Báo cáo khóa học: Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide doc

Eur J Biochem 271, 895–906 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03992.x Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide Enrico Monzani1, Raffaella Roncone1, Monica Galliano2, Willem H Koppenol3 and Luigi Casella1 Dipartimento di Chimica Generale, 2Dipartimento di Biochimica, Universita` di Pavia, Italy; 3Institute of Inorganic Chemistry, ETH Hoănggerberg, Zuărich, Switzerland Peroxidases perform the nitration of tyrosine and tyrosyl residues in proteins, in the presence of nitrite and hydrogen peroxide The nitrating species is still unknown but it is usually assumed to be nitrogen dioxide In the present investigation, the nitration of phenolic compounds derived from tyrosine by lactoperoxidase and horseradish peroxidase was studied, with the aim of elucidating the mechanism of the reaction The results indicate that nitrogen dioxide cannot be the only nitrating species and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme-generated nitrogen dioxide and another through a more reactive species, assumed to be complexed peroxynitrite, which is generated by reaction of hydrogen peroxide with the enzyme–nitrite complex The importance of the two pathways depends on peroxide and nitrite concentrations With lactoperoxidase, nitration through the highly reactive intermediate is preferred except at very low nitrite concentration, while with horseradish peroxidase, the nitrogen dioxide driven mechanism is preferred except at very high nitrite concentration The preferred mechanism for the two enzymes is that operative in the physiological nitrite concentration range It is well documented that reactive nitrogen species derived from nitrogen monoxide (NO) are involved in many pathological conditions [1,2] Although NO performs many important physiological functions, ranging from neurotransmission to blood pressure regulation, and is involved in the defence mechanism against microorganisms [3,4], overproduction of NO can have damaging effects [4,5] Nitrite is a major product of nitrogen monoxide metabolism [6] and markedly increased nitrite levels have been detected in situations, e.g during inflammatory processes, where NO is overproduced [7,8] However, nitrite does not accumulate in vivo because it is oxidized (to nitrate) by the Fe2+-O2 form of hemoglobin (oxyHb) or the Fe2+-O2 form of myoglobin (oxyMb), producing the Fe3+ forms of these (metHb and metMb) [6], respectively, or by other inflammatory oxidants such as hypochlorous acid [9], causing the formation of reactive nitrogen species [10] An additional pathway for nitrite oxidation that is receiving increasing attention is its reaction with peroxidases in the presence of hydrogen peroxide [11–16] This reaction produces reactive nitrogen species that have been shown to degrade chlorophyll [11], nitrate tyrosine [12] and tyrosyl residues in proteins [13,14] The latter reactions are of particular importance because, in addition to those involving the more typical peroxidase catalyzed oxidations of chloride and thiocyanate [12], they may serve a defensive function against microorganisms Several mechanisms for the peroxidase catalyzed phenol nitration in the presence of nitrite/hydrogen peroxide have been proposed but, in spite of recent efforts, the nature of the nitrating species has not been fully clarified yet The currently favored pathway [15,16] involves one-electron oxidation of nitrite by the peroxide-generated enzyme intermediates known as compound I and compound II [17,18]: 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) Keywords: nitrogen dioxide; peroxidases; peroxynitrite; phenol nitration; reactive nitrogen species E þ H2 O2 ! compound I þ H2 O compound I ỵ NO2 ! compound II ỵ NO2 1ị 2ị compound II ỵ NO2 ỵ 2Hỵ ! E ỵ NO2 þ H2 O ð3Þ where E represents the native (Fe3+) form of the enzyme For myeloperoxidase, the reaction of the enzyme intermediates by nitrite has been studied recently in detail [15] According to this mechanism, NO2 could either nitrate a phenol with a reaction stoichiometry of : or directly react with a peroxidase-generated phenoxy radical according to reaction 5: NO2 ỵ PhOH ! NO2 ỵ PhO ỵ Hỵ 4ị NO2 ỵ PhO ! O2 NPhOH 5ị An alternative pathway, involving a two-electron enzymatic oxidation of nitrite to nitryl cation, a powerful phenol nitrating agent [19]: E ỵ H2 O2 ! compound I ỵ H2 O compound I ỵ NO2 ỵ ỵ 2H ! E ỵ NO2ỵ 1ị ỵ H2 O ð6Þ is considered unlikely in view of the extremely rapid reaction of NO2+ with water, to yield nitrate [20] Ó FEBS 2004 896 E Monzani et al (Eur J Biochem 271) In a recent report, the reaction of lactoperoxidase compound I with nitrite was found to lead directly to nitrate, without formation of NO2 radicals as intermediates [21] Moreover, additional work on eosinophil peroxidase and myeloperoxidase suggested that these proteins perform nitrations in the presence of nitrite and hydrogen peroxide, generating peroxynitrite [22] Therefore, different enzymes may activate nitrite through different mechanisms In this paper we provide new mechanistic insight into the lactoperoxidase and horseradish peroxidase mediated nitration of the representative tyrosine derivatives 1–4 by nitrite/ hydrogen peroxide and, in particular, we address the problem of the possible contribution of peroxynitrite in this reaction Peroxynitrite nitrates phenolic substrates [23,24] and could conceivably be formed by reaction of hydrogen peroxide with a peroxidase-nitrite complex: E ỵ NO2 ! ENO2 ENO2 ỵ H2 O2 ! EOH ỵ O ẳ NOOH ð7Þ ð8Þ The present investigation extends our previous studies on the peroxidase catalyzed oxidation of phenolic compounds by hydrogen peroxide [25–27] The latter reaction competes with phenol nitration and gives rise to the dimeric and oligomeric phenol coupling products shown Materials and methods Materials and instrumentation Bovine lactoperoxidase was purified according to a new procedure, which is an improvement of that reported by Ferrari et al [28] Horseradish peroxidase (HRP) was obtained from Sigma as a freeze-dried powder (RZ ¼ 3.2 at pH 7.0, e402 ẳ 103 mM)1ặcm)1) L-Tyrosine, 3-nitroL-tyrosine, tyramine, 3-(4-hydroxyphenyl)-propionic acid and 4-hydroxybenzonitrile were from Sigma-Aldrich N-Acetyltyramine was prepared