Archives of Biochemistry and Biophysics 484 (2009) 134–145

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Archives of Biochemistry and Biophysics

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The effect of neighboring methionine residue on tyrosine nitration and oxidationin peptides treated with MPO, H2O2, and NO2

� or peroxynitrite and bicarbonate:Role of intramolecular electron transfer mechanism?

Hao Zhang, Jacek Zielonka, Adam Sikora, Joy Joseph, Yingkai Xu, B. Kalyanaraman *

Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, P. O. Box 26509, Milwaukee, WI 53226, USA

a r t i c l e i n f o

Article history:Received 1 October 2008and in revised form 12 November 2008Available online 24 November 2008

Keywords:NitrotyrosineReactive nitrogen speciesElectron transferSpin-trappingEPRRadiolysis

0003-9861/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.abb.2008.11.018

* Corresponding author. Fax: +1 414 456 6512.E-mail address: balarama@mcw.edu (B. Kalyanara

a b s t r a c t

Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site andspecificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects ofmethionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H2O2 and NO2

� andperoxynitrite (ONOO�) or ONOO� and bicarbonate (HCO3

�) in model peptides, tyrosylmethionine(YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides wereanalyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediateswere identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown(Zhang et al., J. Biol. Chem. 280 (2005) 40684–40698) that oxidation and nitration of tyrosyl residuewas inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show thatmethionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighbor-ing tyrosine residue in the presence of ONOO� (or ONOOCO2

�) or MPO/H2O2/NO2� system. Nitration of

tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO�

(formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF,YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteineresidues have been attributed to differences in the direction of intramolecular electron transfer in thesepeptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysisexperiments. Potential implications of the intramolecular electron transfer mechanism in mediatingselective nitration of protein tyrosyl groups are discussed.

� 2008 Elsevier Inc. All rights reserved.

Introduction mental factors (hydrophobicity, CO levels, membrane oxygen con-

Emerging research strongly supports the role of pro-inflamma-tory, reactive oxygen/nitrogen species (ROS/RNS) in cardiovascular,pulmonary, and neurodegenerative diseases [1–9]. Post-transla-tional nitrative modifications of proteins and lipids have beenidentified as biomarkers for RNS in diseases [9–12]. These post-translational modifications include nitration of tyrosyl and lipidmoieties. Two major pathways are responsible for protein and lipidnitration. These include heme peroxidases (e.g., myeloperoxidase,eosinophil peroxidase) using nitrite and H2O2 as substrates and/or peroxynitrite and bicarbonate [13–19]. In addition, the interac-tion between peroxynitrite and metal ions (including heme pro-teins) may act as a biologically relevant source of nitratingspecies [20].

Factors influencing nitration of tyrosyl residues in protein arenot fully known. Several studies have shown that tyrosine nitrationis a selective process that is controlled by various microenviron-

ll rights reserved.

man).

2

centration, acidic environment and amino acid sequence) [21–29].Although no specific amino acid sequence criteria exist for predict-ing tyrosine nitration or lack thereof, it has been suggested thatprotein tyrosyl nitration is (i) enhanced when a tyrosyl moiety issituated closer to a negatively-charged amino acid (i.e., glutamateor aspartate) and (ii) decreased when the tyrosyl residue is presentin the vicinity of a cysteinyl or methionine residue [12,29,30]. De-tailed quantitative analyses of the effects of methionine residue ontyrosyl nitration are, however, lacking. Understanding the biophys-ical/biochemical mechanisms that determine the motif for nitra-tion-sensitive tyrosine residues is clearly pathophysiologicallysignificant [31].

Methionine, along with cysteine and tryptophan, represents oneof the most easily oxidized natural amino acids [32]. It is knownthat methionine can be directly oxidized by peroxynitrite via a rad-ical and non-radical mechanism, leading to the formation of ethyl-ene and methionine sulfoxide as a biologically relevant product[33–36]. Early on, it was suggested that the presence of a methio-nine residue adjacent to tyrosine in peptides and proteins will re-sult in decreased tyrosyl nitration [12,28,30] due to methionine’s

Fig. 1. Structures of peptides and their oxidation and nitration products.

H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145 135

ability to react with peroxynitrite and radicals derived from it [37–43]. However, recent proteomic studies have yielded conflicting re-sults [44–47]. In some cases, the neighboring methionine residueinhibited tyrosine nitration [44,45]; whereas, in other studies,nitrotyrosine formation was enhanced in the proximity of a methi-onine residue [46,47].

To understand how a neighboring methionine residue can influ-ence tyrosyl nitration, we used a model system consisting of sim-ple dipeptides (e.g., tyrosylmethionine (YM)1, YF, etc. (Fig. 1)) andmonitored their nitration/oxidation products in the presence of mye-loperoxidase (MPO), H2O2 and NO2

� or ONOO� and HCO3�. We have

also monitored the products of one-electron oxidation of the pep-tides by Br2

�� or CO3�� using a combination of radiolysis and photo-

lysis techniques. Results indicate that, contrary to the other sulfur-containing amino acid cysteine, methionine did not inhibit tyrosinenitration in the dipeptide. In case of SIN-1 (slow in situ generators ofONOO�), we actually observed stimulation of tyrosine nitration/oxi-dation in YM peptide as compared to YF, YC or tyrosine. This isattributed to a possible intramolecular electron transfer betweenthe methionine sulfide radical cation and the tyrosyl residue result-ing in increased oxidation and nitration of tyrosine. The influence ofintramolecular electron transfer mechanism in protein nitrationreactions is also discussed.

1 Abbreviations used: YM, N-acetyl tyrosylmethionine amide; MYYM, bis-N-acetyltyrosylmethionine amide; Y(NO2)M, N-acetyl nitrotyrosylmethionine; YF, N-acetyltyrosylphenylalanine amide; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid;DMPO, 5,50–dimethyl-1-pyrroline N-oxide; DTPA, diethylenetriaminepentaaceticacid; EPR, electron paramagnetic resonance; MMTS, methylmethanethiosulfonate;MPO, myeloperoxidase; �NO2, nitrogen-di-oxide radical;. NO2Tyr, 3-nitrotyrosine; Tyr,tyrosine; YC, N-acetyl tyrosylcysteine amide; YCysCysY, disulfide formed from YC,FYYF, bis-N-acetyl tyrosylphenylalanine amide; YM=O, N-acetyl tyrosylmethioninesulfoxide amide. Recommended IUPAC nomenclature: NaHCO3, sodium hydrogencarbonate; ONOO�, oxoperoxonitrate (1�); ONOOH, hydrogen oxoperoxonitrate;�NO2, dioxidonitrogen(�); CO3

��, trioxidocarbonate(�1�).

