Nitration and inactivation of tyrosine hydroxylase by peroxynitrite.

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.


Tyrosine hydroxylase (TH) is modified by nitration
However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr 423 to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 ؎ 0.9) ؋ 10 3 M ؊1 s ؊1 at pH 7.4 and 25°C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.
Tyrosine hydroxylase (TH) 1 (EC 1.14.16.2) is a non-heme iron, tetrahydrobiopterin-dependent protein that catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) and represents the rate-limiting step in the biosynthesis of catecholamines (1). Loss of ability to synthesize catecholamines is an important step in the development of Parkinson's disease (PD) and other neurodegenerative diseases (2)(3)(4)(5)(6). Early loss of TH activity followed by a decline in TH protein is thought to contribute to the dopamine deficiency and phenotypic expression in PD and the MPTP mouse model (4). Tyrosine hydroxylase is a selective target for nitration following administration of the parkinsonian toxin MPTP to mice and following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (7). Nitration of one or more tyrosine residues of TH was temporally associated with loss of enzymatic activity. The magnitude of inactivation was proportional to the number of TH molecules that were nitrated in PC12 cells. In the mouse striatum, the tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 h post-MPTP injection (7).
However, a recent report indicated that exposure of recombinant purified TH to peroxynitrite in vitro results not only in nitration of tyrosine residues but also in the formation of covalently linked dimers and oxidation of cysteine residues (8). The same report also indicated that cysteine oxidation rather than tyrosine nitration is responsible for the loss of TH enzymatic activity (8). Cysteine, methionine, tryptophan, and tyrosine appear to be the principal amino acids in proteins modified by peroxynitrite in vitro (9 -14). To resolve the apparent differences, the reaction of peroxynitrite with recombinant purified rat TH in vitro was re-examined, and no evidence of cysteine oxidation was found. Oxidation of one cysteine residue per molecule of TH was observed only at high peroxynitrite concentrations, and three cysteine residues were oxidized in partially unfolded protein. Amino acid analysis failed to show any * 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 1 The abbreviations used are: TH, tyrosine hydroxylase; DAMBI, 4-(dimethylamino)phenylazophenyl-4Ј-maleimide; L-DOPA, L-dihydroxyphenylalanine; DTNB, 5,5Ј-dithiobis(2-nitrobenzene); DTPA, diethylenetriaminepentaacetic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydrophenylpyridine; NEM, N-ethylmaleimide; PD, Parkinson's disease; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; OPA, ortho-phenyldialdehyde; CD, circular dichroism; GdmHCl, guanidinium hydrochloride. alteration of methionine, tryptophan, or any other amino acid residues. Digestion and sequence analysis of peptides indicated that nitration of tyrosine 423 is the primary residue modified by peroxynitrite, which was further confirmed by a significant decrease in nitration of the Tyr 423 3 Phe mutant TH expressed in Escherichia coli. In addition, no loss of TH enzymatic activity was detected after peroxynitrite treatment of the Tyr 423 3 Phe mutant TH. Stopped flow experiments revealed reactivity with the ferrous iron in TH typical of metalloproteins reacting with peroxynitrite (15)(16)(17). The absence of other amino acid modifications at low peroxynitrite concentrations suggests that nitration of tyrosine 423 is responsible for the inactivation of TH by peroxynitrite.

MATERIALS AND METHODS
Purification and Activity Assay of TH-Recombinant tyrosine hydroxylase was isolated from BL21(DE3)pLysS E. coli expressing the full-length cDNA clone isolated from a rat pheochromocytoma library (18). E. coli were grown in LB broth in the presence of 0.1 mM FeSO 4 to midlog phase and then induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for another 2 h. Addition of FeSO 4 is essential to retain functional protein, because in the absence of ferrous iron salt, only 60% of the purified GST-TH fusion contained metal (19). The E. coli pellets were suspended in a 10-fold excess of 0.05 M Tris-HCl, pH 7.2 (w/v), and homogenized according to the method of Wang et al. (20). This mixture was sonicated with four 30-s pulses at 20% power. After centrifugation, the supernatant was discarded and the pellet was resuspended in the same volume. The mixture was then sonicated with ten 30-s pulses. The supernatant was collected by centrifugation and removed. The pellet was resuspended and sonicated once again. The combined supernatants were precipitated with ammonium sulfate. The fraction between 30 and 42% was purified further by column chromatography on heparin-Sepharose CL-6B. Tyrosine hydroxylase was eluted with a KCl gradient (0 -0.7 M) in 0.05 M phosphate buffer, pH 6.5. The purity of peak fractions was evaluated by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue and was found to be greater than 95%.