by reaction between tyramine and acetic anhydride as reported previously [25] Peroxynitrite was prepared according to a literature procedure [29] NO2 was obtained by air oxidation of NO All other chemicals were reagent grade and used as received Hydrogen peroxide solutions were freshly prepared by diluting a 30% (v/v) solution in water and were standardized by iodimetry Optical spectra were measured with an HP 8452 A diode array spectrophotometer (Agilent Technologies, Italy) Stopped-flow experiments were carried out with a SMF-3 Bio-Logic coupled to a diode array J & M TIDAS spectrophotometer (J&M Analytische Mess und Regeltechnik GmbH, Germany) with ms dead time and a 0.5 cm path length cell, or an Applied Photophysics model RS-1000 (Applied Photophysics Ltd, UK) instrument with ms dead time and cm or 0.2 cm path length cells NMR spectra were obtained at 400 MHz with a Bruker AVANCE spectrometer (Bruker BioSpin, Italy) Electrospray ionization MS data were acquired using a Finnigan LCQ ion trap mass spectrometer (Thermo, Italy) Purification of lactoperoxidase (LPO) Fresh untreated bovine milk (10 L) was defatted by centrifugation (twice for h at 10 900 g, °C), the pH of the resulting liquid was adjusted to 6.6, and casein was precipitated by addition of M CaCl2 (60 mL per L of defatted milk) After stirring overnight at °C, the precipitate was eliminated by centrifugation (twice for h at 10 900 g, °C) The whey was dialyzed against 10 mM Tris/HCl (pH 7.0) and applied sequentially through two tandemly linked ion exchange columns packed with DEAE-cellulose (5 · 50 cm) and CM-cellulose (2.6 · 30 cm) preequilibrated with the same buffer At the end of sample loading, the unbound material was washed out with the initial buffer until the absorbance at 280 nm became negligible The columns were then detached from each other and the LPO bound to the cation exchange column was eluted by making the buffer 0.5 M in NaCl This step was followed by gel filtration on Sephadex G-100 in a column (6 · 65 cm) equilibrated with 20 mM Tris buffer, 0.15 M NaCl, pH 7.0 The 412 nm absorbing fractions were pooled and, using an Amicon 30 kDa cut-off filter (Millipore), concentrated and equilibrated in 10 mM phosphate buffer, pH 6.8 All these steps were carried out at °C The protein was then separated from contaminating lactoferrin by ion exchange chromatography on a Mono S HR 10/10 column (Amersham Pharmacia Biotech), equililibrated with the phosphate buffer, and connected to an Akta Purifier system (Amersham Pharmacia Biotech) Elution was achieved at a flow rate of mLỈmin)1 for 20 with the equilibration buffer, followed by a threestep linear gradient: from 0–25% of buffer B (10 mM phosphate buffer, M NaCl, pH 6.8) in 20 min, from 25– 35% in 60 and from 35–100% of buffer B in 20 min, and monitored at 280 and 412 nm The lactoperoxidase containing peak was manually collected and the homogeneity of the protein was checked by SDS/PAGE under reducing and nonreducing conditions in 10% gels The enzyme preparation gave 75 mg of protein with RZ > 0.90 The concentration of enzyme solutions was determined optically using e412 ¼ 114 mM)1Ỉcm)1 Preparation of nitrophenols The nitrated derivatives of 1, and are not commercially available and were therefore prepared in a small scale by LPO mediated reactions 3-(4-Hydroxy-3-nitrophenyl)-propionic acid 3-(4-Hydroxyphenyl)-propionic acid (50 mg) was dissolved in 25 mL of a mM phosphate buffer solution at pH 7.5 containing sodium nitrite (0.08 M) To this solution, dilute solutions of LPO in the same buffer (final concentration · 10)8 M) and hydrogen peroxide (final concentration 8.0 · 10)4 M) were added in small amounts during 0.5 h at 25 °C in order to obtain an intense and persistent yellow color Then, the pH of Ó FEBS 2004 Peroxidase catalyzed nitration (Eur J Biochem 271) 897 Table 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 (%) Calculated Found C 51.19 H 4.30 N 6.63 C 50.95 H 4.27 N 6.60 1H NMR (CDCl3) (d) MS (ESI) (m/z) MS/MS (ESI m/z 210.3) (m/z) IR (NaCl, Nujol mull) (mỈcm)1) 10.5 (broad, OH) 7.95 (d, 1H, phenyl 2-H) 7.44 (dd, 1H, phenyl 6-H) 7.12 (d, 1H, phenyl 5-H) 2.96 (t, 2H,CH2-CO) 2.69 (t, 2H, C-CH2-Ph) 212.3 [M+1] 166.3 [(M-CO2)+1] 1510 m(NO2)as 1338 m(NO2)s 850 m(C-N) Table 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) (mỈcm)1) Calculated Found C 52.74 H 5.53 N 15.38 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 5.59 7.18 (dd, 1H, phenyl 6-H) 166.2 [(M-NH3) +1] 1345 m(NO2)s 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) the solution was brought to about in order to obtain the product in the acidic form The reaction products were extracted several times with dichloromethane and the organic phase was rotary evaporated to dryness The residue was chromatographed on a silica gel column using a mixture of dichloromethane/methanol (95 : 5, v/v) 3-(4-Hydroxy-3nitrophenyl)-propionic acid eluted as the first fraction The product was recovered upon evaporation of the solvent (yield % 40%) Table details the analytical data obtained for 3-(4-hydroxy-3-nitrophenyl)-propionic acid The extinction coefficient of 3-(4-hydroxy-3-nitrophenyl)-propionic acid at 422 nm in phosphate buffer (pH 7.5) is 3600 M)1Ỉcm)1 3-Nitrotyramine N-Acetyltyramine (50 mg) was dissolved in 10 mL of a 10 mM phosphate buffer solution at pH 7.5 containing sodium nitrite (0.25 M) To this solution, several additions 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 the mixture was allowed to react while stirring at room temperature for h After acidification to pH 4, the organic products were extracted with chloroform and the solution was rotary evaporated to dryness to give a red solid The crude 3-nitro-N-acetyltyramine was hydrolyzed by refluxing it in a M solution of sodium hydroxide (10 mL) for h After rotary evaporation, the residue was applied on a silica gel column and chromatographed using a gradient of dichloromethane/methanol The product was recovered after evaporation of the solvent (yield 20%) Table details the analytical data obtained for 3-nitrotyramine The extinction coefficient of 3-nitrotyramine at 422 nm in phosphate buffer pH 7.5 is 2800 M)1Ỉcm)1 4-Hydroxy-3-nitrobenzonitrile 4-Hydroxybenzonitrile (100 mg) was dissolved in 25 mL of a mM phosphate buffer solution at pH 7.5 Dilute