Mechanistic insight into the peroxidase catalyzed nitration of tyrosinederivatives by nitrite and hydrogen peroxide

Enrico Monzani1, Raffaella Roncone1, Monica Galliano2, Willem H. Koppenol3 and Luigi Casella1

1Dipartimento di Chimica Generale, 2Dipartimento di Biochimica, Universita di Pavia, Italy; 3Institute of Inorganic Chemistry,

ETH Honggerberg, Zurich, Switzerland

Peroxidases perform the nitration of tyrosine and tyrosylresidues in proteins, in the presence of nitrite and hydrogenperoxide. The nitrating species is still unknown but it isusually assumed to be nitrogen dioxide. In the presentinvestigation, the nitration of phenolic compounds derivedfrom tyrosine by lactoperoxidase and horseradish peroxi-dase was studied, with the aim of elucidating the mechanismof the reaction. The results indicate that nitrogen dioxidecannot be the only nitrating species and suggest the presenceof two simultaneously operative pathways, one proceedingthrough enzyme-generated nitrogen dioxide and anotherthrough a more reactive species, assumed to be complexedperoxynitrite, which is generated by reaction of hydrogen

peroxide with the enzyme–nitrite complex. The importanceof the two pathways depends on peroxide and nitrite con-centrations. With lactoperoxidase, nitration through thehighly reactive intermediate is preferred except at very lownitrite concentration, while with horseradish peroxidase, thenitrogen dioxide driven mechanism is preferred except atvery highnitrite concentration.The preferredmechanism forthe two enzymes is that operative in the physiological nitriteconcentration range.

Keywords: nitrogen dioxide; peroxidases; peroxynitrite;phenol nitration; reactive nitrogen species.

It is well documented that reactive nitrogen species derivedfrom nitrogen monoxide (NO) are involved in manypathological conditions [1,2]. Although NO performs manyimportant physiological functions, ranging from neuro-transmission to blood pressure regulation, and is involved inthe defence mechanism against microorganisms [3,4], over-production of NO can have damaging effects [4,5]. Nitriteis a major product of nitrogen monoxide metabolism [6]and markedly increased nitrite levels have been detectedin situations, e.g. during inflammatory processes, where NOis overproduced [7,8]. However, nitrite does not accumulatein vivo because it is oxidized (to nitrate) by the Fe2+-O2

form of hemoglobin (oxyHb) or the Fe2+-O2 form ofmyoglobin (oxyMb), producing the Fe3+ forms of these(metHb and metMb) [6], respectively, or by other inflam-matory oxidants such as hypochlorous acid [9], causing theformation of reactive nitrogen species [10].

An additional pathway for nitrite oxidation that isreceiving increasing attention is its reaction with peroxidasesin the presence of hydrogen peroxide [11–16]. This reactionproduces reactive nitrogen species that have been shown todegrade chlorophyll [11], nitrate tyrosine [12] and tyrosylresidues in proteins [13,14]. The latter reactions are ofparticular importance because, in addition to those invol-ving the more typical peroxidase catalyzed oxidations of

chloride and thiocyanate [12], they may serve a defensivefunction against microorganisms. Several mechanisms forthe peroxidase catalyzed phenol nitration in the presence ofnitrite/hydrogen peroxide have been proposed but, in spiteof recent efforts, the nature of the nitrating species hasnot been fully clarified yet. The currently favored pathway[15,16] involves one-electron oxidation of nitrite by theperoxide-generated enzyme intermediates known as com-pound I and compound II [17,18]:

E þ H2O2 ! compound I þ H2O ð1Þ

compound I þ NO2� ! compound II þ NO2

� ð2Þ

compound II þ NO2� þ 2Hþ ! E þ NO2

� þ H2O ð3Þ

where E represents the native (Fe3+) form of the enzyme.For myeloperoxidase, the reaction of the enzyme inter-mediates by nitrite has been studied recently in detail [15].According to this mechanism, NO2

� could either nitratea phenol with a reaction stoichiometry of 2 : 1 or directlyreact with a peroxidase-generated phenoxy radical accord-ing to reaction 5:

NO2� þ PhOH ! NO2

� þ PhO� þ Hþ ð4Þ

NO2� þ PhO� ! O2N�PhOH ð5Þ

An alternative pathway, involving a two-electron enzymaticoxidation of nitrite to nitryl cation, a powerful phenolnitrating agent [19]:

E þ H2O2 ! compound I þ H2O ð1Þcompound I þ NO2

� þ 2Hþ ! E þ NO2þ þ H2O ð6Þ

is considered unlikely in view of the extremely rapid reactionof NO2

+ with water, to yield nitrate [20].

Correspondence to L. Casella, Dipartimento di Chimica Generale,

Via Taramelli 12, 27100 Pavia, Italy.

Fax: + 39 0382 528544, Tel.: + 39 0382 507331,

E-mail: bioinorg@unipv.it

Abbreviations: LPO, lactoperoxidase; HRP, horseradish peroxidase.

(Received 21 November 2003, revised 29 December 2003,

accepted 13 January 2004)

Eur. J. Biochem. 271, 895–906 (2004) � FEBS 2004 doi:10.1111/j.1432-1033.2004.03992.x

In a recent report, the reaction of lactoperoxidasecompound I with nitrite was found to lead directly tonitrate, without formation of NO2

� radicals as intermediates[21]. Moreover, additional work on eosinophil peroxidaseand myeloperoxidase suggested that these proteins performnitrations in the presence of nitrite and hydrogen peroxide,generating peroxynitrite [22]. Therefore, different enzymesmay activate nitrite through different mechanisms. In thispaper we provide new mechanistic insight into the lacto-peroxidase and horseradish peroxidase mediated nitrationof the representative tyrosine derivatives 1–4 by nitrite/hydrogen peroxide and, in particular, we address theproblem of the possible contribution of peroxynitrite inthis reaction. Peroxynitrite nitrates phenolic substrates[23,24] and could conceivably be formed by reaction ofhydrogen peroxide with a peroxidase-nitrite complex:

E þ NO2� ! E�NO2

� ð7Þ

E�NO2� þ H2O2 ! E�OH� þ O¼N�O�OH ð8Þ

The present investigation extends our previous studies onthe peroxidase catalyzed oxidation of phenolic compoundsby hydrogen peroxide [25–27]. The latter reaction competeswith phenol nitration and gives rise to the dimeric andoligomeric phenol coupling products shown.

Materials and methods

Materials and instrumentation

Bovine lactoperoxidase was purified according to a newprocedure, which is an improvement of that reported byFerrari et al. [28]. Horseradish peroxidase (HRP) wasobtained from Sigma as a freeze-dried powder (RZ ¼ 3.2at pH 7.0, e402 ¼ 103 mM

)1Æcm)1). L-Tyrosine, 3-nitro-L-tyrosine, tyramine, 3-(4-hydroxyphenyl)-propionic acidand 4-hydroxybenzonitrile were from Sigma-Aldrich.N-Acetyltyramine was prepared by reaction between tyr-amine and acetic anhydride as reported previously [25].Peroxynitrite was prepared according to a literature proce-dure [29]. NO2

� was obtained by air oxidation of NO. Allother chemicals were reagent grade and used as received.Hydrogen peroxide solutions were freshly prepared bydiluting a 30% (v/v) solution in water and were standard-ized by iodimetry. Optical spectra were measured with anHP 8452 A diode array spectrophotometer (Agilent Tech-nologies, Italy). Stopped-flow experiments were carried outwith a SMF-3 Bio-Logic coupled to a diode array J & MTIDAS spectrophotometer (J&M Analytische Mess undRegeltechnik GmbH, Germany) with 6 ms dead time and a0.5 cm path length cell, or an Applied Photophysics model

RS-1000 (Applied Photophysics Ltd, UK) instrumentwith 1 ms dead time and 1 cm or 0.2 cm path length cells.NMR spectra were obtained at 400 MHz with a BrukerAVANCE spectrometer (Bruker BioSpin, Italy). Electro-spray ionization MS data were acquired using a FinniganLCQ ion trap mass spectrometer (Thermo, Italy).