Experimental

The following chemicals and enzymes were purchased from dif-ferent sources as indicated: tyrosine, hydrogen peroxide, sodiumnitrite, sodium bicarbonate, 3-nitrotyrosine, and D,L-methioninewere from Sigma; myeloperoxidase was from CalBiochem. Rinkamide methylbenzhydrylamine (MBHA) resin and Fmoc-protectedamino acids were from Calbiochem-Novabiochem (LaJolla). Di-iso-propylcarbodiimide (DIC), 1-hydroxybezotriazole (HOBt), triiso-propylsilane (TIS), methylmethane-thiosulfonate (MMTS), N-methyl-pyrrolidinone (NMP) and piperidine were purchased fromFisher Scientific. Peroxynitrite was synthesized in-house as re-ported previously [48].

Peptide synthesis and purification

The structures of the peptides used in this study are shown inFig. 1. Peptides were chemically synthesized using the standardFmoc solid phase peptide based synthetic procedure on an Ad-vanced Chemtech Model 90 synthesizer (Louisville, Ky) [29]. RinkAmide MBHA resin (loading 0.72 mmol/g) was used as a solid sup-port. Fmoc-protected amino acids were coupled as HOBt-esters. Allamino acids were double-coupled using HOBt/DIC. The followingsteps were performed in the reaction vessel for each double-cou-pling: deprotection of the Fmoc group with 20% piperidine inNMP for 30 min (twice), three NMP washes, two dichloromethane(DCM) washes, first coupling for 1 h with a five-fold excess of Fmocamino acid in 0.5 M HOBt and 0.5 M DIC, second coupling using afresh addition of the same reagent for 1 h, followed by three NMPwashes, and two DCM washes. Final acetylation was performedusing an acetic anhydride/HOBt/DIC mixture for 30 min (twice).The resin was washed twice with DCM, three times with methanol,and then dried at room temperature under vacuum prior to cleav-age. The peptide was deprotected and cleaved from the resin with90% TFA containing TIS for 3 h at room temperature. The resinwas removed by filtration, washed with TFA, and the combinedTFA filtrates were evaporated to dryness under a stream of dry N2

gas. The oily residue was washed three times with cold ether andthe dry crude peptide was dissolved in acetonitrile/H2O (1:1) andlyophilized. The crude peptides were purified by a semi-preparativereverse phase-HPLC on a RP-C18 (10 mm� 250 mm) column usinga CH3CN/water gradient (5–25% CH3CN over 60 min) containing0.1% TFA at a flow rate of 3 ml/min with UV absorption detectionat 280 nm. The identity of YM peptide was verified by its UV absorp-tion spectrum and ESI-MS (M + H+: 356).

Synthesis of nitrated YM

Nitrated YM peptide (Y(NO2)M) was prepared as follows: TheYM peptide (1 mM) was mixed with MPO/H2O2 and NO2

� in aphosphate buffer (100 mM, pH 7.4) containing DTPA (100 lM)for 20 min. The reaction was terminated by adding catalase(100 U/ml). The resulting Y(NO2)M peptide was purified using asemi-preparative HPLC. Nitrated peptides show characteristicUV–Vis absorption spectra. Upon adding NaOH, the absorptionpeak at 350 nm was shifted to 430 nm. The structure of Y(NO2)Mwas verified by LC-MS spectrometry on an Agilent 1100 seriesLC-MS (M + H+; 401). An extinction coefficient of 4100 M�1 cm�1

(pH > 11) at 430 nm was used for quantitation of Y(NO2)M.

Synthesis of YM sulfoxide

The peptide YM (1 mM) was mixed with H2O2 (2 mM) in phos-phate buffer (100 mM, pH 7.4) containing DTPA (1 mM) at roomtemperature for 12 h. The resulting YM sulfoxide was purified byHPLC and its identity was further verified by ESI-MS (M + H+; 372).

A

B

C

YM

YM +MPO/H2O2

YM +MPO/H2O2/nitrite

YM

YM +MPO/H2O2

YM +MPO/H2O2/nitrite

(NO2)YM (20 μM)

YM

YM +MPO/H2O2

YM +MPO/H2O2/nitrite

MYYM (1 μM)

YM MYYM (NO2)YMYM(S=O)

MYYM

uk

Time (min)

Time (min)Time (min)

Time (min)

0 5 10 15 20

0 5 10 15 20

0 5 10 15 20

Fig. 2. HPLC analyses of products formed from tyrosylmethionine in MPO/H2O2/NO2

� system. YM (250 lM) was incubated with MPO (30 nM), H2O2 (100 lM) andNaNO2 (500 lM) for 30 min at room temperature. The reaction was terminated byadding catalase and the resulting mixture was analyzed by HPLC using differentdetection conditions. (A) YM and (NO2)YM were detected as major components at9.5 and 18 min using the HPLC/UV detection at 280 nm. (B) Major peak due to(NO2)YM was detected at 18 min using a detection wavelength of 350 nm. (C) Thedityrosyl product, MetTyrTyrMet, was detected as a major product using the HPLC-fluorescence mode (kmax = 290 nm, kmin = 410 nm). In addition, there was anunidentified peak (uk).

136 H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145

Synthesis of carbonato tetrammine cobalt(III) perchlorate

Carbonato tetrammine cobalt(III) perchlorate [Co(NH3)4CO3]-ClO4 was synthesized in two steps, as described elsewhere: (1) syn-thesis of [Co(NH3)4CO3]Cl [49] and (2) exchange of chloride anionwith perchlorate anion [50].

Nitration and oxidation of YM peptide induced by MPO/H2O2/NO2�

Typically, peptides (250 lM) were incubated with NaNO2

(500 lM), H2O2 (100 lM) and MPO (30 nM) in a phosphate buffer(100 mM, pH 7.4) containing DTPA (100 lM) at room temperaturefor 30 min. Reactions were terminated by adding catalase (200 U/ml) enzyme and directly analyzed by HPLC. Repeated injectionsin 24 h showed no significant oxidation of YM under these exper-imental conditions.

Nitration and oxidation of YM peptide induced by peroxynitrte

YM (250 lM) was rapidly mixed with with peroxynitrite (20–500 lM) in a phosphate buffer (100 mM, pH 7.4) containing DTPA(100 lM) and incubated at room temperature for 10 min, and ana-lyzed by HPLC. In control experiments peroxynitrite was firstadded to the buffer followed by the addition of YM after 10 min.