solutions of LPO (final concentration 5.6 · 10)8 M), hydrogen peroxide (0.88 mM) and sodium nitrite (6 mM) were added in small portions to the substrate solution during h at 25 °C Formation of the product was accompanied by the development of a yellow and persistent color in the solution Then, the pH of the mixture was brought to about in order to obtain the product in the protonated form The solution was extracted several times with ethyl acetate and the organic phase was rotary evaporated to dryness The residue was chromatographed on a silica gel column using dichloromethane as eluent 4-Hydroxy-3-nitrobenzonitrile eluted as the first fraction The product was recovered upon evaporation of the solvent (yield % 5%) Table details the analytical data obtained for 4-hydroxy-3-nitrobenzonitrile The extinction coefficient of 4-hydroxy-3-nitrobenzonitrile at 422 nm in phosphate buffer (pH 7.5) used in the kinetic experiments is 1700 MỈcm)1 and the wavelength of maximum absorption is at 400 nm (e 2200 M)1Ỉcm)1) Kinetic experiments of phenol nitration The kinetics of the enzymatic phenol nitration were studied spectrophotometrically using a magnetically stirred, thermostated optical cell of cm path length, in 200 mM phosphate buffer, pH 7.5 The temperature was maintained at 25 ± 0.1 °C The reactions were conveniently followed through the increase of absorbance at 422 nm, due to the formation of the nitrophenolic derivatives, in the initial phase of the reactions The conversion of the data from Ds)1 to MỈs)1 was performed using the e422 values for 1–4; for 3-nitro-L-tyrosine, the extinction coefficient e422 ¼ 4000 M)1Ỉcm)1 was used In order to reduce the effect of noise in the absorbance readings, the difference between the absorbance at 422 nm and that at 820 nm, where the absorption remains negligible during the assay, was monitored Preliminary experiments were Ó FEBS 2004 898 E Monzani et al (Eur J Biochem 271) Table Analytical data of 4-hydroxy-3-nitrobenzonitrile Elemental analysis, 1H-NMR, MS (ESI) and IR characterization of 4-hydroxy3-nitrobenzonitrile Elemental analysis (%) Calculated Found C 51.23 H 2.46 N 17.07 C 50.80 H 2.54 N 16.89 1H NMR (CDCl3) (d) MS (ESI) (m/z) IR (NaCl, Nujol mull) (mỈcm)1) 10.9 8.5 7.8 7.3 165.1 [M +1] 1510 m(NO2)as 1320 m(NO2)s 870 m(C-N) (broad, OH) (d, 1H, phenyl, 2H) (dd, H, phenyl, 6H) (d, H, phenyl, 5H) performed with substrates 1–4 to find appropriate conditions to follow the reactions and, in particular, to establish conditions of saturation of hydrogen peroxide, in order to avoid inconvenient excess of this reagent Steady-state kinetics were then studied as a function of both the phenol and nitrite concentrations For each substrate, the conditions required to study the rate dependence on the reactants concentrations were found through the following steps: (a) determination of the peroxide concentration that maximizes the nitration rate with high substrate and nitrite concentrations (typically starting from [phenol] ¼ 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 concentration maintaining [H2O2] as optimized in the previous step and high [NO2–]; (c) study of the dependence of the rate versus nitrite concentration maintaining [H2O2] and [PhOH] saturating as found in step b; (d) when the substrate and nitrite concentrations that maximize the rate did not fit with those used in step a, the whole procedure was repeated starting with different [NO2–] and [phenol] in an iterative way The kinetic studies were then performed with the following concentrations of the reactants: (a) dependence of the rate versus phenol concentration with LPO (50 nM): [1] ¼ 0–10 mM, [H2O2] ¼ 0.42 mM, [NO2–] ¼ 94 mM; [2] ¼ 0–1.0 mM, [H2O2] ¼ 0.84 mM, [NO2–] ¼ 78 mM; [3] ¼ 0–20 mM, [H2O2] ¼ 0.84 mM, [NO2–] ¼ 30 mM; [4] ¼ 0–10 mM, [H2O2] ¼ 1.3 mM, [NO2–] ¼ 0.2 M; (b) dependence of the rate versus phenol concentration with HRP (30 nM): [1] ¼ 0–20 mM, [H2O2] ¼ 1.60 mM, [NO2–] ¼ 2.1 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] ¼ 0.42 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] ¼ 1.6 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 at low nitrite concentration The nitration of tyrosine by LPO and HRP was also studied at a pathophysiological concentration of nitrite With LPO (0.50 lM) the concentrations of the reactants were: [H2O2] ¼ 0.84 mM, [3] ¼ 0.59 mM, [NO2–] ¼ 100 lM With HRP (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 Steady state kinetic experiments of catalytic oxidation of by LPO or HRP and hydrogen peroxide were performed in 200 mM phosphate buffer (pH 7.5) at 25 ± 0.1 °C, according to the following procedure To the solution containing the enzyme (56 nM LPO or 71 nM HRP) and variable amounts of (0–50 mM) in an optical quartz cell of cm path length, hydrogen peroxide (0.2 mM) was added to the fixed final volume of 1.6 mL The progress of the reaction was followed by monitoring the absorbance changes at 322 nm due to the formation of the oxidative coupling dimer of The initial rates were determined from the linear part of the trace at 322 nm To convert the rates from Ds)1 to MỈs)1 it was necessary to determine the extinction coefficient of the dimeric product of the reaction This e322 value was obtained from a plot of absorbance versus number of moles of hydrogen peroxide consumed in the HRP-catalyzed oxidation of 4, where hydrogen peroxide was the limiting reagent The following reagent concentrations were used: [HRP] 33 nM, [4] 0.3 mM, and [H2O2] from 3.8 · 10)5 to 1.5 · 10)4 M, the other conditions were the same as in the kinetic experiments From this analysis the e322 value of 5600 M)1Ỉcm)1 was obtained Reduction of compound II by substrates The second-order catalytic constant for the reaction between HRP compound II and nitrite was determined in 200 mM phosphate buffer (pH 7.5) at 25.0 ± 0.1 °C Compound II was prepared by incubation of the protein solution (7.0 lM) with a small excess (two mol equivalents) of hydrogen peroxide for The transformation to the iron(III) species was followed by monitoring the absorbance changes of the protein with time (readings every 0.1 s), using a variable excess of nitrite (from 40 lM to 1.6 mM) The compound II reduction to iron(III) followed a first-order behavior In order to decrease the noise in the readings, the determination