Purification of lactoperoxidase (LPO)

Fresh untreated bovine milk (10 L) was defatted by centrif-ugation (twice for 1 h at 10 900 g, 4 �C), the pH of theresulting liquid was adjusted to 6.6, and casein was precipi-tated by addition of 2 M CaCl2 (60 mL per L of defattedmilk). After stirring overnight at 4 �C, the precipitate waseliminated by centrifugation (twice for 1 h at 10 900 g,4 �C). The whey was dialyzed against 10 mM Tris/HCl(pH 7.0) and applied sequentially through two tandemlylinked ion exchange columns packed with DEAE-cellulose(5 · 50 cm) andCM-cellulose (2.6 · 30 cm)preequilibratedwith the same buffer. At the end of sample loading, theunboundmaterialwaswashedoutwith the initial bufferuntilthe absorbance at 280 nm became negligible. The columnswere then detached from each other and the LPO bound tothe cation exchange column was eluted by making the buffer0.5 M in NaCl. This step was followed by gel filtration onSephadex G-100 in a column (6 · 65 cm) equilibrated with20 mM Tris buffer, 0.15 M NaCl, pH 7.0. The 412 nmabsorbing fractions were pooled and, using an Amicon30 kDa cut-off filter (Millipore), concentrated and equili-brated in 10 mM phosphate buffer, pH 6.8. All these stepswere carried out at 4 �C. The protein was then separatedfrom contaminating lactoferrin by ion exchange chromato-graphy on a Mono S HR 10/10 column (Amersham Phar-maciaBiotech), equililibratedwith thephosphate buffer, andconnected toanAktaPurifier system (AmershamPharmaciaBiotech). Elution was achieved at a flow rate of 3 mLÆmin)1

for 20 min with the equilibration buffer, followed by a three-step linear gradient: from 0–25% of buffer B (10 mM

phosphate buffer, 1 M NaCl, pH 6.8) in 20 min, from 25–35% in 60 min and from 35–100% of buffer B in 20 min,and monitored at 280 and 412 nm. The lactoperoxidasecontaining peak was manually collected and the homo-geneity of the protein was checked by SDS/PAGE underreducing and nonreducing conditions in 10% gels. Theenzyme preparation gave 75 mgof protein with RZ > 0.90.The concentration of enzyme solutions was determinedoptically using e412 ¼ 114 mM

)1Æcm)1.

Preparation of nitrophenols

The nitrated derivatives of 1, 2 and 4 are not commerciallyavailable and were therefore prepared in a small scale byLPO mediated reactions.

3-(4-Hydroxy-3-nitrophenyl)-propionic acid. 3-(4-Hydro-xyphenyl)-propionic acid (50 mg) was dissolved in 25 mL ofa 5 mM phosphate buffer solution at pH 7.5 containingsodium nitrite (0.08 M). To this solution, dilute solutions ofLPO in the same buffer (final concentration 3 · 10)8

M) andhydrogen peroxide (final concentration 8.0 · 10)4

M) wereadded in small amounts during 0.5 h at 25 �C in order toobtainan intenseandpersistentyellowcolor.Then, thepHof

896 E. Monzani et al. (Eur. J. Biochem. 271) � FEBS 2004

the solution was brought to about 4 in order to obtain theproduct in the acidic form. The reaction products wereextracted several times with dichloromethane and theorganic phase was rotary evaporated to dryness. The residuewas chromatographed on a silica gel column using a mixtureof dichloromethane/methanol (95 : 5, v/v). 3-(4-Hydroxy-3-nitrophenyl)-propionic acid eluted as the first fraction. Theproductwas recovereduponevaporationof the solvent (yield� 40%). Table 1 details the analytical data obtained for3-(4-hydroxy-3-nitrophenyl)-propionic acid. The extinctioncoefficient of 3-(4-hydroxy-3-nitrophenyl)-propionic acid at422 nm in phosphate buffer (pH 7.5) is 3600 M

)1Æcm)1.

3-Nitrotyramine. N-Acetyltyramine (50 mg) was dissolvedin 10 mL of a 10 mM phosphate buffer solution at pH 7.5containing sodium nitrite (0.25 M). To this solution, severaladditions of a dilute solution of LPO in the same buffer(final concentration 3.0 · 10)8

M) and hydrogen peroxide(final concentration 8.0 · 10)4

M) were made and themixture was allowed to react while stirring at roomtemperature for 1 h. After acidification to pH 4, the organicproducts were extracted with chloroform and the solutionwas rotary evaporated to dryness to give a red solid. Thecrude 3-nitro-N-acetyltyramine was hydrolyzed by refluxingit in a 1 M solution of sodium hydroxide (10 mL) for 1 h.After rotary evaporation, the residue was applied on asilica gel column and chromatographed using a gradient ofdichloromethane/methanol. The product was recoveredafter evaporation of the solvent (yield 20%). Table 2 detailsthe analytical data obtained for 3-nitrotyramine. Theextinction coefficient of 3-nitrotyramine at 422 nm inphosphate buffer pH 7.5 is 2800 M

)1Æcm)1.

4-Hydroxy-3-nitrobenzonitrile. 4-Hydroxybenzonitrile(100 mg) was dissolved in 25 mL of a 5 mM phosphatebuffer solution at pH 7.5. Dilute solutions of LPO (final

concentration 5.6 · 10)8M), hydrogen peroxide (0.88 mM)

and sodium nitrite (6 mM) were added in small portions tothe substrate solution during 1 h at 25 �C. Formation of theproduct was accompanied by the development of a yellowand persistent color in the solution. Then, the pH of themixture was brought to about 4 in order to obtain theproduct in the protonated form. The solution was extractedseveral times with ethyl acetate and the organic phase wasrotary evaporated to dryness. The residue was chromato-graphed on a silica gel column using dichloromethaneas eluent. 4-Hydroxy-3-nitrobenzonitrile eluted as the firstfraction. The product was recovered upon evaporation ofthe solvent (yield � 5%). Table 3 details the analytical dataobtained for 4-hydroxy-3-nitrobenzonitrile. The extinctioncoefficient of 4-hydroxy-3-nitrobenzonitrile at 422 nm inphosphate buffer (pH 7.5) used in the kinetic experimentsis 1700 MÆcm)1 and the wavelength of maximum absorptionis at 400 nm (e 2200 M

)1Æcm)1).

