In this paper we provide new mechanistic insight into the lacto-peroxidase and horseradish lacto-peroxidase mediated nitration of the representative tyrosine derivatives 1–4 by nitrite/
Trang 1Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide
Enrico Monzani1, Raffaella Roncone1, Monica Galliano2, Willem H Koppenol3and Luigi Casella1
1
Dipartimento di Chimica Generale,2Dipartimento di Biochimica, Universita` di Pavia, Italy;3Institute of Inorganic Chemistry, ETHHo¨nggerberg, Zu¨rich, 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
peroxi-dase 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 con-centrations 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
Keywords: nitrogen dioxide; peroxidases; peroxynitrite; phenol nitration; reactive nitrogen species
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
neuro-transmission to blood pressure regulation, and is involved in
the defence mechanism against microorganisms [3,4],
over-production 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
inflam-matory 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
invol-ving 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 com-pound I and comcom-pound II [17,18]:
Eþ H2O2! compound I þ H2O ð1Þ compound Iþ NO2 ! compound II þ NO2 ð2Þ compound IIþ 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 nitrate
a phenol with a reaction stoichiometry of 2 : 1 or directly react with a peroxidase-generated phenoxy radical accord-ing to reaction 5:
NO2 þ PhOH ! NO2 þ PhOþ Hþ ð4Þ
An alternative pathway, involving a two-electron enzymatic oxidation of nitrite to nitryl cation, a powerful phenol nitrating agent [19]:
Eþ H2O2! compound I þ H2O ð1Þ compound Iþ NO2 þ 2Hþ! E þ NO2 þþ H2O ð6Þ
is considered unlikely in view of the extremely rapid reaction
of NO+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)
Trang 2In 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
lacto-peroxidase and horseradish lacto-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:
ENO2 þ H2O2! EOHþ O ¼ NOOH 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 (RZ¼ 3.2
at pH 7.0, e402¼ 103 mM )1Æcm)1) L-Tyrosine,
3-nitro-L-tyrosine, tyramine, 3-(4-hydroxyphenyl)-propionic acid
and 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 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
standard-ized by iodimetry Optical spectra were measured with an
HP 8452 A diode array spectrophotometer (Agilent
Tech-nologies, 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 6 ms dead time and a
0.5 cm path length cell, or an Applied Photophysics model
RS-1000 (Applied Photophysics Ltd, UK) instrument with 1 ms dead time and 1 cm or 0.2 cm path length cells NMR spectra were obtained at 400 MHz with a Bruker AVANCE spectrometer (Bruker BioSpin, Italy) Electro-spray 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 centrif-ugation (twice for 1 h at 10 900 g, 4C), the pH of the resulting liquid was adjusted to 6.6, and casein was precipi-tated by addition of 2MCaCl2(60 mL per L of defatted milk) After stirring overnight at 4C, the precipitate was eliminated by centrifugation (twice for 1 h at 10 900 g,
4C) 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 0.5Min NaCl This step was followed by gel filtration on Sephadex G-100 in a column (6· 65 cm) equilibrated with
20 mM Tris buffer, 0.15M NaCl, pH 7.0 The 412 nm absorbing fractions were pooled and, using an Amicon
30 kDa cut-off filter (Millipore), concentrated and equili-brated in 10 mMphosphate buffer, pH 6.8 All these steps were carried out at 4C The protein was then separated from contaminating lactoferrin by ion exchange chromato-graphy on a Mono S HR 10/10 column (Amersham Phar-macia Biotech), equililibrated with the phosphate buffer, and connected to an Akta Purifier system (Amersham Pharmacia Biotech) 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, 1MNaCl, 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 lactoperoxidase containing peak was manually collected and the homo-geneity 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 e412¼ 114 mM )1Æcm)1
Preparation of nitrophenols The nitrated derivatives of 1, 2 and 4 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-Hydro-xyphenyl)-propionic acid (50 mg) was dissolved in 25 mL of
a 5 mM phosphate buffer solution at pH 7.5 containing sodium nitrite (0.08M) To this solution, dilute solutions of LPO in the same buffer (final concentration 3· 10)8M) and hydrogen peroxide (final concentration 8.0· 10)4M) were added in small amounts during 0.5 h at 25C in order to obtain an intense and persistent yellow color Then, the pH of