Tyrosine hydroxylase was assayed by the release of [ 3 H]H 2 O from [ 3 H]tyrosine in the presence of catalase (21,22). Approximately 5 g of purified tyrosine hydroxylase was assayed in a volume of 30 l containing the following additions: 50 mM MES, pH 5.5, 1 mM 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (Alexis), 1500 units/ml catalase, 5 mM dithiothreitol, 5 mM FeSO 4 , 0.10 mM tyrosine, 500,000 dpm of L-[ring-3,5-3 H]tyrosine (specific activity 50 Ci/mmol, PerkinElmer Life Sciences). The reaction was incubated for 2 min at 37°C. At the end of the incubation, 300 l of a cold suspension containing 7.5% activated charcoal in 1 N HCl was added to each tube. After vortexing and centrifugation, 100 l of supernatant was counted by liquid scintillation spectrometry. Blanks contained buffer instead of enzyme.
Treatment of TH with Peroxynitrite-Peroxynitrite was synthesized from sodium nitrite and acidified hydrogen peroxide, as previously described (23), and treated with manganese dioxide to remove residual hydrogen peroxide. The concentration of peroxynitrite was determined spectrophotometrically by the measurement of absorption at 302 nm in 1 N NaOH (⑀ ϭ 1670 M Ϫ1 .cm Ϫ1 ). Dilutions in 0.1 N NaOH were made immediately before use to achieve the desired concentrations.
The protein was first dialyzed against 50 mM phosphate buffer at pH 7.4, and protein concentration was determined by the Bradford assay. Tyrosine hydroxylase was then diluted to a final concentration of 18 M in 0.1 M phosphate buffer containing 0.1 mM DTPA, pH 7.4, and finally treated with one bolus of 25-500 M peroxynitrite (1% v/v) at room temperature while stirring. In reverse order experiments, peroxynitrite was added to the buffer before TH addition.
Analysis of TH Sulfhydryl Content-The effect of peroxynitrite on TH sulfhydryl content was determined by ThioGlo-1 assay (24), by 5,5Јdithiobis(2-nitrobenzene) (DTNB) reaction (25) and by labeling with 4-(dimethylamino)phenylazophenyl-4Ј-maleimide (DAMBI) (26). A 26 mM solution of ThioGlo-1 (Covalent Associates Inc., MA) was prepared in acetonitrile. The protein (0.2 M) was incubated with 7 M guanidine HCl overnight in phosphate buffer, 50 mM, pH 7.4, before ThioGlo-1 addition. ThioGlo-1 (10 M) was added to the denatured TH, and the incubation was continued for another 7 h at room temperature. The reaction was monitored by fluorescence spectroscopy with the excitation wavelength set at 384 nm and the emission at 513 nm. The emission values of a series of N-acetyl-L-cysteine standards modified with Thio-Glo-1 were used to establish a standard curve of fluorescence.
Amino Acid Analysis-Amino acid analysis of TH was performed by hydrolyzing the protein either in the gas phase (HCl) or with methanesulfonic acid. Gas phase acid hydrolysis was achieved with 6 N HCl and 1% (w/v) phenol under vacuum (ϳ40 millitorr) at 115°C for 22 h, as described by Meltzer et al. (27). Protein hydrolysates were analyzed by reverse-phase HPLC (Varian 9012) on an Inertsil C18 ODS3 column (250 ϫ 4.6 mm) using pre-column derivatization with phenylisothiocyanate (PITC) and absorbance detection at 254 nm (Varian 9050). Samples were dissolved in mobile phase, injected onto the column equilibrated with 10 mM potassium phosphate buffer, pH 6.5 (phase A) at 40°C, and eluted at 1 ml/min by an increase of mobile phase B (75% acetonitrile/25% phase A) to 11% at 15 min, to 14% at 17 min, to 36% at 39 min, and to 45% at 48 min. Amino acids were quantified using the chromatographic peak area, compared with both an amino acid standard (Pierce), and hydrolysis of a known concentration of bovine serum albumin hydrolyzed under similar conditions. Methane sulfonic acid hydrolysis of protein samples was used for the determination of tryptophan, methionine, and methionine sulfoxide, which are usually unstable under conditions of HCl hydrolysis. Samples were hydrolyzed with 4 M methane sulfonic acid at 115°C for 22 h in an evacuated chamber (ϳ40 millitorr). After hydrolysis, samples were neutralized by the addition of an equal volume of 3.5 M NaOH. They were analyzed by reverse-phase HPLC on a Supelcosil LC-18T column (150 ϫ 4.6 mm), following pre-column derivatization with ortho-phenyldialdehyde (OPA), and fluorescence detection (330 and 450 nm, excitation and emission wavelength, respectively) as described previously (28). Samples were injected onto the column equilibrated at 35°C with mobile phase A, containing 95% 25 mM sodium acetate buffer (pH 5.8) and 5% tetrahydrofuran, and eluted at 0.7 ml/min by the following gradient of mobile phase B (95% methanol and 5% tetrahydrofuran): 0 -15% B between 0 and 0.5 min, 15-45% B from 10 to 20 min, and 45-100% B between 30 and 40 min. Amino acids were quantified as described above for the PITC method. Circular Dichroism (CD)-CD spectra were obtained using a Jasco J720 instrument at room temperature in a 0.2-mm path length cell with the concentration of protein being ϳ0.2 mg/ml. Curves were baselinecorrected and smoothed by the algorithm provided by Jasco. Mean residue ellipticity, [⌰] MR , is expressed in deg ϫ cm 2 /dmol using mean residue masses of 110.