One-electron oxidation of peptides by dibromine radical anion

The peptides (100 lM) were incubated with KBr (100 mM) inphosphate buffer (50 mM, pH 7.4) saturated with N2O. Dibromineradical anion (Br2

��) was generated in situ by radiolysis of solutionscontained in HPLC vials using X-rays (dose rate of 3 Gy/min) [51].Under these conditions Br2

�� was the major species responsible forpeptide consumption. After irradiation, samples were analyzed byHPLC.

One-electron oxidation of peptides by carbonate radical anion

The peptides (100 lM) were incubated with phosphate buffer(50 mM, pH 7.4) in aqueous solution containing 4 mM[Co(NH3)4CO3]ClO4. The carbonate radical anion (CO3

��) was gener-ated in situ by irradiation of the samples placed in quartz cuvetteswith UV light [50,51]. Photolysis was carried out using a 300 W Xehigh pressure compact arc lamp (Eimac Model VIX 300 UV). Thelight was passed through a water filter prior to sample irradiation.Under those conditions CO3

��was the major species responsible forpeptide consumption. After photolysis the samples have been ana-lyzed by HPLC.

HPLC analyses of nitration and oxidation products

Typically, a 20 ll of sample was injected into a HPLC system(HP1100) equipped with C-18 column (250 mm� 2.0 mm) equili-brated with a 5% CH3CN in 0.1% TFA. Products were separated bya linear increase of CH3CN concentration to 25% in 60 min at a flowrate of 0.2 ml/min. The elution was monitored using the on-lineUV–Vis absorption and fluorescence detectors. YM, dityrosyl YMand nitrated YM were eluted at 9.5, 17 and 18 min, respectively.

Nitration of tyrosine with or without methionine by MPO/H2O2/NaNO2 or peroxynitrite was performed under the same conditionsand the products analyzed as reported previously [29].

EPR spin-trapping experiments

Incubations consisting of a peptide or tyrosine (1 mM), MPO(150 nM), and DBNBS (20 mM) in a phosphate buffer (100 mM,pH 7.4) containing DTPA (100 lM) were rapidly mixed with H2O2

(1 mM). Samples were subsequently transferred to a 100 ll capil-lary tube and ESR spectra were taken within 30 s after startingthe reaction in a Bruker EMX spectrometer. Typical spectrometerparameters were: scan range, 100 G; field set, 3505 G; time con-stant, 0.64 ms; scan time, 10 s; modulation amplitude, 2.0 G; mod-ulation frequency, 100 kHz; receiver gain, 5 � 105; and microwavepower, 20 mW. The spectra shown were the average of 30 scans.

Results

MPO/H2O2/NaNO2-dependent oxidation and nitration oftyrosylmethionine

The effects of a methionine residue on tyrosine nitration andoxidation were determined by measuring the MPO/H2O2/NaNO2-dependent nitration/oxidation of YM peptides (Fig. 2).

HPLC with UV–Vis absorption detection at 280 nm for tyrosylresidues and at 350 nm for nitro tyrosyl residues were employed.The HPLC/fluorescence (kexc = 290 nm, kemi = 410 nm) was used for

H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145 137

detecting the dityrosyl product of YM (Fig. 2A–C). HPLC productanalyses of incubations containing YM (250 lM), MPO (30 nM),H2O2 (100 lM), DTPA (100 lM) and NaNO2 (500 lM) in a phos-phate buffer (100 mM, pH 7.4) for 30 min at room temperatureshowed a significant depletion of YM peptide and formation ofa new major product (retention time, 18 min). Based on its UVabsorption maximum (k = 350 nm) and its pH-dependent changes,this new product was assigned to a methionyl nitrotyrosine((NO2)YM). Additional evidence in favor of this structural assign-ment was obtained from the ESI-MS analysis of this product(M + H+; 401). Another minor product (retention time, 17 min)was also detected by fluorescence that was assigned to a dityrosylproduct (Fig. 2A and C). This structural assignment was furtherconfirmed by the ESI/MS analysis (M + H+; 709). Incubations ofYM (250 lM) and MPO (30 nM), H2O2 (100 lM) in the absenceof NaNO2 also yielded the same dityrosyl product (Fig. 2A). In thiscase, an additional product was detected (retention time, 5 min)which was assigned to an YM sulfoxide structure on the basisof MS analysis. From these results, we conclude that only thetyrosyl residue but not the methionine residue undergoes nitra-tion and oxidation induced in the MPO/H2O2/NO2

�/YM system.This is in stark contrast to oxidation of YC peptides in MPO/H2O2/NO2

� which yielded a corresponding disulfide (i.e., YCCY)

0

50

100

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200

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300

Control H2O2 H2O2+

MPO

H2O2+

MPO+NO2-

YF

(μM

)

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Dit

yros

ine

(μM

)

0

4

8

12

16

MPO+H2O2+ NO2-

MPO+H2O2

A B

C D

p<0.01

p<0.005

Fig. 3. Substrate depletion and product formation from oxidation/nitration of dipeptidesMPO (30 nM), H2O2 (100 lM) and NaNO2 (500 lM) in a phosphate buffer (100 mM, pHterminated by adding catalase (200 U/ml) and reaction mixtures analyzed by HPLC usivarious incubating conditions. (B) Same as (A) except that YF was substituted by YM. (CMPO/H2O2/NO2

� oxidation. The incubation conditions are as indicated in (A) and (B). (DNO2

� mediated nitration. Experimental conditions are as in (A) and (B).

as a major product with little or no nitrated product formation[29].

As MPO/H2O2/NaNO2-dependent nitration/oxidation of YM pep-tide yielded exclusively tyrosyl nitration and oxidation products,we compared the product profiles with another dipeptide, tyrosine-phenylalanine (YF). Incubation of YF with MPO/H2O2/NaNO2

� re-sulted in the depletion of YF at nearly the same rate as that ofYM (Fig. 3A and B). Incubation of YF or YM with hydrogen peroxidealone showed no significant depletion of these peptides, whichindicated that direct oxidation of YM or YF peptide by H2O2 is neg-ligible. As with YM, incubation of YF with MPO/H2O2 also resultedin its oxidation. However, the extent of depletion of YM peptide inboth systems is the same as that of YF, suggesting that the rate ofoxidation of both dipeptides by MPO is similar. Under similar con-ditions, the dityrosine yields of YM (i.e., MYYM) induced by MPO/H2O2 or MPO/H2O2/NaNO2 were significantly higher than those ofYF (FYYF) (Fig. 3C). Nitration of YM by MPO/H2O2/NaNO2 wasslightly increased as compared to nitration of YF (Fig. 3D). Theseresults clearly demonstrate that tyrosine nitration and oxidationinduced by MPO/H2O2/NO2

� system was not inhibited by a neigh-boring methionine residue.