Kinetic experiments of phenol nitration

The kinetics of the enzymatic phenol nitration were studiedspectrophotometrically using a magnetically stirred,thermostated optical cell of 1 cm path length, in 200 mM

phosphate buffer, pH 7.5. The temperature was main-tained at 25 ± 0.1 �C. The reactions were convenientlyfollowed through the increase of absorbance at 422 nm,due to the formation of the nitrophenolic derivatives, inthe initial phase of the reactions. The conversion of thedata from DAÆs)1 to MÆs)1 was performed using the e422

values for 1–4; for 3-nitro-L-tyrosine, the extinctioncoefficient e422 ¼ 4000 M

)1Æcm)1 was used. In order toreduce the effect of noise in the absorbance readings, thedifference between the absorbance at 422 nm and that at820 nm, where the absorption remains negligible duringthe assay, was monitored. Preliminary experiments were

Table 1. Analytical data of 3-(4-hydroxy-3-nitrophenyl)-propionic acid. Elemental analysis, 1H-NMR, MS (ESI) and IR characterization data of

3-(4-hydroxy-3-nitrophenyl)-propionic acid.

Elemental analysis (%)1H NMR (CDCl3)

(d)MS (ESI)

(m/z)

MS/MS (ESI m/z 210.3)

(m/z)

IR (NaCl, Nujol mull)

(mÆcm)1)Calculated Found

C 51.19 C 50.95 10.5 (broad, OH) 212.3 [M+1] 166.3 [(M-CO2)+1] 1510 m(NO2)as

H 4.30 H 4.27 7.95 (d, 1H, phenyl 2-H) 1338 m(NO2)sN 6.63 N 6.60 7.44 (dd, 1H, phenyl 6-H) 850 m(C-N)

7.12 (d, 1H, phenyl 5-H)

2.96 (t, 2H,CH2-CO)

2.69 (t, 2H, C-CH2-Ph)

Table 2. Analytical data of 3-nitrotyramine. Elemental analysis, 1H-NMR, MS (ESI) and IR characterization data of 3-nitrotyramine.

Elemental analysis (%)1H NMR

(D2O)

(d)MS (ESI)

(m/z)

MS/MS

(ESI m/z 210.3)

(m/z)

MS-MS-MS

(ESI, m/z 166.2)

(m/z)

IR (NaCl,

Nujol mull)

(mÆcm)1)Calculated Found

C 52.74 C 52.13 7.72 (d, 1H, phenyl 2-H) 183.2 [M +1] 166.2 [(M-NH3)+1] 120.2 [(M-NH3-NO2)+1] 1522 m(NO2)as

H 5.53 H 5.59 7.18 (dd, 1H, phenyl 6-H) 166.2 [(M-NH3) +1] 1345 m(NO2)sN 15.38 N 14.95 6.70 (d, 1H, phenyl 5-H) 858 m(C-N)

3.15 (t, 2H, CH2-N)

2.78 (t, 2H, C-CH2-Ph)

� FEBS 2004 Peroxidase catalyzed nitration (Eur. J. Biochem. 271) 897

performed with substrates 1–4 to find appropriate condi-tions to follow the reactions and, in particular, to establishconditions of saturation of hydrogen peroxide, in order toavoid inconvenient excess of this reagent. Steady-statekinetics were then studied as a function of both the phenoland nitrite concentrations. For each substrate, the condi-tions required to study the rate dependence on thereactants concentrations were found through the followingsteps: (a) determination of the peroxide concentration thatmaximizes the nitration rate with high substrate and nitriteconcentrations (typically starting from [phenol] ¼ 2 mM

and [NO2–] ¼ 0.2 M for LPO or 2.0 M for HRP); (b) study

of the dependence of the rate versus substrate concentra-tion maintaining [H2O2] as optimized in the previous stepand high [NO2

–]; (c) study of the dependence of the rateversus nitrite concentration maintaining [H2O2] and[PhOH] saturating as found in step b; (d) when thesubstrate and nitrite concentrations that maximize the ratedid not fit with those used in step a, the whole procedurewas repeated starting with different [NO2

–] and [phenol] inan iterative way.

The kinetic studies were then performed with thefollowing concentrations of the reactants: (a) dependenceof the rate versus phenol concentration with LPO (50 nM):[1] ¼ 0–10 mM, [H2O2] ¼ 0.42 mM, [NO2

–] ¼ 94 mM; [2] ¼0–1.0 mM, [H2O2] ¼ 0.84 mM, [NO2

–] ¼ 78 mM; [3] ¼0–20 mM, [H2O2] ¼ 0.84 mM, [NO2

–] ¼ 30 mM; [4] ¼0–10 mM, [H2O2] ¼ 1.3 mM, [NO2

–] ¼ 0.2 M; (b) depend-ence of the rate versus phenol concentration with HRP(30 nM): [1] ¼ 0–20 mM, [H2O2] ¼ 1.60 mM, [NO2

–] ¼2.1 M; [2] ¼ 0–10 mM, [H2O2] ¼ 3.0 mM, [NO2

–] ¼ 2.1 M;[3] ¼ 0–1.0 mM, [H2O2] ¼ 0.24 mM, [NO2

–] ¼ 2.1 M;[4] ¼ 0–24 mM, [H2O2] ¼ 0.40 mM, [NO2

–] ¼ 2.1 M; (c)dependence of the rate versus nitrite concentration(0–0.4 M) with LPO (50 nM): [1] ¼ 1.0 mM, [H2O2] ¼0.42 mM; [2] ¼ 1.0 mM, [H2O2] ¼ 0.84 mM; [3] ¼ 0.6 mM,[H2O2] ¼ 0.84 mM; [4] ¼ 5.0 mM, [H2O2] ¼ 1.3 mM;(d) dependence of the rate versus nitrite concentration(0–3.5 M) with HRP (30 nM): [1] ¼ 13.0 mM, [H2O2] ¼1.6 mM; [2] ¼ 5.0 mM, [H2O2] ¼ 3.0 mM; [3] ¼ 0.70 mM,[H2O2] ¼ 0.24 mM; [4] ¼ 17.0 mM, [H2O2] ¼ 0.40 mM.

Nitration of 3 at low nitrite concentration

The nitration of tyrosine by LPO and HRP was also studiedat a pathophysiological concentration of nitrite. WithLPO (0.50 lM) the concentrations of the reactants were:[H2O2] ¼ 0.84 mM, [3] ¼ 0.59 mM, [NO2

–] ¼ 100 lM. WithHRP (0.50 lM) the concentrations of the reactants were:[H2O2] ¼ 0.24 mM, [3] ¼ 0.69 mM, [NO2

–] ¼ 100 lM.

Peroxidase catalyzed oxidation of 4

Steady state kinetic experiments of catalytic oxidation of 4by LPO or HRP and hydrogen peroxide were performed in200 mM phosphate buffer (pH 7.5) at 25 ± 0.1 �C, accord-ing to the following procedure. To the solution containingthe enzyme (56 nM LPO or 71 nM HRP) and variableamounts of 4 (0–50 mM) in an optical quartz cell of 1 cmpath length, hydrogen peroxide (0.2 mM) was added to thefixed final volume of 1.6 mL. The progress of the reactionwas followed by monitoring the absorbance changes at322 nm due to the formation of the oxidative couplingdimer of 4. The initial rates were determined from the linearpart of the trace at 322 nm. To convert the rates fromDAÆs)1 to MÆs)1 it was necessary to determine the extinctioncoefficient of the dimeric product of the reaction. This e322

value was obtained from a plot of absorbance versusnumber of moles of hydrogen peroxide consumed in theHRP-catalyzed oxidation of 4, where hydrogen peroxidewas the limiting reagent. The following reagent concentra-tions were used: [HRP] 33 nM, [4] 0.3 mM, and [H2O2] from3.8 · 10)5 to 1.5 · 10)4

M, the other conditions were thesame as in the kinetic experiments. From this analysis thee322 value of 5600 M

)1Æcm)1 was obtained.