Analysis of 3-Nitrotyrosine Content-After treatment with peroxynitrite, TH (0.5 g) was subjected to a 10%-SDS-polyacrylamide gel electrophoresis, transferred overnight to nitrocellulose membrane, and probed with either a polyclonal anti-TH (Calbiochem) or an affinitypurified polyclonal anti-3-nitrotyrosine antibody. The blots were blocked with 10% dry milk in Tris-buffered saline containing 0.5% Tween 20 for 1 h, prior to antibody addition. The polyclonal anti-TH antibody was used at 1:10,000 dilution in 1% dry milk for 2 h, and the anti-3-nitrotyrosine antibody was used at 1:5000 dilution for 3 h. The anti-3-nitrotyrosine antibody was pre-conjugated with the secondary antibody in 3% dry milk overnight at 4°C. After several washings, probed membranes were incubated with the horseradish peroxidaseconjugated secondary antibody at 1:5000 dilution. After washing, immunoreactive signals were revealed using a chemiluminescence assay (ECL kit, Amersham Pharmacia Biotech). Samples containing untreated or peroxynitrite-treated TH were incubated with freshly prepared solution of Streptomyces griseus protease (Pronase, Sigma Chemical Co.) to yield 0.1 mg of protease/mg of protein. Pronase-treated samples were incubated for 18 h at 50°C and centrifuged through a 10,000 molecular weight cut-off filter. Samples were then dried down, re-suspended in 40 l of water of HPLC quality, and frozen at Ϫ80°C until analysis. HPLC with electrochemical detection was performed on an ESA model 5600 CoulArray instrument equipped with eight-channel detector operating in the oxidative mode at specified potentials (channel/potential (in mV) ϭ 1, 400; 2, 450; 3, 500; 4, 640; 5, 660; 6, 680; 7, 810; and 8, 810). Analyte separation was conducted on a TOSOHAAS (Montgomeryville, PA) reverse-phase ODS 80-T M C-18 analytical column (4.6-mm inner diameter ϫ 25 cm; 5-m particle size) using isocratic elution consisted of 50 mM sodium acetate, 50 mM citric acid, pH 4.7, with 10% methanol. The concentration of 3-nitrotyrosine was determined from the area under the curve using a standard curve of authentic 3-nitrotyrosine from the seventh electrode.
Stopped-flow Experiments-The kinetics of peroxynitrite reaction with TH were studied with or without removing the iron from TH. Accordingly, the same preparation of the enzyme was divided in two parts. One fraction was incubated with ferrous ammonium sulfate (1 mM) for 60 min at 4°C to obtain the holoenzyme with full complement of iron, and the other was dialyzed against 1,10-phenantroline (1 mM) in 100 mM potassium phosphate buffer, pH 7.35 at 4°C, to obtain the apoenzyme (29). Moreover, fractions of the TH holoenzyme were incu-bated either with ␤-mercaptoethanol (10 mM) or with N-ethylmaleimide (NEM, 10 mM) for 30 min at 4°C. Enzyme preparations were then extensively dialyzed against 100 mM potassium phosphate buffer, pH 7.35 at 4°C. Protein concentration was determined by measuring the absorbance at 280 nm (A 1% ϭ 10.4 cm Ϫ1 ) (29), which correlated well with Bradford protein measurements. The cysteine content in the enzyme was determined by the reaction of 4 M protein with 0.2 mM of 5,5Ј-dithiobis(2-nitrobenzene) (DTNB) in the absence or presence of 6 M guanidine at pH 7.8 and room temperature for 90 min. The cysteine concentration was determined by the absorbance at 412 nm (⑀ ϭ 13,600 M Ϫ1 ⅐cm Ϫ1 ) (25). The rate constant at 37°C could not be evaluated due to protein aggregation, which significantly interfered with the absorbance determinations.