To further understand the effect of methionine on tyrosyl oxida-tion/nitration by MPO/H2O2/NO2

�, we incubated tyrosine (250 lM)

0

50

100

150

200

250

300

Control H2O2 H2O2+

MPO

H2O2+

MPO+NO2-

YM

(μM

)

YF YM

NO

2Tyr

(μM

)

0

30

60

90

120

150

180

in in MPO/H2O2/NO2� system. (A) The dipeptide, YF (250 lM), was incubated with

7.4) containing DTPA (100 lM) for 30 min at room temperature. The reaction wasng conditions described in Fig. 2. Bar graphs show the concentrations of YF under) The concentrations of dipeptides obtained from YF and YM during MPO/H2O2 and) The concentrations of nitrotyrosines obtained from YF and YM during MPO/H2O2/

Tyr (mM) 0.25 0.25 0.25Met(mM) 0 0.25 1.00

Tyr

osin

e(%

of

Con

trol

)

020

406080100120

Tyr (mM) 0.25 0.25 0.25Met (mM) 0 0.25 1.00

Dit

yros

ine

(% o

f C

ontr

ol)

020

406080100120

Tyr (mM) 0.25 0.25 0.25Met (mM) 0 0.25 1.00

(NO

2)T

yr(%

of

Con

trol

)

0

20

40

60

80

100

120

A

B

C

Fig. 4. The effect of methionine on tyrosine oxidation/nitration in MPO/H2O2/NO2�

system. (A) Incubation mixtures contained Tyr (250 lM), MPO (30 nM), H2O2

(100 lM), DTPA (100 lM) and varying amounts of Met for 30 min at roomtemperature. Graphs show Tyr concentrations measured after the incubationperiod. (B) Incubation conditions are as in (A). Graphs show dityrosine levelsmeasured after 30 min of incubation. (C) Same as in (A) except that nitrotyrosinewas measured following the incubation period.

138 H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145

with MPO, H2O2 and various amounts of methionine (0, 0.25 and1 mM) and observed that oxidation of tyrosine to dityrosine byMPO/H2O2 was not affected by methionine (Fig 4A). This indicatesthat methionine did not react with the tyrosyl radicals formed inthis system (Fig. 4A). In addition, nitration of tyrosine by MPO/H2O2/NaNO2 was also not affected by methionine (Fig. 4C), sug-gesting that the nitrogen-dioxide radical (�NO2) formed in this sys-tem reacts with tyrosine more rapidly than with methionine.Collectively, these results show that the exogenously-addedmethionine does not affect tyrosyl nitration or oxidation in MPO/H2O2/NO2

� system.

Peroxynitrite-dependent oxidation and nitration of tyrosylmethioninepeptide

Fig. 5A (left) shows the HPLC traces obtained from an incuba-tion mixture containing YM (250 lM), DTPA (100 lM), peroxyni-trite (100 lM), and bicarbonate (25 mM) in a phosphate buffer(100 mM, pH 7.4). HPLC traces were recorded 10 min after theaddition of peroxynitrite. Predominant products were (NO2)YM(eluting at 18 min with absorption at 280 and 350 nm) and MYYM(the dityrosyl product detected using fluorescence parameters:kexc = 290 nm, kemi = 410 nm) (Fig. 5A, middle and right). Incuba-tion of YM (250 lM) with different amounts of peroxynitriteshowed a dose-dependent increase in formation of the nitratedproduct (Fig. 5B) and the corresponding dityrosyl product (MYYM)(Fig. 5C).

As with MPO/H2O2/NO2� system, we compared the yields of

nitration/oxidation products of YM peptide with those obtainedfrom YF peptide and with tyrosine treated with peroxynitrite(Fig. 6). Oxidation of YM (250 lM) and Y (250 lM) with 100 lMperoxynitrite yielded 10.5 ± 1.2 lM of (NO2)YM and 11.1 ± 0.8 lMof Y(NO2) and treatment of FY with peroxynitrite yielded6.3 ± 0.8 lM of (NO2)YF (Fig. 6A). Under these conditions, higheryields of MYYM (1.19 ± 0.08 lM) were obtained as compared toFYYF (0.61 ± 0.05 lM) and YY (0.78 ± 0.06 lM). As it is well-knownthat bicarbonate can enhance the nitration yields of tyrosine in-duced by peroxynitrite through intermediate formation of carbon-ate radical and nitrogen-dioxide radical [19,21,27,32,52], weinvestigated the effects of bicarbonate on nitration and oxidationproducts of YM. Incubation of YM (250 lM) with 100 lM peroxyni-trite in the presence of 25 mM bicarbonate yielded 16.8 ± 0.7 lM of(NO2)YM similar to those formed from YF and tyrosine (Fig. 6A).However, under the same conditions, the amount of dityrosylproduct formed from YM (MYYM) was significantly higher thanthe dityrosyl products (YY and FYYF) formed from oxidation of Yor YF peptide (Fig. 6B).

To better understand the effects of methionine on peroxyni-trite-induced tyrosine nitration under physiologically-relevantconditions, we used SIN-1 that simultaneously generated equalamounts of �NO and superoxide. SIN-1 has been widely used tosimulate the in vivo system, in which peroxynitrite is formed byreaction of �NO with O2

��. Incubation of YM (250 lM) with SIN-1(250 lM) yielded the corresponding nitration product, (NO2)YM,in a much higher yield as compared to that formed from tyrosineor YF (Fig. 7A). There was a 4-fold and 7-fold increase in the forma-tion of (NO2)YM and MYYM as compared to Y(NO2) and YY(Fig. 7B). Similar results were obtained in the presence of bicarbon-ate. In agreement with literature reports, the nitration efficiency ofthe tyrosyl residues was much lower in the case of SIN-1 (Fig. 7Aand B) than in the case of bolus peroxynitrite (Fig. 6) or slowly in-fused (5 lM/min) peroxynitrite (data not shown). From these stud-ies, we conclude that a neighboring methionine residue actuallyenhances the nitration and oxidation of the adjacent tyrosyl groupin the presence of in situ-generated peroxynitrite with or withoutbicarbonate.

Previous data suggest that peroxynitrite is scavenged by methi-onine [38]. We investigated the effects of free methionine on tyro-sine nitration and oxidation induced by peroxynitrite (Fig. 8A–D).In agreement with literature data, addition of methionine dose-dependently inhibited tyrosine oxidation and nitration inducedby peroxynitrite (Fig. 8C and D). Methionine also inhibited thenitration and oxidation of tyrosine in the presence of bicarbonate(Fig. 8A and B). Thus, these results clearly indicate the differentialeffects of free methionine and the peptidyl methionyl residue ontyrosyl nitration. Whereas exogenously-added methionine inhib-ited peroxynitrite-mediated nitration of tyrosine, the methionylresidue in YM dipeptide did not inhibit nitration of adjacent tyro-sine but in fact stimulated nitration of adjacent tyrosyl group ascompared to YF. This is a potentially new finding.