Reduction of compound II by substrates

The second-order catalytic constant for the reactionbetween HRP compound II and nitrite was determinedin 200 mM phosphate buffer (pH 7.5) at 25.0 ± 0.1 �C.Compound II was prepared by incubation of the proteinsolution (7.0 lM) with a small excess (two mol equivalents)of hydrogen peroxide for 1 min. The transformation to theiron(III) species was followed by monitoring the absorbancechanges of the protein with time (readings every 0.1 s), usinga variable excess of nitrite (from 40 lM to 1.6 mM). Thecompound II reduction to iron(III) followed a first-orderbehavior. In order to decrease the noise in the readings, thedetermination of the observed rate constants (kobs) wasperformed following the reaction at the two wavelengthswhere the spectral changes are largest and interpolatingtheir difference in absorbance (A400–A420 nm) with a first-order equation. The replot of kobs versus [NO2

–] was linearand the slope gave the catalytic constant.

In a similar way, the second-order catalytic constants forthe reaction between LPO or HRP compound II and therepresentative phenols 3 and 4were determined. The enzymecompound II derivatives (2 lM) were prepared as describedbefore. Solutions of the substrates (1–10 mM) in an appro-priate volume of 200 mM phosphate buffer (pH 7.5) were

Table 3. Analytical data of 4-hydroxy-3-nitrobenzonitrile. Elemental analysis, 1H-NMR, MS (ESI) and IR characterization of 4-hydroxy-

3-nitrobenzonitrile.

Elemental analysis (%)1H NMR (CDCl3)

(d)MS (ESI)

(m/z)

IR (NaCl, Nujol mull)

(mÆcm)1)Calculated Found

C 51.23 C 50.80 10.9 (broad, OH) 165.1 [M +1] 1510 m(NO2)as

H 2.46 H 2.54 8.5 (d, 1H, phenyl, 2H) 1320 m(NO2)sN 17.07 N 16.89 7.8 (dd, 1 H, phenyl, 6H) 870 m(C-N)

7.3 (d, 1 H, phenyl, 5H)

898 E. Monzani et al. (Eur. J. Biochem. 271) � FEBS 2004

prepared from fresh stock solutions. The reactions werecarriedoutunderpseudo-first-order conditions and followedby monitoring the disappearance of compound II with time(readings every 0.1 s). The rate constants (kobs) weredetermined from the changes in the difference of absorbance(A402–A420) with time, which were fitted to a first-orderequation. The replots of kobs versus [phenol] were linear andthe slopes gave the catalytic constants.

Stopped-flow experiments

The reaction betweenLPO, nitrite andH2O2 was followed ina stopped-flow apparatus using an optical cell of path length0.5 cm; one of the syringes was filled with a solution of theenzyme (5.4 lM) and NaNO2 (20 or 300 mM) in 200 mM

phosphate buffer (pH 7.5) at 25 �C. The other syringe wasfilled with H2O2 (1.7 mM). Mixing of the two solutions in thereaction cuvette reduced the concentration of the reactantsto one half. Control experiments were carried out withoutperoxide and with either one tenth or twofold concentrationof the oxidant. In analogous experiments performed withHRP, a path length of 0.2 cm was used; one of the syringeswas filled with a solution of the enzyme (75 lM) and NaNO2

(2 M or 50 mM) in the same buffer as above. The othersyringe was filled with H2O2 (2.0 mM). Control experimentswere carried out without peroxide.

Nitrate assay

The determination of nitrate formed competitively by theenzymatic reaction with nitrite and hydrogen peroxide invarious conditions was carried out using a Metrohm IC ionchromatograph (Metrohn AG, Switzerland) with a Super-Sep column at a 1 mLÆmin)1 flow rate. All the experimentswere performed in triplicate. In a typical experiment, 10 mM

sodium nitrite was allowed to react for 20 min with 0.8 mM

hydrogen peroxide in the presence of 5 mM 2 and 10 nM

LPO in 20 mM phosphate buffer (higher buffer concentra-tions reduce the sensitivity of nitrate determination) pH 7.5,at 25 �C. Then, the sample was diluted tenfold in doubledistilled water and injected into the column. Other experi-ments were performed without enzyme, in the absence orpresence of substrate (55 mM), and with 140 mM nitrite and2 mM hydrogen peroxide.

Binding experiments

The binding of nitrite to LPO and HRP was studiedspectrophotometrically, by following the spectral changesupon addition of small aliquots of a concentrated NaNO2

solution in 200 mM phosphate buffer (pH 7.5) to the enzymesolution in the same buffer, at 25 ± 0.1 �C. No attemptswere made to keep the ionic strength constant. With LPO(6 · 10)6

M),a1 Mstocknitrite solutionandanopticalcellof1 cmpath lengthwereused. In thecaseofHRP(6 · 10)5

M),the bindingprocess exhibited biphasic behavior and, in orderto reach saturation in the second step, the titration was per-formed using a more concentrated solution of NaNO2 (4 M)in a cellwitha smaller path length (0.1 cm).The spectral datawere analyzed, after subtractionof the absorptiondue to freenitrite, as described previously [30] to obtain equilibriumconstants and stoichiometry of adduct formation.

Differential pulse voltammetry

Polarographic experiments on substrates 2 and 4 wereperformed at room temperature in 200 mM phosphatebuffer (pH 7.5), using an Amel model 591/ST Polarographcoupled with an Amel 433 Trace Analyzer, with a glassycarbon electrode and an Ag/AgCl/KCl saturated referenceelectrode. The scans were performed from 300 to 1200 mVusing a differential pulse voltammetry of 100 mVÆs)1 and apulse amplitude of 50 mV. The redox potential measuredpolarographically corresponds to the transformation of thephenols to the corresponding phenoxide radicals; the valuesof 840 mV (versus Ag/AgCl/KCl saturated) for 4 and790 mV for 2 were found. Voltammeric oxidation ofphenols causes passivation of the electrode surface thatresults in rapidly diminishing voltammetric curve responseand enlarged peaks. For this reason, the absolute values ofthe oxidation potentials of the compounds investigated maybe affected by experimental conditions (electrode surface,pH and concentration of the solutions). However, thedifferences between the values of the oxidation potentialsfound are significant because they were obtained in the sameexperimental conditions.

HPLC analysis of the nitration products

The product mixtures derived from the chemical orenzymatic nitration of compounds 1–4 and phenylaceticacid (5) were analyzed by HPLC using a Jasco MD-1510instrument with diode array detection and a Supelco LC18reverse-phase semipreparative column (250 · 10 mm;Sigma-Aldrich). Elution was carried out using 0.1%trifluoroacetic acid in distilled water (solvent A) and 0.1%trifluoroacetic acid in acetonitrile (solvent B), with a flowrate of 5 mLÆmin)1. Elution started with 100% solvent Afor 4 min, followed by a linear gradient from 100% A to100% B in 20 min. Spectrophotometric detection of theeluate was performed in the range 200–600 nm.

Reaction of 2 and 4 with peroxynitrite

Solutions of the phenol (2 or 4) (1 mM) in 200 mM

phosphate buffer (pH 7.5) were treated with five- or tenfoldmolar excess peroxynitrite for 5 min at room temperature.The reaction mixtures were analyzed by HPLC as describedabove. The retention times of 2 and 4 were 11.5 and12.7 min, respectively, and those of the correspondingnitration products, 3-(4-hydroxy-3-nitrophenyl)-propionicacid and 4-hydroxy-3-nitrobenzonitrile, were 13.9 and15.7 min, respectively. The identity of the products waschecked by comparison with the spectra of authenticsamples. Yields of nitration products were estimated fromthe extinction coefficients of the phenolic derivatives and thepeak areas in the HPLC chromatograms.

