. Beyer Jr., W.F. Hallewell Z.A. Tainer J.A. Cell. 1992; 71: 107-118Abstract Full Text PDF PubMed Scopus (422) Google Scholar), only Tyr-34 was nitrated by peroxynitrite to cause inactivation of the enzyme. This tyrosine has a specific position in the three-dimensional structure of human Mn-SOD. Structural studies on human Mn-SOD (18Borgstahl G.E. Parge H.E. Hickey M.J. Beyer Jr., W.F. Hallewell Z.A. Tainer J.A. Cell. 1992; 71: 107-118Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 27Wagner U.G. Pattridge K.A. Ludwig M.L. Stallings W.C. Werber M.M. Oefner C. Frolow F. Sussman J.L. Protein Sci. 1993; 2: 814-825Crossref PubMed Scopus (57) Google Scholar), along with studies on other bacterial Fe-SODs (28Stoddard B.L. Howell P.L. Ringe D. Petsko G.A. Biochemistry. 1990; 29: 8885-8893Crossref PubMed Scopus (141) Google Scholar, 29Lah M.S. Dixon M.M. Pattridge K.A. Stallings W.C. Fee J.A. Ludwig M.L. Biochemistry. 1995; 34: 1646-1660Crossref PubMed Scopus (317) Google Scholar, 30Cooper J.B. McIntyre K. Badasso M.O. Wood S.P. Zhans Y. Garbe T.R. Youns D. J. Mol. Biol. 1995; 246: 531-544Crossref PubMed Scopus (89) Google Scholar, 31Lim J.-H. Yu Y.G. Han Y.S. Cho S.-J. Ahn B.-Y. Kim S.-H. Cho Y. J. Mol. Biol. 1997; 270: 259-274Crossref PubMed Scopus (117) Google Scholar), Mn-SODs (29Lah M.S. Dixon M.M. Pattridge K.A. Stallings W.C. Fee J.A. Ludwig M.L. Biochemistry. 1995; 34: 1646-1660Crossref PubMed Scopus (317) Google Scholar, 32Parker M.W. Blake C.F. J. Mol. Biol. 1988; 199: 649-661Crossref PubMed Scopus (154) Google Scholar, 33Ludwig M.L. Metzger A.L. Pattridge K.A. Stalling W.C. J. Mol. Biol. 1991; 219: 335-358Crossref PubMed Scopus (198) Google Scholar), and cambialistic SOD (34Schmidt M. Meier B. Parak F. J. Biol. Inorg. Chem. 1996; 1: 532-541Crossref Scopus (72) Google Scholar), show that Tyr-34 is conserved in all these SODs and that the phenolic oxygen atom of Tyr-34 is located 5.4 Å from manganese (18Borgstahl G.E. Parge H.E. Hickey M.J. Beyer Jr., W.F. Hallewell Z.A. Tainer J.A. Cell. 1992; 71: 107-118Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 27Wagner U.G. Pattridge K.A. Ludwig M.L. Stallings W.C. Werber M.M. Oefner C. Frolow F. Sussman J.L. Protein Sci. 1993; 2: 814-825Crossref PubMed Scopus (57) Google Scholar) at the vertex of the substrate funnels (18Borgstahl G.E. Parge H.E. Hickey M.J. Beyer Jr., W.F. Hallewell Z.A. Tainer J.A. Cell. 1992; 71: 107-118Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 29Lah M.S. Dixon M.M. Pattridge K.A. Stallings W.C. Fee J.A. Ludwig M.L. Biochemistry. 1995; 34: 1646-1660Crossref PubMed Scopus (317) Google Scholar). These funnels are proposed to lead the substrate, O⨪2, from the bulk solvent toward the metal ions by an electrostatic guidance mechanism (35Stalling W.C. Metzger A.L. Pattridge K.A. Fee J.A. Ludwig M.L. Free Radical Res. Commun. 1991; 12–13: 259-268Crossref Scopus (101) Google Scholar). Peroxynitrite may be introduced to manganese through the position of Tyr-34 by the same mechanism as O⨪2. Although we cannot clarify whether the active site manganese participates in the nitration reaction of Tyr-34 or not, we are in favor of the mechanism of a manganese-catalyzed nitration for the following reason: the proportion of nitrated tyrosine residue, which was estimated by the decrease of tyrosine contents in the amino acid analysis of the peroxynitrite-inactivated Mn-SOD (−0.80 mol/mol of subunit) and by the proportion of the new peak in the first HPLC (83%, Fig. 2 B), closely coincides with the manganese content of the native Mn-SOD (0.81 g atom/mol of subunit). Therefore, peroxynitrite