One-electron oxidation of YM and other peptides

To investigate the mechanism of the nitration/oxidation oftyrosyl residue in YM peptide, we performed steady-state radiol-ysis and photolysis techniques to generate short-lived oxidizingradicals. We used dibromine radical anion (Br2

��) as a one-elec-tron oxidant (Figs 1S–4S) [51]. In the case of YM peptide(Fig. 1S) it is likely that the methionine residue is the primary siteof attack, as the rate constant of the reaction of methionine withBr2�� (k = 2 � 109 M�1 s�1) is two orders of magnitude higher than

the reaction of Br2�� with tyrosine (k = 2 � 107 M�1 s�1). Although

Time (min)0 5 10 15 20

Time (min)0 5 10 15 20

YM

(NO2)YM

(NO2)YM

MYYMMYYM

Peroxynitrite0 200 400 600 800 1000

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MY

YM

(µ

M)

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M (

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MYYM (1 μM)YM

YM/ONOO_/CO2

(NO2)YM (20 μM)

Time (min)0 5 10 15 20

Time (min)0 5 10 15 200 5 10 15 20

Fig. 5. HPLC ananlysis of nitration and oxidation products formed from tyrosylmethionine and peroxynitrite and bicarbonate (A) YM (250 lM) was incubated withperoxynitrite (100 lM) in a phosphate buffer (pH 7.4, 100 mM) containing DTPA (100 lM) for 10 min and analyzed by HPLC. (bottom trace) Incubations contained YM aloneand (top trace) Incubations contained YM, peroxynitrite, and bicarbonate (25 mM). (B) Dose-dependent formation of nitrated YM under incubation conditions shown in (A).(C) Dose-dependent formation of dityrosine formed from YM.

H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145 139

the primary site of reaction is a methionyl residue, the final prod-uct of the reaction is dityrosyl tetrapeptide (MYYM) (Fig. 1S), indi-cating a fast intramolecular electron transfer from tyrosyl residueto the one-electron oxidized methionine moiety. MYYM peptideas the major product was also detected in the presence of otherone-electron oxidants generated radiolytically, including N3

� and(SCN)2

�� or the carbonate radical (CO3��) generated by photolysis

of the [Co(NH3)4CO3]+ complex (Fig. 2S). Those radicals are ex-pected to oxidize mostly the tyrosyl residue (with N3

�) or bothresidues to a similar extent (with (SCN)2

�� and CO3��); however,

in all cases the corresponding dityrosyl peptide was the majorproduct, again supporting the role of intramolecular electrontransfer from tyrosine to the one-electron oxidized methionine.The dityrosine-type peptide has been observed as a major productalso in the case of tyrosine and YF peptide, when oxidized by Br2

��

(Fig. 3S and 5S), N3�, (SCN)2

�� and CO3�� (Fig. 4S and 6S). On the

other hand, the one-electron oxidation of YC peptide leads exclu-sively to a disulfide product, regardless of the oxidant used(Fig. 7S and 8S). With Br2

�� (Fig. 7S) it is likely that the primarysite of attack in YC peptide is the cysteinyl residue, as the rateconstant of the reaction of cysteine with Br2

�� is an order of mag-nitude higher (k = 1.8 � 108 M�1 s�1) than of the reaction of tyro-sine with Br2

�� (see above). Thus the disulfide product could beexpected in the absence of an intramolecular electron transfermechanism. On the other hand, in the case of azidyl radical(N3

�), the primary site of attack is expected to be the tyrosyl res-idue, as the rate constant of the reaction of N3

� with cysteine(k = 1.4 � 107 M�1 s�1) is an order of magnitude lower than ofthe reaction with tyrosine (k = 1.0 � 108 M�1 s�1). As the finalproduct is also a disulfide, the occurrence of an intramolecularelectron transfer from the cysteinyl moiety to the tyrosyl radicalis plausible. This conclusion is in agreement with the observedprotective effect of the cysteinyl residue on tyrosyl nitration inYC-type peptides [29].

EPR spin-trapping of radical intermediates

To further understand the paradoxical differences between theexogenously-added free methionine and the endogenously presentmethionyl residue on tyrosyl nitration, we used the EPR spin-trap-ping to detect radical intermediates formed in MPO/H2O2/NO2

�

and peroxynitrite/bicarbonate systems. Incubations containingtyrosine and MPO/H2O2 in the presence of DBNBS yielded athree-line EPR spectrum (Fig. 9A). This spectrum was assigned toa DBNBS-tyrosyl adduct (aN = 13.6 G) [53]. In the absence ofMPO, the DBNBS adduct formation was minimal (Fig. 9B). Additionof methionine (1 and 5 mM) had no effect on the DBNBS-tyrosyladduct formation (Fig. 9D and E), suggesting that methionine didnot effectively inhibit the tyrosyl radical formation. The DBNBS ad-duct was also minimal in incubations containing methionine(1 mM), MPO (150 nM), DBNBS (25 mM), H2O2 (1 mM) and DTPA(100 lM) (Fig. 9C). This is consistent with the report that methio-nine is a poor substrate for MPO/H2O2 [54]. These results are inagreement with the HPLC results shown in Fig. 4.

Fig. 10A shows the EPR spectrum obtained from incubationscontaining tyrosine (2 mM), DTPA (100 lM), DBNBS (25 mM) andperoxynitrite (2.5 mM) which was attributed to the DBNBS-tyrosyladduct. The intensity of the EPR signal was enhanced in the pres-ence of bicarbonate (Fig. 10B). The computer simulation showsthe presence of the two radical adduct species (at C-3 and C-5 posi-tions of the tyrosyl group) (aN = 13.6 G and aN = 13 G, aH = 6 G), aspreviously reported [53]. Unlike the MPO/H2O2 oxidizing system,peroxynitrite or peroxynitrite/bicarbonate-mediated oxidation ofmethionine yielded a DBNBS-methionine radical adduct (DBNBS-Met) (aN = 14 G, aH(2) = 11 G) (Fig. 10C). On the basis of publishedresults [55], this spectrum was assigned to the DBNBS-CH2 S(CH2)2-CH(NH2)COOH and/or the DBNBS-(CH2)2CH(NH2)COOH adduct[55–57]. Bicarbonate enhanced this DBNBS-methionine adduct sig-nal (Fig. 10D). Decomposed peroxynitrite did not stimulate any

(NO

2)T

yr (

μM)

0

4

8

12

16

20

25 mM NaHCO3

0 mM NaHCO3

25 mM NaHCO3

0 mM NaHCO3B

A

YMTyr YF

Dit

yros

ine

(μM

)

0.0

0.4

0.8

1.2

1.6

Tyr YF YM

p<0.05

p<0.005

p<0.005

p<0.001

Fig. 6. Relative formation of nitrotyrosine and dityrosine from different dipeptidesin peroxynitrite/bicarbonate system. Dipeptides (250 lM) were incubated withperoxynitrite (100 lM) in a phosphate buffer (pH 7.4, 100 mM) containing DTPA(100 lM) for 5 min and reaction contents analyzed by HPLC. (A) nitrated product ofY, YF, and YM, and (B) dityrosyl product of Tyr, YF, YM.