Nitration of phenylacetic acid, 5

CO2-free peroxynitrite. A solution of 5 was purged withargon for 20 min and then reacted with tenfold molar excessperoxynitrite. HPLC analysis of the reaction mixtureshowed, as well as unreacted 5, five minor peaks withretention times of 9.9, 10.1, 10.4, 12.2 and 12.4 min.

� FEBS 2004 Peroxidase catalyzed nitration (Eur. J. Biochem. 271) 899

Peroxynitrite-CO2. A solution of 5 (1 mM) in 200 mM

phosphate buffer (pH 7.5), equilibrated with atmosphericCO2, was treated with five- or tenfold molar excessperoxynitrite for 5 min at room temperature, and thenanalyzed by HPLC. The chromatogram contained onlyone peak corresponding to 5, with a retention time of11.6 min.

Peroxidase/H2O2/NO2–. The catalytic nitrations of 5

were performed under the following experimental condi-tions: HRP (30 nM), [5] ¼ 1 mM, [H2O2] ¼ 0.4 mM,[NaNO2] ¼ 0, 0.025, 0.25 or 2 M; LPO (50 nM),[5] ¼ 1 mM, [H2O2] ¼ 1.3 mM, [NaNO2] ¼ 0, 0.025 or0.25 M. Analysis of the product mixtures resulting fromthe reactions carried out in the presence of high nitriteconcentrations (2 M for HRP and 0.25 M for LPO) showedextensive modification of substrate 5. The HPLC chroma-togram showed two main peaks at 11.1 and 11.3 min,and minor peaks for unreacted 5 and six other productswith retention times of 9.9, 10.4, 11.8, 12.0, 12.2 and12.4 min.

Formation of phenol dimers by the HRP/H2O2/NO2–

system

The phenol dimers formed by the HRP/H2O2/NO2– system

during the nitration of 1 and 2 were analyzed by HPLC inthe same conditions as reported in a previous work [31]. Thereactions were performed in 200 mM phosphate buffer(pH 7.5) at 25 �C in the presence of 30 nM HRP andvariable nitrite concentration (0, 0.2, and 1.0 M). Theother reagents were as follows: with [1] ¼ 13.0 mM,[H2O2] ¼ 1.6 mM; with [2] ¼ 5.0 mM, [H2O2] ¼ 3.0 mM.

Results

Steady-state kinetics

The kinetics of phenol nitration were studied by followingthe characteristic absorption near 420 nm of the nitrophen-ols in neutral medium. At this wavelength the interferenceby the phenolic dimers formed according to the normalperoxidase reaction [25–27] is completely negligible. Theenzymatic nitration reaction of phenols 1–4was studied as afunction of both the phenol and nitrite concentrations, withthe other reagents saturating, except for the HRP experi-ments, where saturating nitrite concentrations were toohigh. In these cases, the kinetics were studied at a nitriteconcentration corresponding to � 60% saturation. Withboth LPO and HRP, the rate of the enzymatic reactionexhibits a hyperbolic dependence on the concentration ofthe phenols. A more complex behavior was found whenthe rate dependence was studied as a function of nitriteconcentration. For substrates 1–3, the LPO mediatedreactions exhibited a normal saturation behavior, whilethe HRP mediated reactions were biphasic (Fig. 1). Withsubstrate 4, inhibition was observed for both LPO and HRPat moderate concentrations of nitrite (Figs 2 and 3). Thesaturation behavior found for 1–3 is not due to nitriteinhibition on the peroxide involving step, as this shouldbe connected to a change in the slow step; instead, the ratedoes not increase on increasing peroxide concentration.

These findings indicate that an efficient nitration reactionrequires the interaction of the enzyme with both nitrite andphenol.

Fig. 1. Biphasic behavior of the rate of HRP-mediated phenol nitration

as a function of nitrite concentration. Rate dependence of HRP-cata-

lyzed nitration of 2 on nitrite concentration in 200 mM phosphate

buffer (pH 7.5), at 25 �C. The inset shows an expansion of the plot in

the low nitrite concentration range.

Fig. 2. Inhibition of the LPO-mediated phenol nitration by nitrite. Plot

of the rate of LPO-catalyzed nitration of phenol 4 as a function of

nitrite concentration in 200 mM phosphate buffer (pH 7.5), at 25 �C.

The inset shows an expansion of the plot in the low nitrite concen-

tration range.

Fig. 3. Inhibition of the HRP-mediated phenol nitration by nitrite. Plot

of the rate of HRP-catalyzed nitration of phenol 4 as a function of

nitrite concentration in 200 mM phosphate buffer (pH 7.5), at 25 �C.

The inset shows an expansion of the plot in the low nitrite concen-

tration range.

900 E. Monzani et al. (Eur. J. Biochem. 271) � FEBS 2004

The experimental data can be interpreted considering twosimplified mechanisms, which differ with respect to theactive species involved and for the dependence on theoxidant concentration. The first mechanism, pathway A(Scheme 1), considers that product formation followsreactions 9–14 (corresponding to reactions 1–5 shownabove). Compound I and compound II react with nitriteor the phenol generating free diffusible radicals [16]. Thenitrating agent is thus NO2

�, which can be derived fromeither compound I or compound II. The observation ofsubstrate saturation behavior indicates that, even if NO2

�

could react with free phenol, the reaction is faster when thephenoxy radical is formed by direct reaction with com-pound I or compound II.

E þ H2O2 ! compound I þ H2O (fast) ð9Þ

compound I þ NO2� , compound I=NO2

�

! compound II þ NO2� ð10Þ

compound I þ PhOH , compound I=PhOH

! compound II þ PhO� þ Hþ ð11Þ

compound II þ NO2� , compound II=NO2

�

�!2Hþ

E þ NO2� þ H2O ð12Þ

compound II þ PhOH , compound II=PhOH

�!2Hþ

E þ PhO2� þ H2O ð13Þ

PhO� þ NO2� ! O2N�PhOH ð14Þ

Scheme 1. Pathway A mechanism.

The PhO• produced can also give rise to dimeric couplingproducts through the normal peroxidase catalytic cycle[25–27]:

2PhO� ! dimers ð15Þ

The second mechanism (pathway B), represented inScheme 2, where E–NO2

– is the peroxidase-nitrite complexand Enitr the nitrating active species. This mechanism firstconsiders binding of nitrite to the iron center of the protein.Upon reaction of this complex with hydrogen peroxide, Enitr

is formed in a fast step. While in the absence of the phenol,Enitr degrades to E and nitrate (or performs nitration ofprotein residues), in the presence of bound substrate, theformation of O2N–PhOH competes with the degradation.The interaction of the protein with the substrate can precedethe interaction with peroxide and even with nitrite, withoutaltering the essence of the mechanism.

E þ NO2� , E�NO2

� ð16Þ

E�NO2� þ H2O2 ! Enitr þ H2O ðfastÞ ð17Þ

Enitr þ PhOH , ½Enitr�PhOH� �!2Hþ

E þ O2N�PhOH

þ H2O ð18Þ

Scheme 2. Pathway B mechanism.