of the observed rate constants (kobs) was performed following the reaction at the two wavelengths where the spectral changes are largest and interpolating their difference in absorbance (A400–A420 nm) with a firstorder equation The replot of kobs versus [NO2–] was linear and the slope gave the catalytic constant In a similar way, the second-order catalytic constants for the reaction between LPO or HRP compound II and the representative phenols and were determined The enzyme compound II derivatives (2 lM) were prepared as described before Solutions of the substrates (1–10 mM) in an appropriate volume of 200 mM phosphate buffer (pH 7.5) were Ó FEBS 2004 prepared from fresh stock solutions The reactions were carried out under pseudo-first-order conditions and followed by monitoring the disappearance of compound II with time (readings every 0.1 s) The rate constants (kobs) were determined from the changes in the difference of absorbance (A402–A420) with time, which were fitted to a first-order equation The replots of kobs versus [phenol] were linear and the slopes gave the catalytic constants Stopped-flow experiments The reaction between LPO, nitrite and H2O2 was followed in a stopped-flow apparatus using an optical cell of path length 0.5 cm; one of the syringes was filled with a solution of the enzyme (5.4 lM) and NaNO2 (20 or 300 mM) in 200 mM phosphate buffer (pH 7.5) at 25 °C The other syringe was filled with H2O2 (1.7 mM) Mixing of the two solutions in the reaction cuvette reduced the concentration of the reactants to one half Control experiments were carried out without peroxide and with either one tenth or twofold concentration of the oxidant In analogous experiments performed with HRP, a path length of 0.2 cm was used; one of the syringes was filled with a solution of the enzyme (75 lM) and NaNO2 (2 M or 50 mM) in the same buffer as above The other syringe was filled with H2O2 (2.0 mM) Control experiments were carried out without peroxide Nitrate assay The determination of nitrate formed competitively by the enzymatic reaction with nitrite and hydrogen peroxide in various conditions was carried out using a Metrohm IC ion chromatograph (Metrohn AG, Switzerland) with a SuperSep column at a mLỈmin)1 flow rate All the experiments were performed in triplicate In a typical experiment, 10 mM sodium nitrite was allowed to react for 20 with 0.8 mM hydrogen peroxide in the presence of mM and 10 nM LPO in 20 mM phosphate buffer (higher buffer concentrations reduce the sensitivity of nitrate determination) pH 7.5, at 25 °C Then, the sample was diluted tenfold in double distilled water and injected into the column Other experiments were performed without enzyme, in the absence or presence of substrate (55 mM), and with 140 mM nitrite and mM hydrogen peroxide Binding experiments The binding of nitrite to LPO and HRP was studied spectrophotometrically, by following the spectral changes upon addition of small aliquots of a concentrated NaNO2 solution in 200 mM phosphate buffer (pH 7.5) to the enzyme solution in the same buffer, at 25 ± 0.1 °C No attempts were made to keep the ionic strength constant With LPO (6 · 10)6 M), a M stock nitrite solution and an optical cell of cm path length were used In the case of HRP (6 · 10)5 M), the binding process exhibited biphasic behavior and, in order to reach saturation in the second step, the titration was performed using a more concentrated solution of NaNO2 (4 M) in a cell with a smaller path length (0.1 cm) The spectral data were analyzed, after subtraction of the absorption due to free nitrite, as described previously [30] to obtain equilibrium constants and stoichiometry of adduct formation Peroxidase catalyzed nitration (Eur J Biochem 271) 899 Differential pulse voltammetry Polarographic experiments on substrates and were performed at room temperature in 200 mM phosphate buffer (pH 7.5), using an Amel model 591/ST Polarograph coupled with an Amel 433 Trace Analyzer, with a glassy carbon electrode and an Ag/AgCl/KCl saturated reference electrode The scans were performed from 300 to 1200 mV using a differential pulse voltammetry of 100 mVỈs)1 and a pulse amplitude of 50 mV The redox potential measured polarographically corresponds to the transformation of the phenols to the corresponding phenoxide radicals; the values of 840 mV (versus Ag/AgCl/KCl saturated) for and 790 mV for were found Voltammeric oxidation of phenols causes passivation of the electrode surface that results in rapidly diminishing voltammetric curve response and enlarged peaks For this reason, the absolute values of the oxidation potentials of the compounds investigated may be affected by experimental conditions (electrode surface, pH and concentration of the solutions) However, the differences between the values of the oxidation potentials found are significant because they were obtained in the same experimental conditions HPLC analysis of the nitration products The product mixtures derived from the chemical or enzymatic nitration of compounds 1–4 and phenylacetic acid (5) were analyzed by HPLC using a Jasco MD-1510 instrument with diode array detection and a Supelco LC18 reverse-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 flow rate of mLỈmin)1 Elution started with 100% solvent A for min, followed by a linear gradient from 100% A to 100% B in 20 Spectrophotometric detection of the eluate was performed in the range 200–600 nm Reaction of and with peroxynitrite Solutions of the phenol (2 or 4) (1 mM) in 200 mM phosphate buffer (pH 7.5) were treated with five- or tenfold molar excess peroxynitrite for at room temperature The reaction mixtures were analyzed by HPLC as described above The retention times of and were 11.5 and 12.7 min, respectively, and those of the corresponding nitration products, 3-(4-hydroxy-3-nitrophenyl)-propionic acid and 4-hydroxy-3-nitrobenzonitrile, were 13.9 and 15.7 min, respectively The identity of the products was checked by comparison with the spectra of authentic samples Yields of nitration products were estimated from the extinction coefficients of the phenolic derivatives and the peak areas in the HPLC chromatograms Nitration of phenylacetic acid, CO2-free peroxynitrite A solution of was purged with argon for 20 and then reacted with tenfold molar excess peroxynitrite HPLC