B

Dit

yros

ine

(μM

)

0

1

2

3

45

6

78

(NO

2)T

yr (

μM)

0.0

0.5

1.0

1.5

Tyr

Tyr

YF YM

YF YM

2.0 p<0.005

p<0.005

p<0.005

p<0.005

A25 mM NaHCO3

0 mM NaHCO3

p<0.05

p<0.01

Fig. 7. SIN-1 induced nitrotyrosine and dityrosine formation from dipeptides.Dipeptides (250 lM) were incubated with SIN-1 (250 lM) in a phosphate buffer(pH 7.4, 100 mM) containing DTPA (100 lM) for 10 h at room temperature andsolutions analyzed by HPLC. (A) Relative levels of nitrated products of tyrosine and(B) dityrosyl products of Tyr, YF, and YM.

140 H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145

adduct formation (Fig. 10E). In the absence of either tyrosine ormethionine, the active peroxynitrite induced a three-line nitroxidesignal in the presence of DBNBS alone (Fig. 10F) [53]. Addition ofmethionine (at various concentrations) inhibited the DBNBS-tyro-syl adduct formation. Clearly, the EPR results suggest that methio-nine oxidation by peroxynitrite or peroxynitrite/bicarbonateinhibits tyrosyl radical formation (Fig. 10G and H) as comparedto results (obtained in the absence of methionine) shown inFig. 10A and B. Incubation mixtures containing the YM peptide,DBNBS, DTPA, and peroxynitrite stimulated the formation of theDBNBS-MY adduct (DBNBS-MetTyr), the spectral intensity ofwhich was enhanced with 25 mM of bicarbonate (Fig. 10I–L).Based on the computer simulations (shown in dotted lines inFig. 10L), the spectrum was assigned to the DBNBS-MetTyr, formedfrom trapping of a radical derived from the methionine residue inYM. The structure was further confirmed by identifying the pron-ase-induced degradation of the primary adduct. Fig. 11 showsthe effect of pronase on the DBNBS-MetTyr adduct formation.Upon adding the enzyme, pronase, to incubations containing tyro-sylmethionine, DBNBS, and peroxynitrite (after the formation ofthe DBNBS-MetTyr adduct), the EPR spectrum (Fig. 11A) changedto that of the DBNBS-Met adduct resulting from a peptidic cleavageof the DBNBS-MetTyr (Fig. 11C).

Although, the yield of both nitration and oxidation of YM (form-ing Y(NO2)M and MYYM) was increased by MPO/H2O2/NO2

� andperoxynitrite/bicarbonate, we were unable to detect the corre-sponding tyrosyl radical formed from YM (i.e., �YM). At this timewe do not have an explanation for the lack of detection of the

DBNBS-tyrosylmethionine adduct (formed from trapping of �YMby DBNBS) by EPR. However, we compared the effects of DBNBSon the nitration and oxidation products formed from Y, YF, YM,and YC (MTSL modified). DBNBS effectively inhibited the dimericproducts (YY, FYYF, CYYC) formed from oxidation of the parenttyrosines by MPO/H2O2 (data not shown). Concomitantly, the cor-responding DBNBS-tyrosyl adducts were detected by EPR [29]. Incontrast, DBNBS had little or no effect on MYYM formation inMPO/H2O2 system (data not shown), indicating that DBNBS eitherdoes not effectively trap the tyrosylmethionine radical or theresulting spin adduct is not stable enough to be detected by EPR.However, the failure to trap �YM by DBNBS remains enigmatic.Additional computational analysis of the stability of tyrosyl radicalin YM peptide is, therefore, warranted.

Discussion

Results from this study show that the presence of a methionineresidue adjacent to a tyrosyl group does not inhibit tyrosyl nitra-tion and oxidation catalysed by MPO/H2O2/NO2

�. In contrast, themethionine residue next to a tyrosyl group enhances its nitrationand oxidation reactions induced by in situ-generated peroxynitriteand peroxynitrite/bicarbonate systems. This is tentatively attrib-uted to an intramolecular radical transfer from methionine sulfideradical cation to the adjacent tyrosyl group. The possibility of anelectron transfer from tyrosyl residue to one-electron oxidizedmethionine moiety has been confirmed by the steady-state radiol-ysis and photolysis experiments, showing the formation of thecorresponding dityrosyl product in the presence of different one-electron oxidants, including Br2

��, N3�, (SCN)2

�� and CO3��. In con-

trast, no dityrosyl product has been detected after one-electronoxidation of the YC peptide; instead, only the disulfide productwas formed exclusively. We have used the EPR spin-trapping andHPLC/mass spectrometry techniques to identify intermediatesand products, respectively. Schemes 1 and 2 summarize the

Methionine (mM)0.0 0.2 0.4 0.6 0.8 1.0

Dit

yros

ine

(% o

f C

omtr

ol)

0

20

40

60

80

100

120

Methionine (mM)0.0 0.2 0.4 0.6 0.8 1.0

(NO

2)T

yr (

% o

f C

omtr

ol)

0

20

40

60

80

100

120

Methionine (mM)0.0 0.2 0.4 0.6 0.8 1.0

(NO

2)T

yr(%

of

Com

trol

)

0

20

40

60

80

100

120

Methionine (mM)0.0 0.2 0.4 0.6 0.8 1.0

Dit

yros

ine

(% o

f C

omtr

ol)

0

20

40

60

80

100

120A

B

Fig. 8. The effects of methionine on tyrosine nitration/oxidation induced by peroxynitrite and peroxynitrite/bicarbonate. (A) Tyr (250 lM) and Met were mixed withperoxynitrite (100 lM) in the presence and absence of bicarbonate (25 mM) in a phosphate buffer (pH 7.4, 100 mM), incubated for 5 min and analyzed by HPLC. (A)Dityrosine (Y-Y) and nitrotyrosine (NO2Y) were measured in incubations containing peroxynitrite, tyrosine, and methionine at concentrations as indicated. (B) Same as (A)but without bicarbonate.