may react with manganese to form an active nitrating species, probably nitronium ion (NO2+), similar to the case of Cu,Zn-SOD (6Ischiropoulos H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1423) Google Scholar, 26Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar). This cation then reacts with the adjacent tyrosine residue directly. We cannot rule out the possibility that the nitration of tyrosine 34 could take place without metal catalysis, because tyrosine 34 is located in the gateway of O⨪2 and may have a chance to react directly with peroxynitrite. Ramezanian et al. (25Ramezanian M.S. Padmaja S. Koppenol W.H. Chem. Res. Toxicol. 1996; 93: 232-240Crossref Scopus (155) Google Scholar) reported that peroxynitrite could nitrate tyrosine by the reactions catalyzed by metals or without metals. Recently Gow et al. (36Gow A. Duran D. Thom S.P. Ischiropoulos H. Arch. Biochem. Biophys. 1996; 333: 42-48Crossref PubMed Scopus (279) Google Scholar), Denicolaet al. (37Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (505) Google Scholar), and Lemercier et al. (38Lemercier J.-N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (110) Google Scholar) reported that peroxynitrite nitrates tyrosine and phenol by a mechanism that is catalyzed by carbon dioxide at physiological concentrations (36Gow A. Duran D. Thom S.P. Ischiropoulos H. Arch. Biochem. Biophys. 1996; 333: 42-48Crossref PubMed Scopus (279) Google Scholar, 37Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (505) Google Scholar) or at low concentrations such as an amount achieved by equilibrating with air in an aqueous reaction solution for nitration (38Lemercier J.-N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (110) Google Scholar). Further study is required to elucidate the mechanism of the nitration of Tyr-34 in human Mn-SOD. Because the nitrated enzyme gave only a slightly faster protein band compared with that of the native enzyme in polyacrylamide gel electrophoresis (data not shown) and retained over 75% of the original manganese contents, the nitrated enzyme may still have the same gross structure as the native enzyme. Therefore, a change in Tyr-34 function may more likely cause the inactivation of Mn-SOD than a change in the gross protein structure. Recent mutational studies on Tyr-34 ofE. coli Mn-SOD (39Whittaker M.M. Whittaker J.W. Biochemistry. 1997; 36: 8923-8931Crossref PubMed Scopus (107) Google Scholar) and Fe-SOD (40Hunter T. Ikebukuro K. Bannister W.H. Bannister J.V. Hunter G.J. Biochemistry. 1997; 36: 4925-4933Crossref PubMed Scopus (62) Google Scholar, 41Sorkin D.L. Duong D.K. Miller A.-F. Biochemistry. 1997; 36: 8202-8208Crossref PubMed Scopus (43) Google Scholar) proposed three major functions of Tyr-34 as follows. 1) Tyr-34 may not be essential for catalysis and may function to maintain the appropriate protonation state of the proton cluster, which is required to supply a proton in the second cycle of the dismutation reaction of the enzyme, consisting of Tyr-34, Gln-143, and coordinated solvent (Fig.4) (39Whittaker M.M. Whittaker J.W. Biochemistry. 1997; 36: 8923-8931Crossref PubMed Scopus (107) Google Scholar, 40Hunter T. Ikebukuro K. Bannister W.H. Bannister J.V. Hunter G.J. Biochemistry. 1997; 36: 4925-4933Crossref PubMed Scopus (62) Google Scholar, 41Sorkin D.L. Duong D.K. Miller A.-F. Biochemistry. 1997; 36: 8202-8208Crossref PubMed Scopus (43) Google Scholar). 2) Tyr-34 may be required to make a proper active site environment by forming a hydrogen bond network through Tyr-34, Gln-143, and the coordinated solvent (Fig. 4) (41Sorkin D.L. Duong D.K. Miller A.