analysis of the reaction mixture showed, as well as unreacted 5, five minor peaks with retention times of 9.9, 10.1, 10.4, 12.2 and 12.4 900 E Monzani et al (Eur J Biochem 271) Ó FEBS 2004 Peroxynitrite-CO2 A solution of (1 mM) in 200 mM phosphate buffer (pH 7.5), equilibrated with atmospheric CO2, was treated with five- or tenfold molar excess peroxynitrite for at room temperature, and then analyzed by HPLC The chromatogram contained only one peak corresponding to 5, with a retention time of 11.6 Peroxidase/H2O2/NO2– The catalytic nitrations of were performed under the following experimental conditions: HRP (30 nM), [5] ¼ mM, [H2O2] ¼ 0.4 mM, [NaNO2] ¼ 0, 0.025, 0.25 or M; LPO (50 nM), [5] ¼ mM, [H2O2] ¼ 1.3 mM, [NaNO2] ¼ 0, 0.025 or 0.25 M Analysis of the product mixtures resulting from the reactions carried out in the presence of high nitrite concentrations (2 M for HRP and 0.25 M for LPO) showed extensive modification of substrate The HPLC chromatogram showed two main peaks at 11.1 and 11.3 min, and minor peaks for unreacted and six other products with retention times of 9.9, 10.4, 11.8, 12.0, 12.2 and 12.4 Fig Biphasic behavior of the rate of HRP-mediated phenol nitration as a function of nitrite concentration Rate dependence of HRP-catalyzed nitration of 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 Formation of phenol dimers by the HRP/H2O2/NO2– system The phenol dimers formed by the HRP/H2O2/NO2– system during the nitration of and were analyzed by HPLC in the same conditions as reported in a previous work [31] The reactions were performed in 200 mM phosphate buffer (pH 7.5) at 25 °C in the presence of 30 nM HRP and variable nitrite concentration (0, 0.2, and 1.0 M) The other 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 following the characteristic absorption near 420 nm of the nitrophenols in neutral medium At this wavelength the interference by the phenolic dimers formed according to the normal peroxidase reaction [25–27] is completely negligible The enzymatic nitration reaction of phenols 1–4 was studied as a function of both the phenol and nitrite concentrations, with the other reagents saturating, except for the HRP experiments, where saturating nitrite concentrations were too high In these cases, the kinetics were studied at a nitrite concentration corresponding to % 60% saturation With both LPO and HRP, the rate of the enzymatic reaction exhibits a hyperbolic dependence on the concentration of the phenols A more complex behavior was found when the rate dependence was studied as a function of nitrite concentration For substrates 1–3, the LPO mediated reactions exhibited a normal saturation behavior, while the HRP mediated reactions were biphasic (Fig 1) With substrate 4, inhibition was observed for both LPO and HRP at moderate concentrations of nitrite (Figs and 3) The saturation behavior found for 1–3 is not due to nitrite inhibition on the peroxide involving step, as this should be connected to a change in the slow step; instead, the rate does not increase on increasing peroxide concentration Fig Inhibition of the LPO-mediated phenol nitration by nitrite Plot of the rate of LPO-catalyzed nitration of phenol 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 concentration range Fig Inhibition of the HRP-mediated phenol nitration by nitrite Plot of the rate of HRP-catalyzed nitration of phenol 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 concentration range These findings indicate that an efficient nitration reaction requires the interaction of the enzyme with both nitrite and phenol Ó FEBS 2004 Peroxidase catalyzed nitration (Eur J Biochem 271) 901 The experimental data can be interpreted considering two simplified mechanisms, which differ with respect to the active species involved and for the dependence on the oxidant concentration The first mechanism, pathway A (Scheme 1), considers that product formation follows reactions 9–14 (corresponding to reactions 1–5 shown above) Compound I and compound II react with nitrite or the phenol generating free diffusible radicals [16] The nitrating agent is thus NO2  , which can be derived from either compound I or compound II The observation of substrate saturation behavior indicates that, even if NO2  could react with free phenol, the reaction is faster when the phenoxy radical is formed by direct reaction with compound I or compound II E ỵ H2 O2 ! compound I ỵ H2 O (fast) 9ị compound I ỵ NO2 , compound I=NO2 ! compound II ỵ NO2 10ị compound I ỵ PhOH , compound I=PhOH ! compound II ỵ PhO ỵ Hỵ 11ị compound II ỵ NO2 , compound II=NO2 2Hỵ E ỵ NO2 ỵ H2 O ! 12ị compound II ỵ PhOH , compound II=PhOH 2Hỵ E ỵ PhO2 ỵ H2 O ! 13ị PhO ỵ NO2 ! O2 NÀPhOH ð14Þ Scheme Pathway A mechanism The PhO• produced can also give rise to dimeric coupling products through the normal peroxidase catalytic cycle [25–27]: 2PhO ! dimers ð15Þ The second mechanism (pathway B), represented in Scheme 2, where E–NO2– is the peroxidase-nitrite complex and Enitr the nitrating active species This mechanism first considers 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 of protein residues), in the presence of bound substrate, the formation of O2N–PhOH competes with the degradation The interaction of the protein with the substrate can precede the interaction with peroxide and even with nitrite, without altering the essence of the mechanism E ỵ NO2 , ENO2 ENO2 ỵ H2 O2 ! Enitr ỵ H2 O fastị 2H 16ị 17ị ỵ Enitr þ PhOH , ½Enitr ÀPhOHŠ À E þ O2 NÀPhOH ! ỵ H2 O 18ị Scheme Pathway B mechanism Species derived from Enitr may also react with free PhOH, generating phenoxyl radical and thus dimers (according to reaction 15) Although we could not determine the rate of the competitive dimer formation due to the strong nitrite absorption in the same region as the dimers absorb (% 300 nm), HPLC analysis of the product mixture after reaction of or with the system peroxidase/NO2–/H2O2 shows that, while phenol dimers are formed at every nitrite concentration, the relative amount of dimers versus nitrophenol strongly decreases upon increasing [NO2–] (data not shown) This indicates that reaction 15 competes with reactions 14 and 18 only at low nitrite concentration The two nitrating mechanisms can be simultaneously operative, the first one predominating at low and the second at high nitrite