Trang 3the solution was brought to about 4 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-3-nitrophenyl)-propionic acid eluted as the first fraction The
product was recovered upon evaporation of the solvent (yield
40%) Table 1 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 3600M )1Æcm)1
3-Nitrotyramine N-Acetyltyramine (50 mg) was dissolved
in 10 mL of a 10 mMphosphate buffer solution at pH 7.5
containing sodium nitrite (0.25M) To this solution, several
additions of a dilute solution of LPO in the same buffer
(final concentration 3.0· 10)8M) and hydrogen peroxide
(final concentration 8.0· 10)4M) were made and the
mixture was allowed to react while stirring at room
temperature for 1 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 1Msolution of sodium hydroxide (10 mL) for 1 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 2 details
the analytical data obtained for 3-nitrotyramine The
extinction coefficient of 3-nitrotyramine at 422 nm in
phosphate buffer pH 7.5 is 2800M )1Æcm)1
4-Hydroxy-3-nitrobenzonitrile 4-Hydroxybenzonitrile
(100 mg) was dissolved in 25 mL of a 5 mM phosphate
buffer 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 to the substrate solution during 1 h at 25C 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 4 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 chromato-graphed 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 3 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 1700MÆcm)1and the wavelength of maximum absorption
is at 400 nm (e 2200M )1Æcm)1)
Kinetic experiments of phenol nitration The kinetics of the enzymatic phenol nitration were studied spectrophotometrically 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 ± 0.1C 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 DAÆs)1 to MÆs)1 was performed using the e422 values for 1–4; for 3-nitro-L-tyrosine, the extinction coefficient e422¼ 4000M )1Æcm)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
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 (CDCl 3 ) (d)
MS (ESI) (m/z)
MS/MS (ESI m/z 210.3) (m/z)
IR (NaCl, Nujol mull) (mÆcm)1)
7.12 (d, 1H, phenyl 5-H) 2.96 (t, 2H,CH 2 -CO) 2.69 (t, 2H, C-CH 2 -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 (D 2 O) (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)
C 52.74 C 52.13 7.72 (d, 1H, phenyl 2-H) 183.2 [M +1] 166.2 [(M-NH 3 )+1] 120.2 [(M-NH 3 -NO 2 )+1] 1522 m(NO 2 ) as
3.15 (t, 2H, CH 2 -N) 2.78 (t, 2H, C-CH 2 -Ph)
Trang 4performed with substrates 1–4 to find appropriate
condi-tions to follow the reaccondi-tions 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
condi-tions 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 2 mM
and [NO2]¼ 0.2Mfor LPO or 2.0Mfor HRP); (b) study
of the dependence of the rate versus substrate
concentra-tion 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 0.2M; (b)
depend-ence of the rate versus phenol concentration with HRP
(30 nM): [1]¼ 0–20 mM, [H2O2]¼ 1.60 mM, [NO2]¼
2.1M; [2]¼ 0–10 mM, [H2O2]¼ 3.0 mM, [NO2]¼ 2.1M;
[3]¼ 0–1.0 mM, [H2O2]¼ 0.24 mM, [NO2]¼ 2.1M;
[4]¼ 0–24 mM, [H2O2]¼ 0.40 mM, [NO2]¼ 2.1M; (c)
dependence of the rate versus nitrite concentration
(0–0.4M) 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
(0–3.5M) 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 3 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:
[HO]¼ 0.24 mM, [3]¼ 0.69 mM, [NO ]¼ 100 lM
Peroxidase catalyzed oxidation of 4 Steady state kinetic experiments of catalytic oxidation of 4
by LPO or HRP and hydrogen peroxide were performed in
200 mMphosphate buffer (pH 7.5) at 25 ± 0.1C, accord-ing to the followaccord-ing procedure To the solution containaccord-ing the enzyme (56 nM LPO or 71 nM HRP) and variable amounts of 4 (0–50 mM) in an optical quartz cell of 1 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 4 The initial rates were determined from the linear part of the trace at 322 nm To convert the rates from DAÆs)1toMÆs)1it 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 concentra-tions were used: [HRP] 33 nM, [4] 0.3 mM, and [H2O2] from 3.8· 10)5to 1.5· 10)4M, the other conditions were the same as in the kinetic experiments From this analysis the
e322value of 5600M )1Æcm)1was 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 ± 0.1C Compound II was prepared by incubation of the protein solution (7.0 lM) with a small excess (two mol equivalents)