The kinetics of peroxynitrite decay in the presence of TH at pH 7.4 and 25°C were followed in a stopped-flow spectrophotometer (Applied Photophysics, SF17MV) at 302 nm. An initial rate approach was used to analyze the data, the first 1 s was fitted to a linear plot, and the apparent rate constant of peroxynitrite decay was determined as the ratio between the slope (ϪdA/dt) and the difference between the initial and final absorbance (A o Ϫ A f ). After subtracting the apparent rate constant of peroxynitrite decay in the absence of enzyme and dividing by the enzyme concentration, we determined the second order rate constant of peroxynitrite reaction with the enzyme. To assure the accuracy of the determination, 200 absorbance measurements were acquired during the first 1 s of the reaction, and 200 points were acquired until more than 99.9% peroxynitrite had decomposed (1-20 s) (13,30).
Identification of the Nitrated Tyrosine Residue(s)-Un-reacted control or nitrated TH (18 M) was exhaustively dialyzed against 100 mM ammonium bicarbonate, pH 7.8. The TH samples were then treated with endoproteinase AspN from Pseudomonas fragi (Calbiochem, La Jolla, CA) or sequencing grade-modified trypsin (Promega, WI) at a ratio 1:100 w/w (proteinase/TH) overnight at 37°C. The samples were dried down and resuspended in 0.1% trifluoroacetic acid. The peptides were analyzed by a Hewlett Packard HPLC system with a diode array detector using an octadecyl silica gel reverse-phase column (5 m, 4.6 ϫ 250 mm, Jupiter, Phenomenex, Torrance, CA) and 0.1% trifluoroacetic acid in ultra pure water (solvent A) and acetonitrile as solvent B. Peptides were eluted using an increasing linear gradient of B from 0% to 45% in 60 min with a flow rate of 1 ml/min. The HPLC detector was set at 210, 275, and 365 nm. The peptides with absorbance at 365 nm were collected and subjected to conventional Edman degradation at the Protein Core Facility at the Wistar Institute (Philadelphia, PA).
Expression of Mutant TH and Treatment with Peroxynitrite-To elucidate a possible role for Tyr 423 as a substrate for nitration, the mutant Tyr 423 3 Phe were created with the QuikChange site-directed mutagenesis kit from Stratagene. Wild type TH (25 ng) was used as the DNA template to create Tyr 423 3 Phe. The conditions utilized are outlined in previous studies (31,32). The primers used (but not previously reported) were as follows: TH Y423F Sense, 5Ј-GCA GCT GTG CAG CCC TTC CAA GAT CAA ACC TAC C-3Ј; Antisense, 5Ј-G GTA GGT TTG ATC TTG GAA GGG CTG CAC AGC TGC-3Ј. The underlined nucleotides highlight the mutated codons for amino acid 423. The DNA Sequencing and Gene Analysis Facility of the Molecular Genetics Program (Wake Forest University School of Medicine) performed a complete DNA sequencing by using a PerkinElmer Life Sciences/Applied Biosystems 377 Prism automated DNA sequencer. Complete DNA sequencing was done to verify the presence of the appropriate mutation in the coding sequences of all recombinant proteins. This also established that the non-polymerase chain reaction-based mutagenesis (Tyr 423 3 Phe) did not introduce extraneous mutations. All recombinant proteins were expressed using the approach detailed above for the wild type TH. After ammonium sulfate precipitation, the partially purified wild type and Tyr 423

RESULTS