concentration When peroxide concentration is high, the steps involving H2O2 can be considered fast In these conditions, pathway A can be described as a ping-pong mechanism [32], leading to the rate equation: r¼ 1ỵ kcat ẵE Knitrite KPhOH M ỵ M ẵNO2 ½PhOHŠ ð19Þ where kcat represents the turnover rate of enzymatic nitration, and Knitrite and KPhOH are the Michaelis constants M M for nitrite and the phenol, respectively The fraction of the enzyme involved in pathway B is ruled by the nitrite concentration, through the pre-equilibrium binding of reaction 16 Thus, because the rate determining step of the turnover is reaction 18, the initial rate equation for pathway B is: r¼ 1ỵ kcat ẵE  ỵ Knitrite M ½NO2 Š KPhOH M  ð20Þ ½PhOHŠ where here Knitrite is connected to the reciprocal of the M binding constant of reaction 16 The primary kinetic data can be further simplified to conventional Michaelis–Menten kinetics under conditions nitrite in which either the term KM =½NO2 Š or the term PhOH KM =½PhOHŠ become negligible, i.e as stated above, operating with saturating (or almost saturating) nitrite or phenol concentrations, respectively It should be noted that the biphasic behavior observed in the plot of rate versus [NO2–] (Fig 1), is due both to the presence of the two nitration mechanisms and to dimer production in the low nitrite concentration range The kinetic parameters for the catalytic reactions by LPO and HRP are collected in Tables and The actual enzymatic turnover rates are somewhat larger because part of the enzyme is engaged in the nonproductive nitrate formation For the nitrite inhibited reactions of substrate an estimate of the bimolecular rate constants corresponding to the linear part of the plots in Fig 2, at low nitrite concentration, was obtained (Table 5) Also, from the decreasing portion of the rate versus nitrite concentration plots, the following values of the inhibition constants were estimated: for LPO KI ¼ 20 ± M)1, for HRP KI ¼ 50 ± M)1 For comparison purposes we determined the kinetic parameters for the LPO and HRP catalyzed oxidation of to dimeric coupling products in the presence of hydrogen peroxide, as this particular phenolic substrate was not included in our previous studies [25–27] The following results were obtained (pH 7.5): with LPO, kcat ¼ 16 ± s)1 Ó FEBS 2004 902 E Monzani et al (Eur J Biochem 271) Table 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 LPO HRP PhOH PhOH KM (mM) 0.12 0.14 0.11 15 ± ± ± ± PhOH kcat =KM (M)1Ỉs)1) kcat (s)1) 0.01 0.02 0.01 380 130 75 60 ± ± ± ± 10 3.2 9.4 6.8 3.8 · · · · 106 105 105 103 PhOH KM (mM) 6.8 1.1 0.8 40 ± ± ± ± PhOH kcat =KM (M)1Ỉs)1) kcat (s)1) 0.7 0.2 390 510 17 80 ± ± ± ± 10 30 10 5.7 4.6 2.0 2.0 · · · · 104 105 104 103 Table 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 LPO Phenol HRP nitrite KM (mM) kcat (s)1) nitrite kcat =KM (M)1Ỉs)1) 48 ± 30 ± 16 ± 380 ± 15 135 ± 80 ± 7.9 4.5 5.0 3.6 · · · · nitrite KM (mM) and KM ¼ 11 ± mM; with HRP, kcat ¼ 19 ± s)1 and KM ¼ 20 ± mM Nitrite binding Nitrite forms six coordinated, low-spin adducts with the iron(III) centers of HRP [33] and LPO [34] When studied in the conditions used in our kinetic experiments (pH 7.5), the spectra of these adducts displayed the following optical features: for HRPNO2, kmax ẳ 416 (e 100 mM)1ặcm)1), 534 (e 13.9 mM)1Ỉcm)1) and 576 nm (e 9.8 mM)1Ỉcm)1); for LPONO2, kmax ẳ 424 (e 101 mM)1ặcm)1), 546 (e 11.4 mM)1Ỉcm)1) and 588 nm (e 8.5 mM)1Ỉcm)1) Spectra taken during titration of an LPO solution with nitrite exhibited several isosbestic points, at 420, 482, 524 and 600 nm Fitting of the data gave a binding constant Kb ¼ 22.0 ± 0.5 M)1 In the case of HRP, the changes in the protein spectrum with the addition of the ligand are biphasic, with modest changes at low nitrite concentrations, and not show isosbestic points This behavior can be accounted for by the binding of two nitrite ions to HRP, the first one affects marginally the heme environment, probably through electrostatic interactions with polar amino acid residues in the active site, while the second anion binds to the iron An estimate of the binding constant for the latter step gave Kb ¼ 1.3 M)1 (data not shown) nitrite kcat =KM (M)1Ỉs)1) 1600 ± 200 1600 ± 170 1200 ± 200 103 103 103 104 kcat (s)1) 500 ± 30 580 ± 30 13 ± 3.0 · 102 3.6 · 102 11 3.5 · 104 Table Competitive production of nitrate during enzymatic nitration Nitrate produced by the LPO/NO2–/H2O2 system in the presence of different concentrations of nitrite, phenol and hydrogen peroxide, after 20 reaction in 20 mM phosphate buffer, pH 7.5, at 25 °C LPO (lM) Phenol (mM) NO2– (mM) H2O2 (mM) NO3– (mM) 0.1 0.1 0.1 0.1 0.1 5 55 10 10 10 10 10 10 140 10 0.8 0.8 0.8 0.8 0.8 0.01 0.67 0.39 0.40 0.40 1.36 ± ± ± ± ± ± 0.01 0.02 0.01 0.02 0.03 0.01 produced corresponds to 80% of the hydrogen peroxide oxidizing equivalents If is added at a concentration that gives saturation in the steady-state kinetic experiments, the amount of nitrate produced decreases to 50% of the peroxide 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 saturation (10 mM) to an almost saturating value (140 mM) does not change the yield of NO3– These findings indicate that nitrate is formed by degradation of peroxidase-generated reactive nitrogen species; in the presence of substrate, nitrophenol formation competes with NO3– formation Nitrate production Both nitrating species formed according to mechanisms A and B can undergo competitive degradation to nitrate Table shows the amount of nitrate produced in various experimental conditions In the absence of enzyme, nitrate formation at pH 7.5 is negligible within the time of the experiment In the presence of LPO, the amount of nitrate Reduction of peroxidase compound II by substrates In a recent study, reduction of LPO compound II by nitrite was reported to be fast (3.5 · 105 M)1Ỉs)1 at pH 7.2) [21] We found that nitrite is much less efficient in the reduction of HRP compound II, because the bimolecular rate constant for this reaction is 6.6 ± 0.4 M)1Ỉs)1 at pH 7.5 Ĩ FEBS 2004 (a value of 13.3 M)1Ỉs)1 was reported previously for this reaction at pH 7.0 [35]) Data on the rate of reduction of LPO compound II [36] and HRP compound II by several phenols [37,38] are available in the literature, although sometimes they disagree, possibly