-F. Biochemistry. 1997; 36: 8202-8208Crossref PubMed Scopus (43) Google Scholar). 3) Tyr-34 may control the accessibility of both substrate and inhibitor molecule to the active site metal. Nitration of the Tyr-34 residue may result in a change of the pK a of the phenol group of the residue from around 10 to 7.5, which may cause deprotonation of the phenol group of the tyrosine residue and break the hydrogen bonding network of coordinated solvent Gln-143–Tyr-34 at physiological pH (Fig. 4). Because the substrate is believed to bind the active site metal through a position between His-30 and Tyr-34, the nitro group of the modified tyrosine could have a steric hindrance effect for access of the substrate to the active site. A combination of these effects originating from nitration of Tyr-34 may result in the loss of the enzymatic activity of human Mn-SOD by peroxynitrite. Finally, we can suggest a pathophysiological significance of the nitration of Tyr-34 of human Mn-SOD. Although physiological concentrations (<1 μm) of NO reversibly inhibit cytochrome oxidase in an oxygen concentration-dependent manner and therefore NO was proposed as a regulator of mitochondrial respiration under these conditions (12Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (674) Google Scholar, 42Takehara Y. Nakahara H. Inai Y. Yabuki M. Hamazaki K. Yoshioka T. Inoue M. Horton A.A. Utsumi K. Cell Struct. Funct. 1996; 21: 251-258Crossref PubMed Scopus (37) Google Scholar), a higher concentration of NO (5–200 μm) is proposed to cause mitochondrial dysfunction (13Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (608) Google Scholar). In the latter case, NO will compete with Mn-SOD for mitochondrially produced O⨪2, production of which may be enhanced by the inhibition of cytochrome oxidase with NO, to form peroxynitrite (13Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (608) Google Scholar). Nitration and inactivation of Mn-SOD by peroxynitrite could be a turning point in this mitochondrial dysfunction, because inactivation of Mn-SOD leads to accumulation of O⨪2 and as a consequence more accumulation of peroxynitrite. Both accumulated O⨪2 and peroxynitrite may cause oxidative damage of mitochondrial components. Nitration and inactivation of Mn-SOD was observed in a tissue homogenate of transplanted allogripha during chronic rejection (16MacMillan-Crow L.A. Crow J.P. Kerby J.D. Beckman J.S. Thompson J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11853-11858Crossref PubMed Scopus (717) Google Scholar). Therefore, the presence of Tyr-34-nitrated Mn-SOD could be a marker for NO- and/or peroxynitrite-induced mitochondrial dysfunction under some pathological conditions such as inflammation. To evaluate this possibility, a study of the exact correlation between the NO concentration-dependent nitration of Tyr-34 of Mn-SOD and dysfunction of mitochondrial respiration by using isolated mitochondria is progressing in our laboratory. We thank Dr. Moshe Werber of Bio-Technology General (Israel) Ltd. for kindly supplying human recombinant Mn-SOD. We thank Prof. Takashi Matsumoto for measurement of the metal contents by atomic spectrometry. We are pleased to thank Prof. Joseph S. Beckman for helpful discussions. We are also grateful to Dr. Joseph A. Gardner for critical manuscript review." @default.
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• W2024994588 title "Inactivation of Human Manganese-superoxide Dismutase by Peroxynitrite Is Caused by Exclusive Nitration of Tyrosine 34 to 3-Nitrotyrosine" @default.
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