Species derived from Enitr may also react with free PhOH,generating phenoxyl radical and thus dimers (according toreaction 15). Although we could not determine the rate of

the competitive dimer formation due to the strong nitriteabsorption in the same region as the dimers absorb(� 300 nm), HPLC analysis of the product mixture afterreaction of 1 or 2 with the system peroxidase/NO2

–/H2O2

shows that, while phenol dimers are formed at every nitriteconcentration, the relative amount of dimers versus nitro-phenol strongly decreases upon increasing [NO2

–] (data notshown). This indicates that reaction 15 competes withreactions 14 and 18 only at low nitrite concentration.

The two nitrating mechanisms can be simultaneouslyoperative, the first one predominating at low and thesecond at high nitrite concentration. When peroxideconcentration is high, the steps involving H2O2 can beconsidered fast. In these conditions, pathway A can bedescribed as a ping-pong mechanism [32], leading to therate equation:

r ¼ kcat½E�1 þ Knitrite

M

½NO2�� þ

KPhOHM

½PhOH�ð19Þ

where kcat represents the turnover rate of enzymaticnitration, andKnitrite

M andKPhOHM are the Michaelis constants

for nitrite and the phenol, respectively.The fraction of the enzyme involved in pathway B is

ruled by the nitrite concentration, through the pre-equilib-rium binding of reaction 16. Thus, because the ratedetermining step of the turnover is reaction 18, the initialrate equation for pathway B is:

r ¼ kcat½E�1 þ Knitrite

M

½NO2��

� �� 1 þ KPhOH

M

½PhOH�

� � ð20Þ

where here KnitriteM is connected to the reciprocal of the

binding constant of reaction 16.The primary kinetic data can be further simplified to

conventional Michaelis–Menten kinetics under conditionsin which either the term Knitrite

M =½NO2� or the termKPhOH

M =½PhOH� become negligible, i.e. as stated above,operating with saturating (or almost saturating) nitrite orphenol concentrations, respectively. It should be noted thatthe biphasic behavior observed in the plot of rate versus[NO2

–] (Fig. 1), is due both to the presence of the twonitration mechanisms and to dimer production in the lownitrite concentration range. The kinetic parameters for thecatalytic reactions by LPO and HRP are collected inTables 4 and 5. The actual enzymatic turnover rates aresomewhat larger because part of the enzyme is engaged inthe nonproductive nitrate formation. For the nitrite inhib-ited reactions of substrate 4 an estimate of the bimolecularrate constants corresponding to the linear part of the plotsin Fig. 2, at low nitrite concentration, was obtained(Table 5). Also, from the decreasing portion of the rateversus nitrite concentration plots, the following values ofthe inhibition constants were estimated: for LPOKI ¼ 20 ± 7 M

)1, for HRP KI ¼ 50 ± 5 M)1.

For comparison purposes we determined the kineticparameters for the LPO and HRP catalyzed oxidation of4 to dimeric coupling products in the presence of hydrogenperoxide, as this particular phenolic substrate was notincluded in our previous studies [25–27]. The followingresultswereobtained(pH 7.5):withLPO,kcat ¼ 16 ± 2 s)1

� FEBS 2004 Peroxidase catalyzed nitration (Eur. J. Biochem. 271) 901

andKM ¼ 11 ± 1 mM; with HRP, kcat ¼ 19 ± 2 s)1 andKM ¼ 20 ± 4 mM.

Nitrite binding

Nitrite forms six coordinated, low-spin adducts with theiron(III) centers of HRP [33] and LPO [34]. When studied inthe conditions used in our kinetic experiments (pH 7.5), thespectra of these adducts displayed the following opticalfeatures: for HRP–NO2

–, kmax ¼ 416 (e 100 mM)1Æcm)1),

534 (e 13.9 mM)1Æcm)1) and 576 nm (e 9.8 mM

)1Æcm)1);for LPO–NO2

–, kmax ¼ 424 (e 101 mM)1Æcm)1), 546

(e 11.4 mM)1Æcm)1) and 588 nm (e 8.5 mM

)1Æcm)1). Spectrataken during titration of an LPO solution with nitriteexhibited several isosbestic points, at 420, 482, 524 and600 nm. Fitting of the data gave a binding constantKb ¼ 22.0 ± 0.5 M

)1. In the case of HRP, the changes inthe protein spectrum with the addition of the ligand arebiphasic, with modest changes at low nitrite concentrations,and do not show isosbestic points. This behavior can beaccounted for by the binding of two nitrite ions to HRP, thefirst one affects marginally the heme environment, probablythrough electrostatic interactions with polar amino acidresidues in the active site, while the second anion binds tothe iron. An estimate of the binding constant for the latterstep gave Kb ¼ 1.3 M

)1 (data not shown).

Nitrate production

Both nitrating species formed according to mechanisms Aand B can undergo competitive degradation to nitrate.Table 6 shows the amount of nitrate produced in variousexperimental conditions. In the absence of enzyme, nitrateformation at pH 7.5 is negligible within the time of theexperiment. In the presence of LPO, the amount of nitrate

produced corresponds to 80% of the hydrogen peroxideoxidizing equivalents. If 2 is added at a concentration thatgives saturation in the steady-state kinetic experiments, theamount of nitrate produced decreases to 50% of theperoxide added. Further addition of phenol, up to 55 mM,does not affect the yield of NO3

– in the reaction. In addition,increasing nitrite concentration from a value below satura-tion (10 mM) to an almost saturating value (140 mM) doesnot change the yield of NO3

–. These findings indicate thatnitrate is formed by degradation of peroxidase-generatedreactive nitrogen species; in the presence of substrate,nitrophenol formation competes with NO3

– formation.

Reduction of peroxidase compound II by substrates

In a recent study, reduction of LPO compound II by nitritewas reported to be fast (3.5 · 105

M)1Æs)1 at pH 7.2) [21].

We found that nitrite is much less efficient in the reductionof HRP compound II, because the bimolecular rateconstant for this reaction is 6.6 ± 0.4 M

)1Æs)1 at pH 7.5

Table 5. Kinetic data as a function of nitrate concentration for the enzymatic nitration of tyrosine derivatives 1–4. Steady-state kinetic parameters

determined for the LPO and HRP mediated nitration of 1–4 by nitrite and hydrogen peroxide as a function of nitrite concentration, in 0.2 M

phosphate buffer, pH 7.5 at 25 �C.

Phenol

LPO HRP

KnitriteM

(mM)

kcat

(s)1)

kcat=KnitriteM

(M)1Æs)1)

KnitriteM

(mM)

kcat

(s)1)

kcat=KnitriteM

(M)1Æs)1)

1 48 ± 5 380 ± 15 7.9 · 103 1600 ± 200 500 ± 30 3.0 · 102

2 30 ± 3 135 ± 5 4.5 · 103 1600 ± 170 580 ± 30 3.6 · 102

3 16 ± 1 80 ± 2 5.0 · 103 1200 ± 200 13 ± 1 11

4 3.6 · 104 3.5 · 104

Table 6. Competitive production of nitrate during enzymatic nitration.

Nitrate produced by the LPO/NO2–/H2O2 system in the presence of

different concentrations of nitrite, phenol 2 and hydrogen peroxide,

after 20 min reaction in 20 mM phosphate buffer, pH 7.5, at 25 �C.

