Copper-catalyzed tyrosine nitration

Article abstract of DOI:10.1021/ja206980q

Tyrosine nitration, often observed during neurodegenerative disorders under nitrative stress, is usually considered to be induced chemically either by nitric oxide and oxygen forming nitrogen dioxide or by the decomposition of peroxynitrite. It can also be induced enzymatically by peroxidases or superoxide dismutases in the presence of both hydrogen peroxide and nitrite forming nitrogen dioxide and/or peroxynitrite. In this study, the role of cupric ions for catalyzing tyrosine nitration in the presence of hydrogen peroxide and nitrite, by a chemical mechanism rather similar to enzymatic pathways where nitrite is oxidized to form nitrogen dioxide, was investigated by development of a microreactor also capable of acting as an emitter for electrospray ionization mass spectrometry analysis. Indeed, cupric ions and peptide-cupric ion complexes are found to be excellent Fenton catalysts, even better than Fe(III) or heme, for the formation of ^?OH radicals and/or copper(II)-bound ^?OH radicals from hydrogen peroxide. These radicals are efficiently scavenged by nitrite anions to form ^?NO₂ and by tyrosine to form tyrosine radicals, leading to tyrosine nitration in polypeptides. We also show that cupric ions can catalyze tyrosine nitration from nitric oxide, oxygen, and hydrogen peroxide as the formation of tyrosine radicals is increased in the presence of diffusible and/or copper(II) bound hydroxyl radicals. This study shows that copper has a polyvalent role in the processes of tyrosine nitration.