Lack of Cysteine Oxidation after Exposure of TH to Peroxynitrite-Purified recombinant rat TH was reacted with different concentrations of peroxynitrite, and fractions of the treated and control protein were analyzed for activity and for the concentration of reduced cysteine residues. Exposure to peroxynitrite resulted in a dose-dependent inactivation of TH activity ( Table  I). The concentration of cysteine residues in TH was determined by three different methods: ThioGlo-1, a maleimidederivatized naphthopyranone, 5,5Ј-dithiobis(2-nitrobenzene) (DTNB), and 4-(dimethylamino)phenylazophenyl-4Ј-maleimide (DAMBI) labeling (24 -26). Data in Table I show that in the unreacted protein seven cysteine residues are detected per TH monomer as predicted from the rat TH sequence. No evidence for cysteine oxidation was observed after treatment with up to 250 M peroxynitrite whereas the TH enzymatic activity was already inhibited by more than 50%. With higher peroxynitrite concentration (500 M), one cysteine residue per monomer is oxidized. The same results are obtained with DTNB and DAMBI labeling (not shown). These data suggest that TH activity was inhibited after exposure to peroxynitrite without evidence for oxidation of cysteine residues. Moreover, in addition to cysteine oxidation, the reaction of proteins in simple buffers with peroxynitrite has been shown to modify methionine, tryptophan, and tyrosine residues (10 -14). Hydrolysis using methane sulfonic acid to analyze tryptophan and methionine as well as methionine sulfoxide failed to show any evidence for tryptophan and methionine oxidation (Table II). Complete amino acid analysis of peroxynitrite exposed TH, using gas phase HCl hydrolysis and PITC pre-column derivatization, did not detect any significant modification of any other amino acid residue (data not shown).
Peroxynitrite-induced Cysteine Oxidation in Partially Unfolded Tyrosine Hydroxylase-Tyrosine hydroxylase was partially unfolded with either 7 M urea or 6 M of guanidinium hydrochloride (GdmHCl) for 1 h at room temperature before peroxynitrite treatment. Exposure of the urea or GdmHCl partially unfolded protein to 500 M peroxynitrite resulted in the oxidation of approximately three cysteine residues as 4.1 Ϯ 1 and 3.2 Ϯ 0.4 cysteine residues are detected in the reacted protein, respectively. Another fraction of the protein was analyzed by Western blotting with anti-TH antibodies, which revealed formation of SDS and heat-stable TH polymers (Fig.  1A). The polymerized TH was hydrolyzed, and the hydrolysates were monitored by fluorescence ( ex ϭ 283 nm and em ϭ 410 nm) to determine the presence of dityrosine as reported previously (33). The hydrolysates of TH treated with 6 M GdmHCl and reacted with 500 M peroxynitrite contain 1 Ϯ 0.3 M dityrosine indicating that one tyrosine residue in TH was oxidized to form dityrosine. The degree of cysteine and tyrosine oxidation in the unfolded protein was proportional to the peroxynitrite concentration (Fig. 1B). There was a significant degree of TH unfolding upon treatment with GdmHCl as revealed by the changes in the CD spectrum of the protein; the spectrum of the untreated protein and protein reacted with 1 M GdmHCl is shown in Fig. 2. Computer-assisted analysis of the CD spectra for the composition of different secondary structural elements indicated the presence of ϳ58% ␣-helical conformation in the monomeric TH similar to values reported previously (34,35). The ␣-helical content of TH decreased to 11% upon Gdm-HCl treatment with a concomitant increase in random structure from 30 to 54%. Collectively these data suggested that the degree of cysteine oxidation and the formation of stable dityrosine-containing oligomers appeared to be facilitated by the unfolding of the protein.
Tyrosine Nitration following Exposure of TH to Peroxynitrite-Exposure of TH to a range of peroxynitrite concentrations induced a dose-dependent increase in the nitration of the protein, which correlated with the loss of enzymatic function (Fig. 3). The proportional loss of TH activity and nitration is similar to the data described in PC12 cells exposed to peroxynitrite (7). Assuming that one tyrosine residue is nitrated per TH molecule, then exposure of TH at 1, 2, and 3 mg/ml to 100 M peroxynitrite resulted in the nitration of 59, 22, and 20% TH molecules, which correlated with 52 Ϯ 0.1, 29 Ϯ 2, and 18 Ϯ 1.3% loss of TH enzymatic activity, respectively.
