Peroxynitrite-induced Nitration of Tyrosine Hydroxylase IDENTIFICATION OF TYROSINES 423, 428, AND 432 AS SITES OF MODIFICATION BY MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY AND TYROSINE-SCANNING MUTAGENESIS*

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption timeof-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of 45, 90, and 135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30–60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7–30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr, Tyr, and Tyr as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues. Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine. TH is inhibited by the dopamine neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in PC12 cells and in mice after in vivo treatment (1), suggesting that losses in TH activity that are seen in this model of Parkinson’s disease may occur early in the process of dopamine neuronal degeneration. The mechanisms by which 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine damages dopamine neurons are complex and are thought to involve, at least in part, the production of peroxynitrite (ONOO ) (2). TH is inhibited by ONOO in vitro (1, 3) and in PC12 cells (4). The ONOO -induced inhibition of TH is associated with nitration of tyrosine residues (1) and oxidation of cysteine residues (3), yet neither the identity of the modified residues in TH nor the relative contribution of these posttranslational modifications to loss of catalytic function is known. Ischiropoulos and colleagues (1) first concluded that Tyr was the site in TH of ONOO -induced nitration and attributed enzyme inhibition to this posttranslational modification. A more recent paper from the same group purports that Tyr, not Tyr, is the actual site mediating enzyme inactivation after nitration by ONOO (5). A Y423F mutant of TH was extensively nitrated by high concentrations of ONOO , but its catalytic activity was not inhibited (5). ONOO is a powerful oxidant that can modify cysteine, tryptophan, methionine, and tyrosine residues in proteins. It can also cause lipid peroxidation and DNA damage and lead to mitochondrial dysfunction, effects that contribute to its cytotoxic potential (6–10). Indeed, the ONOO -induced nitration of free tyrosine or of tyrosine residues in proteins is used increasingly as a molecular marker of ONOO participation in conditions that are associated with cell damage or disease states (11–15). Increased tyrosine nitration of proteins, including the Lewy body constituent -synuclein (16–18), in post-mortem tissue from individuals with Parkinson’s disease (19, 20) suggests that ONOO -induced tyrosine nitration plays a causative role in dopamine neuronal degeneration and in TH dysfunction. Based on the importance of TH to dopamine neuronal function, and considering the possibility that ONOO causes the inhibition of TH as an early event in the process of dopamine * This work was supported by National Institute on Drug Abuse Grant DA 10756, the Joe Young, Sr. Psychiatric Research Fund of the Department of Psychiatry and Behavioral Neurosciences, and a Veterans Affairs Merit Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: 2125 Scott Hall, 540 E. Canfield, Wayne State University School of Medicine, Detroit, MI 48201. Tel. /Fax: 313-577-9737; E-mail: donald.kuhn@wayne.edu. 1 The abbreviations used are: TH, tyrosine hydroxylase; ONOO , peroxynitrite; PEO, polyethylene oxide; DTNB, 5,5 -dithiobis-2-nitrobenzoic acid; pCMB, p-chloromercuribenzoic acid, PMAB, PEO-maleimide-activated biotin; BIAM, N-biotinoyl-N-(iodoacetyl)ethylene diamine; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption-ionization; TOF, time-of-flight; ELISA, enzyme-linked immunosorbent assay; MS, mass spectrometry. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 16, Issue of April 19, pp. 14336–14342, 2002 Printed in U.S.A.