because different conditions were employed We determined here the rate constants of LPO and HRP compound II reduction by the representative substrates 3, obtaining the values of (9.4 ± 0.1) · 103 and (1.1 ± 0.1) · 103 M)1Ỉs)1, respectively, and 4, obtaining (4.5 ± 0.1) · 103 and (9.3 ± 0.3) · 103 M)1Ỉs)1, respectively, at pH 7.5 and 25 °C Stopped-flow experiments Because of the relatively large enzyme concentration required in these experiments, all the attempts to monitor the spectrum of the enzymatic species was prevented, even in the early phase of the reaction, by the very fast development of prominent absorptions of the nitrophenolic products, which cover the protein Soret band Therefore, the spectrum of LPO and HRP could only be monitored when the enzymes were treated with nitrite and hydrogen peroxide in the 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 a few 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 by nitration of some endogenous tyrosine residue(s) The formation rate of the band at 422 nm is the same as the rate of disappearance of the band at 425 nm, with an apparent first-order behavior (kobs ¼ 3.2 ± 0.2 s)1) If a phenol is added to the solution a few seconds after mixing, no substrate nitration is observed The nitration is instead observed if hydrogen peroxide is added together with the substrate These findings indicate that, during the transformation, all the peroxide is consumed When the experiment was performed with a subsaturating nitrite concentration, after mixing the enzyme solution with peroxide, the spectra of the iron(III) form of the enzyme (with a weak shoulder at longer wavelength) was observed The same feature was Fig Spectral changes of LPO upon 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) Peroxidase catalyzed nitration (Eur J Biochem 271) 903 observed using a tenfold larger peroxide concentration With HRP, either using saturating or subsaturating nitrite concentrations, upon addition of hydrogen peroxide, the spectrum of compound II was invariably observed (kmax ¼ 422 nm) Reaction of and with peroxynitrite The reaction of excess peroxynitrite with and was studied in comparative experiments in 200 mM phosphate buffer (pH 7.5) The reactions yield the same nitration products as in the enzymatic reaction, but the behavior of the two phenolic compounds is different Compound was almost completely nitrated by five or 10 molar equivalents peroxynitrite, with estimated conversions of 90 and 98%, respectively In the same conditions, the reactivity of phenol is almost negligible, the maximum yield of 4-hydroxy-3nitrobenzonitrile amounting to less than 5% Nitration of Phenylacetic acid was used as a probe for various nitrating agents in 200 mM phosphate buffer (pH 7.5) Bolus additions of several volumes of nitrogen dioxide to solutions of did not yield any nitration products When was reacted with peroxynitrite in the absence of carbon dioxide several products due to nitration, hydroxylation, or both, were observed In the presence of carbon dioxide, nitration by peroxynitrite was quenched The reactions of peroxidase/ H2O2/NO2– on were found to be dependent on nitrite concentration At low nitrite concentration (25 mM) no reaction was observed; with higher nitrite concentrations (0.25 M for LPO, M for HRP), a complex mixture of products was formed When the reaction was carried out with an intermediate concentration of nitrite, the number of products and their yields were reduced These complex mixtures contained the same products formed by the peroxynitrite reaction Discussion In several diseases, the level of 3-nitrotyrosine increases in human tissues and fluids due to the formation of nitrating agents that modify the tyrosines In vitro, the nitration reaction can occur according to several pathways and with different nitrating agents Therefore, more than a single pathway can also be operative in vivo [40] The ability of the peroxidase/H2O2 system to oxidize NO2– to NO2  is well known [11] and the latter is thought to be responsible for phenol nitrations catalyzed by LPO, myeloperoxidase and HRP [12,16] So far, peroxynitrite has been excluded as nitrating agent by the analysis of 15N CIDNP experiments [16], and due to the absence of hydroxylated phenylalanine residues in the products, while NO2+ has not been considered due to its fast degradation in solution [12] Nonetheless, if the active species does not diffuse into the solution, but reacts with the substrate bound close to the active site, nitration by these species could occur before their degradation is complete Our study focused on the systems of LPO/NO2–/H2O2 and HRP/NO2–/H2O2 Both are able to perform the nitration of phenols with, particularly in the first case, high efficiency Phenol dimers are also formed Ó FEBS 2004 904 E Monzani et al (Eur J Biochem 271) competitively, but the importance of this reaction decreases upon increasing [NO2–] It is worth noting, in this respect, that the kcat/KM values associated with dimer formation in the normal peroxidase cycle [25,26] are much smaller than PhOH the kcat =KM for nitration (Table 1) Therefore, the dimers produced when a large amount of nitrite is present are probably derived from phenoxy radicals generated by the PhOH nitrating species The difference in kcat =KM for phenol PhOH nitration and dimer formation depends largely on the KM values, which are much smaller for nitration (Table 4) than for dimer formation in the normal peroxidase reaction (up to two orders of magnitude smaller for tyrosine) [24–26] This indicates that in the presence of nitrite, the binding sites involved in the two reactions are different A close proximity between the phenol and the porphyrin is necessary for the electron transfer that produces a phenoxy radical in the normal peroxidase reaction, while in the nitration process the 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 not follow the substrate redox potentials (for the couple phenoxy radical/phenol), which decrease in the order > > (with values of Ep of 900, 830 and 810 mV versus Ag/AgCl/KCl saturated in acetate buffer, pH 5, respectively) [27], indicating that these parameters are influenced by the substrate disposition in the enzyme– substrate complex As expected, the kcat values obtained from the rate dependence on nitrite