of hydrogen peroxide for 1 min 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–A420nm) with a first-order equation The replot of kobsversus [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 3 and 4 were determined The enzyme compound II derivatives (2 lM) were prepared as described before Solutions of the substrates (1–10 mM) in an appro-priate volume of 200 mMphosphate 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 (CDCl 3 ) (d)
MS (ESI) (m/z)
IR (NaCl, Nujol mull) (mÆcm)1)
7.3 (d, 1 H, phenyl, 5H)
Trang 5prepared 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 kobsversus [phenol] were linear and
the slopes gave the catalytic constants
Stopped-flow experiments
The reaction between LPO, nitrite and H2O2was 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 25C 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
(2M 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
Super-Sep column at a 1 mLÆmin)1flow rate All the experiments
were 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 mMphosphate buffer (higher buffer
concentra-tions reduce the sensitivity of nitrate determination) pH 7.5,
at 25C Then, the sample was diluted tenfold in double
distilled water and injected into the column Other
experi-ments were performed without enzyme, in the absence or
presence of substrate (55 mM), and with 140 mMnitrite and
2 mMhydrogen 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 mMphosphate buffer (pH 7.5) to the enzyme
solution in the same buffer, at 25 ± 0.1C No attempts
were made to keep the ionic strength constant With LPO
(6· 10)6M), a 1Mstock nitrite solution and an optical cell of
1 cm path length were used In the case of HRP (6· 10)5M),
the binding process exhibited biphasic behavior and, in order
to reach saturation in the second step, the titration was
per-formed using a more concentrated solution of NaNO2(4M)
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
Differential pulse voltammetry Polarographic experiments on substrates 2 and 4 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 mVÆs)1and 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 4 and
790 mV for 2 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 5 mLÆmin)1 Elution started with 100% solvent A for 4 min, followed by a linear gradient from 100% A to 100% B in 20 min Spectrophotometric detection of the eluate 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 tenfold molar excess peroxynitrite for 5 min at room temperature The reaction mixtures were analyzed by HPLC as described above The retention times of 2 and 4 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, 5
CO2-free peroxynitrite A solution of 5 was purged with argon for 20 min 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 min
Trang 6Peroxynitrite-CO2 A solution of 5 (1 mM) in 200 mM
phosphate buffer (pH 7.5), equilibrated with atmospheric
CO2, was treated with five-or tenfold molar excess
peroxynitrite for 5 min at room temperature, and then
analyzed by HPLC The chromatogram contained only
one peak corresponding to 5, with a retention time of
11.6 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 2M; LPO (50 nM),
[5]¼ 1 mM, [H2O2]¼ 1.3 mM, [NaNO2]¼ 0, 0.025 or
0.25M Analysis of the product mixtures resulting from
the reactions carried out in the presence of high nitrite
concentrations (2Mfor HRP and 0.25Mfor LPO) showed
extensive 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 products
with retention times of 9.9, 10.4, 11.8, 12.0, 12.2 and
12.4 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 in
the same conditions as reported in a previous work [31] The
reactions were performed in 200 mM phosphate buffer
(pH 7.5) at 25C in the presence of 30 nM HRP and
variable nitrite concentration (0, 0.2, and 1.0M) 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
nitrophen-ols 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
experi-ments, 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 2 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
These findings indicate that an efficient nitration reaction requires the interaction of the enzyme with both nitrite and phenol
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 m M 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 m M 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 m M phosphate buffer (pH 7.5), at 25 C The inset shows an expansion of the plot in the low nitrite concen-tration range.