LPO

(lM)

Phenol 2

(mM)

NO2–

(mM)

H2O2

(mM)

NO3–

(mM)

0 5 10 0.8 0.01 ± 0.01

0.1 0 10 0.8 0.67 ± 0.02

0.1 5 10 0.8 0.39 ± 0.01

0.1 55 10 0.8 0.40 ± 0.02

0.1 10 140 0.8 0.40 ± 0.03

0.1 10 10 2 1.36 ± 0.01

Table 4. Kinetic data as a function of phenol concentration for the enzymatic nitration of tyrosine derivatives 1–4. Steady-state kinetic parameters

determined for the LPO and HRP mediated nitration of 1–4 by nitrite and hydrogen peroxide as a function of phenol concentration, in 0.2 M

phosphate buffer, pH 7.5 at 25 �C.

PhOH

LPO HRP

KPhOHM

(mM)

kcat

(s)1)

kcat=KPhOHM

(M)1Æs)1)

KPhOHM

(mM)

kcat

(s)1)

kcat=KPhOHM

(M)1Æs)1)

1 0.12 ± 0.01 380 ± 10 3.2 · 106 6.8 ± 0.7 390 ± 10 5.7 · 104

2 0.14 ± 0.02 130 ± 5 9.4 · 105 1.1 ± 0.2 510 ± 30 4.6 · 105

3 0.11 ± 0.01 75 ± 2 6.8 · 105 0.8 ± 1 17 ± 2 2.0 · 104

4 15 ± 1 60 ± 4 3.8 · 103 40 ± 8 80 ± 10 2.0 · 103

902 E. Monzani et al. (Eur. J. Biochem. 271) � FEBS 2004

(a value of 13.3 M)1Æs)1 was reported previously for this

reaction at pH 7.0 [35]). Data on the rate of reduction ofLPO compound II [36] and HRP compound II by severalphenols [37,38] are available in the literature, althoughsometimes they disagree, possibly because different condi-tions were employed. We determined here the rate constantsof LPO and HRP compound II reduction by the represen-tative substrates 3, obtaining the values of (9.4 ± 0.1) · 103

and (1.1 ± 0.1) · 103M)1Æs)1, respectively, and 4, obtain-

ing (4.5 ± 0.1) · 103 and (9.3 ± 0.3) · 103M)1Æs)1,

respectively, at pH 7.5 and 25 �C.

Stopped-flow experiments

Because of the relatively large enzyme concentrationrequired in these experiments, all the attempts to monitorthe spectrum of the enzymatic species was prevented, even inthe early phase of the reaction, by the very fast developmentof prominent absorptions of the nitrophenolic products,which cover the protein Soret band. Therefore, the spectrumof LPO and HRP could only be monitored when theenzymes were treated with nitrite and hydrogen peroxide inthe absence of phenols. Using LPO and saturating nitrite(150 mM), upon addition of hydrogen peroxide (0.85 mM)the Soret band, initially at 425 nm, shifted to 422 nm in afew seconds, with an isosbestic point at 424 nm (Fig. 4).The final spectrum is most likely due to a LPO–NO2

–

derivative in which the protein has been modified bynitration of some endogenous tyrosine residue(s). Theformation rate of the band at 422 nm is the same as therate of disappearance of the band at 425 nm, with anapparent first-order behavior (kobs ¼ 3.2 ± 0.2 s)1). If aphenol is added to the solution a few seconds after mixing,no substrate nitration is observed. The nitration is insteadobserved if hydrogen peroxide is added together with thesubstrate. These findings indicate that, during the transfor-mation, all the peroxide is consumed. When the experimentwas performed with a subsaturating nitrite concentration,after mixing the enzyme solution with peroxide, the spectraof the iron(III) form of the enzyme (with a weak shoulder atlonger wavelength) was observed. The same feature was

observed using a tenfold larger peroxide concentration.With HRP, either using saturating or subsaturating nitriteconcentrations, upon addition of hydrogen peroxide, thespectrum of compound II was invariably observed(kmax ¼ 422 nm).

Reaction of 2 and 4 with peroxynitrite

The reaction of excess peroxynitrite with 2 and 4 wasstudied in comparative experiments in 200 mM phosphatebuffer (pH 7.5). The reactions yield the same nitrationproducts as in the enzymatic reaction, but the behavior ofthe two phenolic compounds is different. Compound 2 wasalmost completely nitrated by five or 10 molar equivalentsperoxynitrite, with estimated conversions of 90 and 98%,respectively. In the same conditions, the reactivity of phenol4 is almost negligible, the maximum yield of 4-hydroxy-3-nitrobenzonitrile amounting to less than 5%.

Nitration of 5

Phenylacetic acid was used as a probe for various nitratingagents in 200 mM phosphate buffer (pH 7.5). Bolus addi-tions of several volumes of nitrogen dioxide to solutions of 5did not yield any nitration products. When 5 was reactedwith peroxynitrite in the absence of carbon dioxide severalproducts due to nitration, hydroxylation, or both, wereobserved. In the presence of carbon dioxide, nitration byperoxynitrite was quenched. The reactions of peroxidase/H2O2/NO2

– on 5 were found to be dependent on nitriteconcentration. At low nitrite concentration (25 mM) noreaction was observed; with higher nitrite concentrations(0.25 M for LPO, 2 M for HRP), a complex mixture ofproducts was formed. When the reaction was carried outwith an intermediate concentration of nitrite, the numberof products and their yields were reduced. These complexmixtures contained the same products formed by theperoxynitrite reaction.

Discussion

In several diseases, the level of 3-nitrotyrosine increases inhuman tissues and fluids due to the formation of nitratingagents that modify the tyrosines. In vitro, the nitrationreaction can occur according to several pathways and withdifferent nitrating agents. Therefore, more than a singlepathway can also be operative in vivo [40]. The ability of theperoxidase/H2O2 system to oxidize NO2

– to NO2� is well

known [11] and the latter is thought to be responsible forphenol nitrations catalyzed by LPO, myeloperoxidase andHRP [12,16]. So far, peroxynitrite has been excluded asnitrating agent by the analysis of 15N CIDNP experiments[16], and due to the absence of hydroxylated phenylalanineresidues in the products, while NO2

+ has not beenconsidered due to its fast degradation in solution [12].Nonetheless, if the active species does not diffuse into thesolution, but reacts with the substrate bound close to theactive site, nitration by these species could occur before theirdegradation is complete. Our study focused on the systemsof LPO/NO2

–/H2O2 and HRP/NO2–/H2O2. Both are able

to perform the nitration of phenols with, particularly in thefirst case, high efficiency. Phenol dimers are also formed

Fig. 4. Spectral changes of LPOupon reaction with nitrite and hydrogen

peroxide. UV/Vis spectral changes observed with time upon reacting

LPO (2.7 lM) and nitrite (150 mM) with H2O2 (0.85 mM) in 200 mM

phosphate buffer (pH 7.5), at 25 �C. The reaction was followed with a

0.1–2.0 s time scale in a stopped-flow apparatus (0.5 cm path length

cell).

� FEBS 2004 Peroxidase catalyzed nitration (Eur. J. Biochem. 271) 903

competitively, but the importance of this reaction decreasesupon increasing [NO2

–]. It is worth noting, in this respect,that the kcat/KM values associated with dimer formation inthe normal peroxidase cycle [25,26] are much smaller thanthe kcat=KPhOH

M for nitration (Table 1). Therefore, the dimersproduced when a large amount of nitrite is present areprobably derived from phenoxy radicals generated by thenitrating species. The difference in kcat=KPhOH

M for phenolnitration and dimer formation depends largely on theKPhOH

Mvalues, which are much smaller for nitration (Table 4) thanfor dimer formation in the normal peroxidase reaction (upto two orders of magnitude smaller for tyrosine) [24–26].This indicates that in the presence of nitrite, the binding sitesinvolved in the two reactions are different. A close proximitybetween the phenol and the porphyrin is necessary for theelectron transfer that produces a phenoxy radical in thenormal peroxidase reaction, while in the nitration processthe phenol does not need to approach the heme as closely,because it may simply interact at the protein surface.