concentration (Table 5) are similar to those obtained varying the phenol concentranitrite tion The corresponding KM values are connected to the affinity of nitrite for the protein site where it is transformed into the nitrating species, in the presence of the phenol Interestingly, these constants resemble the reciprocal of the nitrite binding constants to the proteins According to Schemes and 2, the nitrating species produced by the enzyme is either NO2  , formed by compound I or II and nitrite (pathway A), or Enitr formed by the enzyme–nitrite complex and H2O2 in pathway B For LPO, the reaction of compound I with nitrite is extremely fast (% · 107 M)1Ỉs)1) and produces nitrate instead of NO2  [21] The reaction between LPO–NO2– and H2O2 also does not produce NO2  , as this would yield compound II, but instead a nitrating species with optical features (Soret band at 425 nm) similar to LPO–NO2– We attribute this species to a complexed peroxynitrite: ENO2 ỵ H2 O2 ! EN(O)OO ỵ H2 O NO2+ 21ị would produce because the alternative formation of the iron(III) form of the enzyme, which has markedly different optical features Therefore, the enzymatic nitration by LPO can only proceed through pathway A at low concentrations of nitrite, where one-electron reduction of compound I is due to the phenolic substrate, reduction of compound II is due to nitrite, and nitrophenol is formed by reaction 14 In other conditions, pathway B is preferred by this enzyme For the HRP mediated nitration, NO2  is the major nitrating agent, but also in this case the peroxynitrite pathway cannot be completely excluded Compound I can be competitively reduced by nitrite (k ẳ 6.7 à 105 M)1ặs)1 at pH 6.9 [41]) or the phenol (k % 105)106 M)1Ỉs)1 [42]), but reduction of compound II can only occur by reaction with the phenolic substrate (k % 103)106 M)1Ỉs)1 [43]) to support an efficient mechanism In fact, reduction of HRP compound II by nitrite (reaction 12) is a slow process, with a second-order rate constant of 6.6 ± 0.4 MỈs)1, which is much smaller than the kcat =Knitrite values for nitration of all M the substrates (Table 5) It is thus conceivable that nitrations mediated by HRP proceed through pathway A up to moderate concentrations of nitrite At high nitrite concentration, pathway B becomes dominant also for this enzyme, and the biphasic behavior observed in the rate dependence on nitrite concentration testifies to the change in the mechanism The presence of phenol dimers in the enzymatic nitrations, even at high nitrite concentrations, does not contrast with the complexed peroxynitrite nature of the species Enitr In fact, 15N chemical induced dynamic nuclear polarization experiments showed that nitrophenol formation by reaction between tyrosine and peroxynitrite also occurs through the coupling of nitrogen dioxide and tyrosyl radical [44] In addition, the large amount of nitrate accompanying the LPO catalyzed nitration reaction can be accounted for by the promotion of peroxynitrite isomerization by iron(III) porphyrin systems [45] Cyanophenol is a good mechanistic probe for the enzymatic nitration The higher redox potential makes oxidation and nitration of this compound by peroxidases more difficult than for 1–3 The behavior of differs from PhOH that of the other substrates in two respects The KM value for nitration of is in the same range as those found in the normal LPO and HRP mediated peroxidase reactions This indicates that binds to the enzymes in a similar manner in both types of reactions, i.e close to the heme [25] In addition, the enzymatic nitration is inhibited by excess nitrite, i.e in conditions where the peroxynitrite pathway is favored As shown by independent experiments, peroxynitrite is a poor nitrating agent for this substrate The enzymatic nitration of can therefore proceed only through the NO2  pathway In contrast, phenylacetic acid is a good probe for peroxynitrite It is known that reacts with peroxynitrite to form nitrophenyl and also nitrophenol derivatives, while the reaction is blocked in the presence of CO2 [46] We found that is unreactive both to NO and the peroxidase/ H2O2/NO2) system in conditions where the predominant  mechanism 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 of CO2 This clearly indicates that in the latter conditions a nitrating agent is produced (Enitr) and this behaves like peroxynitrite The lack of effect by CO2 further shows that the reaction occurs within the protein and is due to ironbound peroxynitrite and not to free peroxynitrite The observation that the peroxidase/H2O2/NO2– system can proceed through two competing mechanisms raises the question of whether, at least for LPO, this may have physiological relevance At the low nitrite concentration present in the body most of the enzyme should work through pathway A However, because pathway B is much more efficient, even a small fraction of the enzyme acting through the bound peroxynitrite intermediate could account for a large fraction of the nitrophenol produced In order to assess this point, we can compare the rate of nitration of tyrosine obtained at 100 lM nitrite concentration (a condi- Ó FEBS 2004 tion observed during inflammatory processes [12]) with the rate extrapolated from the kinetic data reported in Table nitrite rate=ẵenzymeextrapolated ẳ kcat =KM ẵNO2 ị: As the latter values are obtained from kinetic measurements at high [NO2–], they refer mostly to pathway B The rate determined for 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 for HRP is more than one order of magnitude larger than that extrapolated (rate/[HRP]exp ¼ 0.021 s)1 versus rate/ [HRP]extrapolated ¼ 0.0011 s)1) These results indicate that at physiological concentration of nitrite, with LPO, 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peroxynitrite and the peroxynitrite-CO2 adduct and of reactions with L-tyrosine and related compounds as studied by 15N chemically induced dynamic nuclear polarization Arch Biochem Biophys 368, 303–318  ... derivative in which the protein has been modified by nitration of some endogenous tyrosine residue(s) The formation rate of the band at 422 nm is the same as the rate of disappearance of the band at 425... 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... 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