Identification of the Nitrated Tyrosine Residues-To identify the site of nitration, TH reacted with peroxynitrite was digested with either AspN or trypsin. The AspN-digested peptides were separated by reverse-phase HPLC, and two major peaks with absorbance at 275 and 365 nm eluted at retention times of 29.1 and 47.7 were collected and sequence analysis of the first 20 amino acids was performed. The following sequence, Asp-Thr-Ala-Ala-Val-Gln-Pro-X-Gln (X represents an unknown amino acid) was obtained for the 29.1-min peak. This sequence corresponds to the expected AspN peptide residues 416 -424 (Asp 416 -Thr-Ala-Ala-Val-Gln-Pro-Tyr-Gln 424 ), which contains a tyrosine residue in position 423. The peak with a retention time of 47.7 min contained two sequences, the major sequence (more than 90% abundance of amino acid residues) corresponding to peptide between residues 44 and 77, which does not contain any tyrosine residues. The minor sequence (less than 10%) was X-Gln-Thr-X-Gln-Pro-Val-X-Phe-Val-Ser-Glu-Ser-Phe, which corresponds to residues 425-439 (Asp 425 -Gln-Thr-Tyr-Gln-Pro-Val-Tyr-Phe-Val-Ser-Glu-Ser-Phe-Asn 439 ), which includes tyrosine residues 428 and 432. The site of nitration was also confirmed by digestion of nitrated TH with trypsin. The first 15-amino acid sequence of a peak eluted at 73 min of the tryptic digestion was Ala-Phe-Asp-Pro-Asp-Thr-Ala-Ala-Val-Gln-Pro-X-Gln-Asp-Gln, which corresponds to the expected peptide spanning residues 412 to 442 that includes tyrosine residues 423, 428, and 432. Collectively, these data suggest that the primary site of nitration is tyrosine residue 423 and to a lesser extent tyrosine residues 428 and 432. To further confirm the site of nitration, Tyr 423 was mu-  1. Polymerization and cysteine oxidation of partially unfolded TH. Tyrosine hydroxylase was treated with urea or guanidinium hydrochloride (GdmHCl) for 1 h at room temperature, centrifuged on a 10,000 molecular weight filter and diluted (18 M) in 0.1 M phosphate buffer containing 0.1 mM DTPA, pH 7.4. A, the native and the partially unfolded TH was then reacted with 500 M peroxynitrite and analyzed by Western blot analysis, using anti-TH antibodies. Lanes 1, 3, and 5 are control, urea-treated, and GdmHCl-treated protein; lanes 2, 4, and 6 are control, urea-treated, and GdmHCl-treated protein reacted with peroxynitrite. The inset table gives the cysteine content. B, tyrosine hydroxylase treated with 6 M GdmHCl was reacted with different concentrations of peroxynitrite and the degree of protein crosslinking and cysteine oxidation was evaluated as in A.

FIG. 2. CD spectrum of TH (1) and of TH-treated with 1 M GdmHCl (2).
The spectrum of the untreated control protein is identical to published spectra of TH (34), and treatment with GdmHCl significantly alters the secondary structure of the protein. The cysteine content after exposure to 100 M peroxynitrite was 6.2 Ϯ 0.8 and 2.3 Ϯ 0.5 for the control and the GdmHCl-treated protein, respectively. tated to Phe in TH, and the sequence of the mutant TH (Tyr 423 3 Phe) expressed in E. coli was confirmed. Wild type and mutant proteins expressed in E. coli were partially purified and exposed to peroxynitrite under identical conditions. Fig. 4 shows that there was less nitration of the partially purified Tyr 423 3 Phe mutant as compared with the wild type protein under the same protein and peroxynitrite concentrations. However, the activity of the Tyr 423 3 Phe mutant TH was not affected by nitration as compared with the wild type protein.
The activity of the Tyr 423 3 Phe mutant TH, similar to the wild type was decreased by Ͼ99% after heat inactivation or removal of the ferrous iron for the active site (Table III). In the semipurified preparation the K m for tyrosine was 37.7 Ϯ 2.1 M for the wild type and 14.3 Ϯ 2.6 M for the Tyr 423 3 Phe mutant TH, which are similar to values reported for purified rat TH (34). The cysteine residues in the wild type and Tyr 423 3 Phe mutant TH appear to be equally reactive toward the alkylating agent N-ethylmaleimide (NEM). Treatment of the wild type and mutant proteins with NEM resulted in a significant inhibition of activity as shown in Table III. Moreover, NEM treatment also inhibited the activity of both the wild type and Tyr 423 3 Phe mutant TH proteins that had been reacted with peroxynitrite prior to NEM exposure (Table III). Collectively these data suggest that the cysteine residues were not modified by peroxynitrite exposure and that nitration of Tyr 423 is the principal reason for the loss of enzymatic activity.