Trang 7The 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
com-pound I or comcom-pound II
Eþ H2O2! compound I þ H2O (fast) ð9Þ
compound Iþ NO2 , compound I=NO2
compound Iþ PhOH , compound I=PhOH
compound IIþ NO2 , compound II=NO2
!2H
þ
compound IIþ PhOH , compound II=PhOH
!2H
þ
Scheme 1 Pathway A mechanism
The PhO•produced can also give rise to dimeric coupling
products through the normal peroxidase catalytic cycle
[25–27]:
The second mechanism (pathway B), represented in
Scheme 2, where E–NO2 is the peroxidase-nitrite complex
and Enitrthe 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
ENO2 þ H2O2! Enitrþ H2OðfastÞ ð17Þ
Enitrþ PhOH , ½EnitrPhOH !2H
þ
Eþ O2NPhOH
Scheme 2 Pathway B mechanism
Species derived from Enitrmay 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 1 or 2 with the system peroxidase/NO2/H2O2 shows that, while phenol dimers are formed at every nitrite concentration, the relative amount of dimers versus nitro-phenol 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 kcat½E
1þ KnitriteM
½NO2 þ
K PhOH M
½PhOH
ð19Þ
where kcat represents the turnover rate of enzymatic nitration, and Knitrite
M and KPhOH
M 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 rate determining step of the turnover is reaction 18, the initial rate equation for pathway B is:
1þ KnitriteM
½NO2
1 þ KPhOHM
½PhOH
where here Knitrite
M 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 conditions
in which either the term Knitrite
M =½NO2 or the term
KPhOH
M =½PhOH 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 4 and 5 The actual enzymatic turnover rates are somewhat larger because part of the enzyme is engaged in the nonproductive nitrate formation For the nitrite inhib-ited reactions of substrate 4 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
KI¼ 20 ± 7M )1, for HRP KI¼ 50 ± 5M )1 For comparison purposes we determined the kinetic parameters for the LPO and HRP catalyzed oxidation of
4 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, kcat¼ 16 ± 2 s)1
Trang 8and KM¼ 11 ± 1 mM; with HRP, kcat¼ 19 ± 2 s)1and
KM¼ 20 ± 4 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 HRP–NO2, kmax¼ 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, kmax¼ 424 (e 101 mM )1Æcm)1), 546
(e 11.4 mM )1Æcm)1) and 588 nm (e 8.5 mM )1Æcm)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
Kb¼ 22.0 ± 0.5M )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 do 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 Kb¼ 1.3M )1(data not shown)
Nitrate production
Both nitrating species formed according to mechanisms A
and B can undergo competitive degradation to nitrate
Table 6 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
produced corresponds to 80% of the hydrogen peroxide oxidizing equivalents If 2 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 satura-tion (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
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 )1Æs)1at 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 )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
K nitrite
M
(m M )
k cat
(s)1)
k cat =K nitrite M
( M )1 Æs)1)
K nitrite M
(m M )
k cat
(s)1)
k cat =K nitrite M
( M )1 Æs)1)
Table 6 Competitive production of nitrate during enzymatic nitration Nitrate produced by the LPO/NO 2 /H 2 O 2 system in the presence of different concentrations of nitrite, phenol 2 and hydrogen peroxide, after 20 min reaction in 20 m M phosphate buffer, pH 7.5, at 25 C LPO
(l M )
Phenol 2 (m M )
NO 2
(m M )
H 2 O 2
(m M )
NO 3
(m M )
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
K PhOH
M
(m M )
k cat
(s)1)
k cat =K PhOH M
( M )1 Æs)1)
K PhOH M
(m M )
k cat
(s)1)
k cat =K PhOH M
( M )1 Æs)1)
Trang 9(a value of 13.3M )1Æs)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
condi-tions were employed We determined here the rate constants
of 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)· 103
M )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 25C
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 (kobs¼ 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
transfor-mation, 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
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 (kmax¼ 422 nm)