A

B

C

D

E

Br

Br

N

O

-O3S

.

CH2

CH COO -+H3N

O

DBNBS-tyrosylradical

20 G

Fig. 9. The effects of methionine on DBNBS-tyrosyl radical adduct formation by MPO/H2O2. (A) Incubations contained Tyr (1 mM), MPO (150 nM), H2O2 (1 mM), DTPA(100 lM), and DBNBS spin trap (20 mM) in a phosphate buffer (pH 7.4, 100 mM). (B) Same as (A) but without tyrosine. (C) Same as (A) but without MPO. (D) Same as (A) butcontaining Met (1 mM), and (E) Same as (A) but containing 5 mM of Met. EPR spectra were obtained immediately after the addition of MPO. EPR spectral conditions aredescribed under Materials and methods.

H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145 141

C

G

H

I

J

K

L

F

D

E

A

B

Br

Br

N

O

-O3S

.

CH2

CH COO -+H3N

O

DBNBS-Tyr

Br

Br

N

O

-O3S

.

CH2

CH COO -+H3N

O

DBNBS-Tyr

Br

Br

N

O

-O3S

.

CH2

CH

CONHCH3CONH

CH2

CH2

CH

CONH2

OHDBNBS-MetTyr

15 G

Br

Br

N

O

-O3S

.

CH2

CH COO -+H3N

CH2

DBNBS-Met

Fig. 10. EPR spectra of DBNBS-derived spin adducts formed from oxidation of tyrosine and tyrosylmethionine by peroxynitrite. (A) Incubations contained Tyr (2 mM), DBNBS(20 mM), DTPA (100 lM) and peroxynitrite (2.5 mM) in a phosphate buffer (pH 7.4, 100 mM). (B) Same as (A) but in the presence of bicarbonate (25 mM). Dotted spectrumrepresents a computer simulation using the parameters described in the text. (C) Same as (A) but in the presence of Met (2 mM). (D) Same as (C) but containing bicarbonate(25 mM). Dotted EPR spectrum is a computer simulation using the parameters as indicated in the text. (E) Incubations contained Tyr (2 mM), Met (2.5 mM), DBNBS (25 mM),DTPA (100 lM), and decomposed peroxynitrite (2.5 mM) in a phosphate buffer (pH 7.4; 100 mM). (F) Peroxynitrite (2.5 mM) was incubated with DBNBS (20 mM) in aphosphate buffer. (G) Same as (A) but in the presence of Met (2 mM). (H). Same as (B) but in the presence of Met (2.5 mM). (I) Same as (A) but tyrosine was replaced by thedipeptide, tyrosylmethionine (YM) (0.5 mM). (J) Same as (I) but with bicarbonate (25 mM). (K) Incubations contained YM (1 mM) and peroxynitrite (2.5 mM). Otherconditions are the same as in (A). (L) Same as (K) but in the presence of bicarbonate (25 mM). Note that the possibility of an alternate chemical structure, DBNBS-CH2S(CH2)2CH(NH2)COOH is not ruled out in spectrum (E).

142 H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145

interactions between tyrosylmethionine and nitrating and oxidizingspecies generated in MPO/H2O2/NO2

� and peroxynitrite/bicarbon-ate systems. Rate constants shown in these schemes were obtainedfrom the previous publications [54–65].

Reactions between peroxynitrite/bicarbonate, methionine, andmethionine-containing peptides

Pioneering works by Pryor et al. and others have shown thatperoxynitrite can oxidize methionine by both one-electron andtwo-electron pathways, but the two-electron oxidation, which re-sults in the formation of methionine sulfoxide predominates[40,42]. The methionine sulfide radical cation was proposed as anintermediate during peroxynitrite-induced one-electron oxidationof methionine [40]. Bicarbonate stimulated peroxynitrite-inducedone-electron oxidation of methionine [40]. This was attributed tothe formation of an intermediate, ONOOCO2

�, which decomposedto form a potent one-electron oxidant and nitrating species (i.e.,CO3

�� and �NO2). The carbon dioxide was shown to stimulate perox-ynitrite-mediated nitration of tyrosine residues and inhibit oxida-tion of methionine residues of glutamine synthetase [39,66]. Thepresent data show that there is negligible formation of the tyrosylmethionine sulfoxide during oxidation of YM by both peroxynitrite

or peroxynitrite and bicarbonate. The major products induced byperoxynitrite were the nitration and oxidation products of YM(e.g., Y(NO2)M and MYYM) which were increased in the presenceof bicarbonate. Previously, it has been shown that the product pro-files of peroxynitrite or peroxynitrite and bicarbonate mediatedoxidation of methionine are different from those obtained frommethionine-containing peptides or proteins [67]. The presence ofa methionine residue stimulates oxidation and nitration of theneighboring tyrosine, suggesting that the one-electron oxidizedform of methionine (i.e., methionine sulfide radical cation) can be‘‘repaired” by tyrosine [64,65], resulting in its oxidation and/ornitration. This situation is different for other oxidants such as thehypochlorous acid (HOCl) generated by MPO/H2O2/Cl�. Methionineresidues inhibit tyrosine chlorination by HOCl in proteins due tostoichiometric scavenging of HOCl by methionine [68,69]. Muta-tion of methionine to another amino acid (e.g., alanine) that doesnot react with HOCl enhanced the yield of chlorotyrosine [68].

Influence of intramolecular electron transfer on biological nitrationand nitrosation

Intracellular nitrosative and nitrative modifications are com-plex and not always predictable. Obtaining a deeper understanding

3460 3480 3500 3520 3540

ABr

Br

N

O

-O3S

.

CH2

CH

CONHCH3CONH

CH2

CH2

CH

CONH2

OH

DBNBS-MetTyr

Br

Br

N

O

-O3S

.

CH2

CH COO-+H3N

CH2

DBNBS-Met

B

C

Fig. 11. The effect of pronase on DBNBS-derived spin adduct formed from oxidationof tyrosylmethionine by peroxynitrite. (A) Incubations contained YM (1 mM),DBNBS (20 mM), DTPA (100 lM), and peroxynitrite (2.5 mM) in a phosphate buffer(pH 7.4, 100 mM). (B) Same as (A) but in the absence of decomposed peroxynitrite.(C) Same as (A) but the enzyme, pronase (1 mg/ml) was added to the incubationmixtures 5 min after the addition of peroxynitrite. Note that the possibility of analternate chemical structure, DBNBS-CH2S(CH2)2CH(NH2)COOH is not ruled out inspectrum (C).