Determination of the Second Order Rate Constant for the Reaction of Peroxynitrite with Tyrosine Hydroxylase-Stoppedflow experiments were performed using TH treated either with ferrous ammonium sulfate to ensure full complement of iron (holoenzyme), or with 1,10-phenantroline, to remove the iron (apoenzyme). Peroxynitrite decomposition profiles at 302 nm were obtained by mixing peroxynitrite (75 M) with 20 M of either the holoenzyme or the apoenzyme. The second order rate constant determined for the holoenzyme was (3.8 Ϯ 0.9) ϫ 10 3 M Ϫ1 s Ϫ1 and (1.6 Ϯ 0.8) ϫ 10 3 M Ϫ1 s Ϫ1 for the apoenzyme, at pH 7.45 and 25 Ϯ 1°C. Moreover, no differences were detected between the second order rate constants obtained for TH reacted with mercaptoethanol, which had 5.3 Ϯ 0.2 cysteine residues per monomer, and TH treated with NEM, which had 1.06 Ϯ 0.05 cysteine residue per monomer (data not shown).
These data suggest that the contribution of the cysteine residues in the reaction of peroxynitrite with TH is relatively small, and the second order rate constants are typical of the reactions of peroxynitrite with metal centers (15)(16)(17)30). DISCUSSION Tyrosine hydroxylase is the rate-limiting step in catecholamine synthesis, and thus the activity of this protein is critical for maintaining dopamine production. Inactivation of TH has been observed in early stages of PD as well as the mouse MPTP model of this disease (4). In the MPTP model of Parkinson's disease (6) previous data revealed that TH is a protein specifically modified by nitration of tyrosine(s) residues. In addition, a temporal association between the number of TH molecules modified and loss of activity was observed (7). Amino acid analysis and fluorescence spectrometry of purified TH had failed to detect any other amino acid modification after nitration of the protein, and thus we proposed that tyrosine nitration is responsible for the inactivation of the protein (7). However, a publication by Kuhn and co-workers (8) indicated that cysteine oxidation and not tyrosine nitration was responsible for the inactivation of purified TH by peroxynitrite in vitro. Although cysteine residues are well-recognized targets for peroxynitrite (9,13,26), this study failed to detect oxidation of cysteine residues after exposure of purified recombinant rat TH to peroxynitrite as determined by three independent methods. Cysteine oxidation was evident only after exposure of the protein to large excess of peroxynitrite or when the protein is partially unfolded (Table I and Figs. 1 and 2). It is possible that the protein used by Khun et al. (8) was partially unfolded during purification or removal of the glutathione S-transferase (GST) tag. It has been reported that nearly 40% of TH purified with the GST tag has no metal in the active site (19), and we have observed that metal free TH apoprotein readily aggregates after dialysis and during storage suggesting some degree of protein unfolding (not shown). Therefore, partial protein unfolding and exposure to high peroxynitrite concentration can account for the oxidation of cysteine residues reported previously (8). Consistent with the observation of Khun et al. (8) we also observed formation of SDS and heat-stable TH dimers after exposure to relative high peroxynitrite concentrations and in partially unfolded protein before and after exposure to peroxynitrite (Fig. 1). The dimers appear to be the result of cross-linking via oxidation of tyrosine residues, because dityrosine was detected in the unfolded and peroxynitrite treated TH. Dityrosine cross-linking has been reported for proteins FIG. 4. Exposure of the partially purified wild type and Tyr 423 3 Phe mutant TH to peroxynitrite. A, wild type and Tyr 423 3 Phe mutant TH analysis by Western blot using a polyclonal anti-TH antibody. B, wild type and Tyr 423 3 Phe mutant TH analysis by Western blot using an anti-3-nitrotyrosine antibody, after treatment with a range of peroxynitrite concentrations. C, TH enzymatic activity (mean and standard deviation for three different preparations of TH) as a function of peroxynitrite concentration. with random secondary structure in solution (33) or globular proteins exposed to high peroxynitrite concentrations (36). Although protein cross-linking may be also responsible for the inactivation of TH, appreciable inactivation of the enzymatic activity is observed in the absence of protein dimers (Fig. 3) and other amino acid modifications suggesting that under these conditions tyrosine nitration is responsible for the loss of function (Tables I and II). Peroxynitrite reactivity with proteins in simple buffers is determined by kinetic factors. The stopped-flow data suggest that the contribution of the ferrous iron in the reaction of peroxynitrite with TH is quite important, accounting for nearly 60% of the rate constant. The rate constant of the apoenzyme is typical of peroxynitrite reaction with protein amino acids (13) but precludes the existence of rapidly reacting cysteine residues, similar to those present in glyceraldehyde-3-phosphate dehydrogenase (37) and peroxiredoxins (38). Regarding the polypeptide chain, cysteine, methionine, and tryptophan are the primary amino acids that react with appreciable second order rate constants with peroxynitrite (13). In the case of TH, these amino acids were poorly oxidized after peroxynitrite exposure, similar to data reported previously for sarcoplasmic reticulum Ca-ATPase (39). Instead, at low peroxynitrite concentration, only tyrosine nitration was observed. At high peroxynitrite concentration, tyrosine nitration and oxidation were observed, which could be derived by the formation of hydroxyl and nitrogen dioxide radicals from peroxynitrite or by direct electrophilic substitution at the ortho position of the aromatic ring catalyzed by transition metals. However, the role of the ferrous iron of tyrosine hydroxylase in the nitration of the protein awaits further investigation.