The kcat values for the enzymatic nitration of 1–3 do notfollow the substrate redox potentials (for the couplephenoxy radical/phenol), which decrease in the order3 > 1 > 2 (with values of Ep of 900, 830 and 810 mVversus Ag/AgCl/KCl saturated in acetate buffer, pH 5,respectively) [27], indicating that these parameters areinfluenced by the substrate disposition in the enzyme–substrate complex. As expected, the kcat values obtainedfrom the rate dependence on nitrite concentration (Table 5)are similar to those obtained varying the phenol concentra-tion. The corresponding Knitrite

M values are connected to theaffinity of nitrite for the protein site where it is transformedinto the nitrating species, in the presence of the phenol.Interestingly, these constants resemble the reciprocal of thenitrite binding constants to the proteins.

According to Schemes 1 and 2, the nitrating speciesproduced by the enzyme is either NO2

�, formed bycompound I or II and nitrite (pathway A), or Enitr formedby the enzyme–nitrite complex and H2O2 in pathway B. ForLPO, the reaction of compound I with nitrite is extremelyfast (� 2 · 107

M)1Æs)1) and produces nitrate instead of

NO2� [21]. The reaction between LPO–NO2

– and H2O2 alsodoes not produce NO2

�, as this would yield compound II,but instead a nitrating species with optical features (Soretband at 425 nm) similar to LPO–NO2

–. We attribute thisspecies to a complexed peroxynitrite:

E�NO2� þ H2O2 ! E�N(O)OO� þ H2O ð21Þ

because the alternative formation of NO2+ would produce

the iron(III) form of the enzyme, which has markedlydifferent optical features. Therefore, the enzymatic nitrationby LPO can only proceed through pathway A at lowconcentrations of nitrite, where one-electron reduction ofcompound I is due to the phenolic substrate, reduction ofcompound II is due to nitrite, and nitrophenol is formed byreaction 14. In other conditions, pathway B is preferred bythis enzyme. For the HRP mediated nitration, NO2

� is themajor nitrating agent, but also in this case the peroxynitritepathway cannot be completely excluded. Compound I canbe competitively reduced by nitrite (k ¼ 6.7 · 105

M)1Æs)1

at pH 6.9 [41]) or the phenol (k � 105)106M)1Æs)1 [42]), but

reduction of compound II can only occur by reaction withthe phenolic substrate (k � 103)106

M)1Æs)1 [43]) to support

an efficient mechanism. In fact, reduction of HRP com-pound II by nitrite (reaction 12) is a slow process, with asecond-order rate constant of 6.6 ± 0.4 MÆs)1, which ismuch smaller than the kcat=K

nitriteM values for nitration of all

the substrates (Table 5). It is thus conceivable that nitrationsmediated by HRP proceed through pathway A up tomoderate concentrations of nitrite. At high nitrite concen-tration, pathway B becomes dominant also for this enzyme,and the biphasic behavior observed in the rate dependenceon nitrite concentration testifies to the change in themechanism.

The presence of phenol dimers in the enzymatic nitra-tions, even at high nitrite concentrations, does not contrastwith the complexed peroxynitrite nature of the species Enitr.In fact, 15N chemical induced dynamic nuclear polarizationexperiments showed that nitrophenol formation by reactionbetween tyrosine and peroxynitrite also occurs through thecoupling of nitrogen dioxide and tyrosyl radical [44]. Inaddition, the large amount of nitrate accompanying theLPO catalyzed nitration reaction can be accounted for bythe promotion of peroxynitrite isomerization by iron(III)porphyrin systems [45].

Cyanophenol 4 is a good mechanistic probe for theenzymatic nitration. The higher redox potential makesoxidation and nitration of this compound by peroxidasesmore difficult than for 1–3. The behavior of 4 differs fromthat of the other substrates in two respects. TheKPhOH

M valuefor nitration of 4 is in the same range as those found in thenormal LPO and HRP mediated peroxidase reactions. Thisindicates that 4 binds to the enzymes in a similar manner inboth types of reactions, i.e. close to the heme [25]. Inaddition, the enzymatic nitration is inhibited by excessnitrite, i.e. in conditions where the peroxynitrite pathway isfavored. As shown by independent experiments, peroxyni-trite is a poor nitrating agent for this substrate. Theenzymatic nitration of 4 can therefore proceed only throughthe NO2

� pathway.In contrast, phenylacetic acid is a good probe for

peroxynitrite. It is known that 5 reacts with peroxynitriteto form nitrophenyl and also nitrophenol derivatives, whilethe reaction is blocked in the presence of CO2 [46]. Wefound that 5 is unreactive both to NO�

2 and the peroxidase/H2O2/NO2

) system in conditions where the predominantmechanism is through NO2

�, i.e. at low [NO2–]. Though, at

high [NO2–], the enzymatic systems produce several nitrated

and hydroxylated products independently of the presence ofCO2. This clearly indicates that in the latter conditions anitrating agent is produced (Enitr) and this behaves likeperoxynitrite. The lack of effect by CO2 further shows thatthe reaction occurs within the protein and is due to iron-bound peroxynitrite and not to free peroxynitrite.

The observation that the peroxidase/H2O2/NO2– system

can proceed through two competing mechanisms raises thequestion of whether, at least for LPO, this may havephysiological relevance. At the low nitrite concentrationpresent in the body most of the enzyme should workthrough pathway A. However, because pathway B is muchmore efficient, even a small fraction of the enzyme actingthrough the bound peroxynitrite intermediate could accountfor a large fraction of the nitrophenol produced. In order toassess this point, we can compare the rate of nitration oftyrosine obtained at 100 lM nitrite concentration (a condi-

904 E. Monzani et al. (Eur. J. Biochem. 271) � FEBS 2004

tion observed during inflammatory processes [12]) with therate extrapolated from the kinetic data reported in Table 5ðrate=½enzyme�extrapolated ¼ kcat=Knitrite

M ½NO2��Þ: As the

latter values are obtained from kinetic measurements at high[NO2

–], they refermostly to pathway B.The rate determinedfor tyrosine nitration by LPO (rate/[LPO]exp ¼ 0.48 s)1)compares with that obtained from extrapolation (rate/[LPO]extrapolated ¼ 0.50 s)1), while the rate determined forHRP is more than one order of magnitude larger thanthat extrapolated (rate/[HRP]exp ¼ 0.021 s)1 versus rate/[HRP]extrapolated ¼ 0.0011 s)1). These results indicate that atphysiological concentrationofnitrite,withLPO, at least partof the nitration reaction occurs through the bound peroxy-nitrite species, whereas with HRP, NO2

Æ is by far the majornitrating agent.

Acknowledgements

This work was supported by the Italian CNR (Target Project

�Biotechnology�) and MIUR (through PRIN), and by the European

INTAS and COST Chemistry programmes. We thank Petr Latal (ETH

Zurich) for the nitrate assay measurements, Reinhard Kissner (ETH

Zurich) and Angelo Taglietti (University of Pavia) for assistance in the

stopped-flow experiments, and Antonella Profumo (University of

Pavia) for the determination of the redox potential of the phenolic

compounds.

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