Scheme 1. Oxidative and nitrative pathways of TyrMet in MPO/H2O2/nitritesystem. In the presence of H2O2, myeloperoxidase (MPO) forms higher oxidants,compounds I and II which oxidizes NO2

� to �NO2. Both compounds I and II and �NO2

will oxidize tyrosine to a tyrosyl radical which reacts with �NO2 to formnitrotyrosine. The rate constants shown for �TyrMet represent the reactivity of freetyrosyl radical.

H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145 143

of the structural motif and sequence specificity that facilitatesnitrative and nitrosative post-translational modification is key tomapping functional regions of proteins that mediate redox signal-ing [70,71]. Although tyrosine nitration and cysteinyl thiol nitrosa-tion are induced by NO and oxygen or NO-derived oxidants, thisprocess appears to be selective as not all tyrosine residues or cys-

Scheme 2. Oxidative and nitrative pathways of TyrMet by peroxynitrite and bicarbonatperoxynitrite anion reacts with CO2 (which, in solution, is in equilibrium with bicarbdecomposes to give nitrogen-dioxide (�NO2), and the carbonate anion radical (CO3

��).following a rapid recombination of �NO2 and tyrosyl radical. The rate constants shown f

teine residues undergo nitration or nitrosation. Previously we pro-posed that intramolecular electron transfer reactions betweentyrosyl radical and cysteine residues in peptides may have a pro-found effect on the selective nitration of tyrosine residues and oxi-dation/nitrosation of cysteinyl residues [29]. Analysis of peptidesequences of S-nitrosylated proteins revealed that S-nitrosationoccurs predominantly when cysteinyl residues are present adja-cent to or closer to a tyrosine residue. The proposed mechanism in-volved a rapid intramolecular electron transfer between the tyrosylradical and cysteinyl thiol forming the thiyl radical that reacts with�NO to form the corresponding S-nitrosated cysteine [29]. In thecase of tyrosylmethionine peptide, the electron transfer to themethionine sulfide radical cation from tyrosine will be favored.Pulse radiolysis studies performed nearly 20 years ago indicate

e. Peroxynitrite anion exists in equilibrium with peroxynitrous acid (pKa = 6.8). Theonate anion) to form a transient intermediate, ONOOCO2

�, which homolyticallyONOOH homolytically decomposes to �NO2 and �OH. Nitration of tyrosine occursor TyrMet represent the reactivity of free amino acids (tyrosine and methionine).

Table 1Sequences of the peptides that have a neighboring methionine adjacent tonitrotyrosine.

Proteins Sequence

Eukaryotic translationinitiation factor 2

MY2SGAGPVLASPAPTTSPIPGYAFKPPPRPDFGTTGR

Nuclear factor 1A, isoform 1 MY2SPLCLTQDEFHPFIEALLPHVRPyruvate kinase ITLDNAY148MEKCDENILWLDYKActinin AIMTY241VSSFYHAFSGAQKAETAANRVacuolar protein sorting 16,

isoform 1MDCY4TANWNPLGDSAFYR

T-complex associated-testisexpressed 1-like (Tcte1l)

MEGY4QRPCDEVGFNADEAHNIVK

Interleukin 1 family member 6 KDIMDLY96NQPEPVKSFLFYHSQSGRHypothetical fimbral

chaperoneTRIKMFY139RPAQHLK

144 H. Zhang et al. / Archives of Biochemistry and Biophysics 484 (2009) 134–145

that tyrosine is a better ‘‘radical sink” for methionine radical cationthan is methionine as an ‘‘radical sink” for tyrosyl radicals (Eo

Tyr�

/

Tyr = +0.94 V [72]; EoMet+�/Met = +1.48 V [73] vs. NHE). Thus, the occur-

rence of a rapid and efficient intramolecular electron transfer toMetS�+ from Tyr-OH should induce tyrosyl radical formation. Thelack of the inhibitory action and in some cases the stimulatory actionof neighboring methionine on the oxidation and nitration of tyrosineresidues is attributed to this electron transfer mechanism, althoughefforts to trap and detect the corresponding tyrosyl radical by DBNBShave been unsuccessful.

Biological implications

It was postulated that the presence of a redox-active amino acid(cysteine, methionine) residue adjacent to a tyrosyl residue in pro-teins could inhibit its ability to undergo nitration [30]. As discussedearlier, inhibition of tyrosyl nitration by neighboring cysteinyl res-idues is attributed to a rapid electron transfer to the tyrosyl radicalfrom the cysteinyl thiols, and not due to direct scavenging of reac-tive nitrogen species (�NO2 and/or ONOO�) by cysteinyl thiols.Tyrosyl nitration appears to be a selective process and not all tyro-syl residues in a protein are nitration-sensitive. From looking at thepeptide sequences of nitrated tyrosine (Table 1), it appears thattyrosyl nitration occurs more frequently when tyrosine residuesare juxtaposed to a methionine residue and that the presence ofmethionine does not inhibit nitration of adjacent tyrosines. Theproposed electron transfer mechanism is one of the predictivetools. As reported previously [30], the presence of a positivelycharged amino acid nearer to tyrosine residues might be anotherstructural motif that facilitates nitration mechanism. However,the use of proteomics methods to identify structural sequencesin post-translational nitrative modifications has recently beenquestioned [74]. Post-translational nitrative modifications ofalpha-synuclein tyrosine were linked to the onset and progressionof neurodegenerative synucleinopathy lesions [8]. The presynapticprotein alpha-synuclein has four tyrosines and four methioninesand has no cysteinyl residues. Previously we reported that nitra-tion and oxidation of alpha-synuclein tyrosine were inhibited inthe presence of added methionine [75]. Preliminary results suggestthat a tyrosyl residue (Tyr-125) was nitrated that is adjacent to amethionine residue (Met-127) during peroxynitrite-inducedmodification of alpha-synuclein (data not shown). Clearly, the roleof methionine on protein tyrosyl nitration deserves furtherinvestigation.

Summary

The presence of a methionine residue adjacent to a tyrosyl moi-ety in peptides does not inhibit nitration and oxidation of tyrosineby MPO/H2O2/NO2

�. The methionine residue actually stimulated

peroxynitrite and peroxynitrite/bicarbonate-mediated nitrationand oxidation of tyrosine in TyrMet peptide. An intramolecularelectron transfer to the methionine sulfide radical cation fromthe tyrosyl residue has been proposed to account for enhancedtyrosyl nitration and oxidation.

Acknowledgment

This research was supported by a grant from National Institutesof Health Grant HL63119.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.abb.2008.11.018.

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