The primary site of nitration was identified by digestion of the nitrated protein and peptide sequencing and further confirmed by mutational analysis (Fig. 4). The primary site of nitration was the tyrosine residue 423 and to a lesser extent tyrosine residues 428 and 432. Mutation of Tyr 423 to Phe resulted in an appreciable decrease in tyrosine nitration and, more importantly, no loss of activity was observed after exposure to peroxynitrite. The specific activity of partially purified Tyr 423 3 Phe mutant was ϳ20 -25% of the wild type protein suggesting that this tyrosine residue is critical for the enzymatic activity of the protein. However, similar to wild type protein, the mutant TH activity is completely lost upon heat inactivation and removal of the metal. Kinetic parameters such as the K Tyr of the Tyr 423 3 Phe mutant were similar to the wild type, and the cysteine residues of both the wild type and Tyr 423 3 Phe mutant were sensitive to alkylation by NEM before or after treatment with peroxynitrite. Therefore, nitration of tyrosine 423 and not cysteine oxidation appears to be responsible for the inactivation of TH by peroxynitrite. However, it remains unclear how nitration of tyrosine residue 423 results in the inactivation of the protein. We speculate that it may relate to the critical positioning of this tyrosine residue near the active site of the protein. The active site of TH is located in the center of the catalytic domain (residues 188 -456) and consists of a 17-Å deep cleft. The active site cleft is 30 Å long and 15 Å wide, and within the active site 10 Å below the enzyme surface His 331 , His 336 , and Glu 376 residues bind ferrous iron needed for the catalysis of tyrosine hydroxylation to L-DOPA (35). The entrance to the active site is guarded by two loops (residues 423-428 and 290 -296), which come within 12 Å of each other. Proline residues in either side of the loop break the ␣-helices, and Tyr 423 starts the five-residue loop that ends with Tyr 428 . The aromatic ring of Tyr 423 is oriented toward the opposite loop on the plane of the entrance to the active site whereas the aromatic rings of Tyr 428 and Tyr 432 are oriented away from the entrance to the active site. The narrow point entering the active site can be viewed as a size selection process by which only small substrates may enter into the active site. The addition of the bulky nitro (NO 2 ) in the ortho position of the tyrosine 423, but not the tyrosine residues 428 and 432, will narrow the distance between the two loops by more than 2.5 Å, which may be sufficient to prevent the entry of the substrate tyrosine in the active site of the protein.
Overall, the data reported herein are consistent with the view that tyrosine nitration is responsible for the inactivation of TH. It is now apparent that nitration and oxidation of proteins is a widespread event in the affected areas in the brain of PD patients (40 -43) as well as in the mouse and baboon MPTP models of this disease (2,7,45). More importantly, specific proteins such as TH and ␣-synuclein that may play a key role in the pathogenesis of PD are targets for modification by nitrating agents (7,42). Efforts to limit the formation of nitrating agents by limiting the production of nitric oxide and superoxide have been successful in protecting mice and baboons from MPTP-induced neuronal death (45)(46)(47). Recently, Pong et al. (48) showed that EUK 134, a superoxide dismutase and catalase mimetic, prevented nitration of TH in cultured dopaminergic neurons after 1-methyl-4-phenylpyridinium challenge. Therefore, development of therapeutic agents that can prevent formation of nitrating agents without interfering with normal neuronal function or compounds that will specifically remove nitrating species may protect proteins from inactivation and provide means of limiting neuronal injury in PD.