Reaction of 2 and 4 with peroxynitrite The reaction of excess peroxynitrite with 2 and 4 was studied in comparative experiments in 200 mMphosphate 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 2 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
4 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 nitrating agents in 200 mMphosphate buffer (pH 7.5) Bolus addi-tions of several volumes of nitrogen dioxide to soluaddi-tions of 5 did not yield any nitration products When 5 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 5 were found to be dependent on nitrite concentration At low nitrite concentration (25 mM) no reaction was observed; with higher nitrite concentrations (0.25M for LPO, 2M 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/H2O2system 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 of15N 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/H2O2and 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
Fig 4 Spectral changes of LPO upon reaction with nitrite and hydrogen
peroxide UV/Vis spectral changes observed with time upon reacting
LPO (2.7 l M ) and nitrite (150 m M ) with H 2 O 2 (0.85 m M ) in 200 m M
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).
Trang 10competitively, but the importance of this reaction decreases
upon increasing [NO2] It is worth noting, in this respect,
that the kcat/KMvalues associated with dimer formation in
the normal peroxidase cycle [25,26] are much smaller than
the kcat=KPhOH
M 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
nitrating species The difference in kcat=KPhOH
M for phenol nitration and dimer formation depends largely on the KPhOH
M 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 kcatvalues for the enzymatic nitration of 1–3 do not
follow the substrate redox potentials (for the couple
phenoxy radical/phenol), which decrease in the order
3 > 1 > 2 (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
concentra-tion The corresponding Knitrite
M 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 1 and 2, the nitrating species
produced by the enzyme is either NO2 , formed by
compound I or II and nitrite (pathway A), or Enitrformed
by the enzyme–nitrite complex and H2O2in pathway B For
LPO, the reaction of compound I with nitrite is extremely
fast ( 2 · 107M )1Æs)1) and produces nitrate instead of
NO2 [21] The reaction between LPO–NO2 and H2O2also
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 þ H2O2! EN(O)OOþ H2O ð21Þ
because the alternative formation of NO2+would produce
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, NO2is the
major nitrating agent, but also in this case the peroxynitrite
pathway cannot be completely excluded Compound I can
be competitively reduced by nitrite (k¼ 6.7 · 105
M )1Æs)1
at pH 6.9 [41]) or the phenol (k 105)106
M )1Æs)1[42]), but reduction of compound II can only occur by reaction with
the 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 a second-order rate constant of 6.6 ± 0.4MÆs)1, which is much smaller than the kcat=Knitrite
M values for nitration of all 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 concen-tration, 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 nitra-tions, even at high nitrite concentranitra-tions, 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 4 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 4 differs from that of the other substrates in two respects The KPhOH
for nitration of 4 is in the same range as those found in the normal LPO and HRP mediated peroxidase reactions This indicates that 4 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, peroxyni-trite is a poor nitrating agent for this substrate The enzymatic nitration of 4 can therefore proceed only through the NO2 pathway
In contrast, phenylacetic acid is a good probe for peroxynitrite It is known that 5 reacts with peroxynitrite
to form nitrophenyl and also nitrophenol derivatives, while the reaction is blocked in the presence of CO2 [46] We found that 5 is unreactive both to NO2and 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 CO2further shows that the 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 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 l nitrite concentration (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. .. 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. .. which the protein has been modified bynitration 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