Full text of DOI:10.1021/ja206980q

ARTICLE
pubs.acs.org/JACS
Copper-Catalyzed Tyrosine Nitration
Liang Qiao,† Yu Lu,† Baohong Liu,‡ and Hubert H. Girault*,†
†Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fꢀedꢀerale de Lausanne, Station 6,
CH-1015 Lausanne, Switzerland
‡Department of Chemistry, Institute of Biomedical Sciences, Fudan University, Shanghai 200433, People’s Republic of China
S Supporting Information
b
ABSTRACT: Tyrosine nitration, often observed during neurodegenerative disorders
under nitrative stress, is usually considered to be induced chemically either by nitric oxide
and oxygen forming nitrogen dioxide or by the decomposition of peroxynitrite. It can also
be induced enzymatically by peroxidases or superoxide dismutases in the presence of both
hydrogen peroxide and nitrite forming nitrogen dioxide and/or peroxynitrite. In this
study, the role of cupric ions for catalyzing tyrosine nitration in the presence of hydrogen
peroxide and nitrite, by a chemical mechanism rather similar to enzymatic pathways where
nitrite is oxidized to form nitrogen dioxide, was investigated by development of a microreactor also capable of acting as an emitter for
electrospray ionization mass spectrometry analysis. Indeed, cupric ions and peptideÀcupric ion complexes are found to be excellent
Fenton catalysts, even better than Fe(III) or heme, for the formation of •OH radicals and/or copper(II)-bound •OH radicals from
hydrogen peroxide. These radicals are efficiently scavenged by nitrite anions to form •NO2 and by tyrosine to form tyrosine radicals,
leading to tyrosine nitration in polypeptides. We also show that cupric ions can catalyze tyrosine nitration from nitric oxide, oxygen,
and hydrogen peroxide as the formation of tyrosine radicals is increased in the presence of diffusible and/or copper(II) bound
hydroxyl radicals. This study shows that copper has a polyvalent role in the processes of tyrosine nitration.
’ INTRODUCTION
and nucleic acids.15,16 Copper at high concentrations can be an
important factor in the pathogenesis of many neurodegenerative
disorders,17,18 and the binding between copper and disease-
associated peptides or proteins has been widely studied.19,20
However, Cu2+-catalyzed nitration has attracted little attention,
especially in mechanistic and pathogenesis studies. To date, most
of the studies about copper related tyrosine nitration have
focused on copperÀzinc superoxide dismutase (Cu,Zn-SOD).
Apart from its principal role in scavenging the superoxide anion
O2À to form hydrogen peroxide and oxygen, it has been shown to
promote the oxidation of nitrite to form nitrogen dioxide.21À24
In this study, electrospray ionization (ESI) mass spectrometry
(MS) was used to analyze the nitration products. Specifically, we
have designed a dedicated polymer microchip reactor that also
acts as an ESI emitter to monitor on-line nitrite-induced peptide
nitration. Both Cu2+ and Cu2+Àpeptide complexes were found
to catalyze the tyrosine nitration in the presence of nitrite and
hydrogen peroxide. To explain the observed copper-mediated
tyrosine nitration, a mechanism was proposed where hydroxyl
Biomolecule oxidation plays an important role in aging and
the development of diseases. There are two major classes of
intermediates involved in the in vivo oxidative reactions, reactive
oxygen species (ROS) and reactive nitrogen species (RNS).1 At
low/moderate concentrations, ROS/RNS are important mes-
sengers for signal transduction, while at high concentrations, they
can induce oxidative damage to DNA, lipids, and proteins, a
phenomenon named as oxidative stress.2,3
RNS can react with proteins to induce tyrosine nitration. This
process is drawing considerable interest as it can alter protein
functions. It can also be used as a diagnostic biomarker for
diseases involving radical reactions, such as cardiovascular,4
Alzheimer’s,5 and Parkinson’s diseases.6 It is widely accepted
that tyrosine nitration occurs in the presence of either peroxyni-
trite anion (ONOOÀ)7 or nitrogen dioxide (•NO2).8 ONOOÀ is
a RNS anion generated in vivo by the very fast combination of
nitric oxide (•NO) and superoxide anion (O2À).9À11 •NO2 can
be generated via several mechanisms, including the oxidation of
•NO by oxygen,12 the decomposition of ONOOÀ,7,11,13 and the
oxidation of nitrite (NO2À) by hydrogen peroxide (H2O2)
catalyzed with peroxidases.14 Peroxidases are often heme-
containing enzymes,14 where the heme is an iron-protoporphyrin
IX able to accept or donate electrons and to interconvert among
the states of Fe(II), Fe(III), and Fe(IV).14
•
•
radicals (•OH) and/or copper(II)-bound OH (Cu2+À OH)
can be generated from Cu2+ and H2O2 through a Fenton-like
reaction. These radicals may then be scavenged by both NO2À to
form •NO2 and tyrosine to form tyrosine radicals (Tyr•), leading
to tyrosine nitration. Cu2+/H2O2 was also found to catalyze the
tyrosine nitration induced by nitric oxide and oxygen. •NO was
oxidized by O2 to form •NO2, and the role of Cu2+/H2O2 was to
Herein, we have investigated cupric ions (Cu2+) or Cu2+À
peptide complexes-catalyzed peptide nitration in the presence of
either nitrite or nitric oxide. Copper is able to favor the genera-
tion of free radicals causing oxidative damage to proteins, lipids,
Received: July 26, 2011
Published: November 02, 2011
r
2011 American Chemical Society
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Scheme 1. (a) Polyimide Microchip with Three Microchannels (White Lines) and One Micro-carbon Electrode (Black Line); and
(b) the Microchip Works as an Electrospray Emitter for On-Line Tyrosine Nitration Studya
a The depths and widths of all microchannels are 50 and 100 μm, respectively.
•
•
generate OH/Cu2+À OH to promote Tyr• formation. This
work shows that cupric ions and peptideÀcupric ion complexes
are very efficient Fenton catalysts having neurotoxicity, for exam-
ple, inducing nitration of α-synuclein relevant to Parkinson’s
disease.6,25
H2O2 and Cu2+ may be added to assist the nitration. To perform the
reaction under anaerobic conditions, nitrogen was used to remove
dissolved oxygen in water, and the reaction was performed under the
protection of nitrogen. After reaction, the sample was diluted 11 times in
the ESI buffer before MS identification.
Mass Spectrometry. Mass spectrometry (MS) and tandem mass
spectrometry (MS/MS) experiments were performed on the Thermo
LTQ Velos. For MS experiments, 3.7 kV of voltage was applied to induce
electrospray ionization. For MS/MS experiments, collision-induced
dissociation (CID) was performed under 20% of instrumental collision
energy.
’ EXPERIMENTAL SECTION
Nitration Induced by Nitrite. Off-line tyrosine nitration was
performed by incubating angiotensin I (Ang I, 0.25 mM, trifluoroacetate
salt), CuCl2 (0.25 mM), H2O2 (0.5 mM), and NaNO2 (1 mM) in H2O
under ambient temperature and pressure in a 1.5 mL Eppendorf tube
under persistent shaking for 4 h. After reaction, the products were
diluted 11 times in an ESI buffer of 50% methanol with 49% H2O and 1%
acetic acid (50%MeOH/49%H2O/1%HAc) before MS identification.
Detailed information about all chemicals used can be found in Support-
ing Information S-1.
On-line nitration was directly performed in a microchip as shown in
Scheme 1 followed with MS identification. The microchip fabrication is
illustrated in Supporting Information S-2 by using a previously reported
scanning laser ablation method.26,27 Solution A of Ang I (0.5 mM,
trifluoroacetate salt), H2O2, and NaNO2 in H2O was pushed by a syringe
pump through channel A. Solution B of Fe2+, Fe3+, heme, Cu2+,
β-amyloid 16 copper(II) complex (Aβ-16ÀCu2+), ethylene-diaminete-
traacetic acid copper(II) complex (EDTAÀCu2+), or 1,4,8,11-tetraaza-
cyclotetradecane copper(II) complex (TCÀCu2+) was pushed through
channel B. At the end of channel A, the ESI buffer was introduced by
channel C to maintain steady ionization conditions. Nitration of Ang I
occurred under ambient temperature and pressure in channel A, mostly
before the introduction of the ESI buffer. By controlling the different
flow rates, the reaction time in channel A can be controlled. On-line
nitration of α-synuclein, leu-enkephalin, or 4-aminophenol was per-
formed similarly just by replacing Ang I in channel A with these
substrates. The Aβ-16ÀCu2+, TCÀCu2+, and EDTAÀCu2+ complexes
were prepared by incubating the Aβ-16 peptide, TC, and EDTA with
CuCl2, respectively, with a ratio of 2:1 (mol:mol) for 1 h under
persistent shaking in ambient conditions.
For the on-line nitration induced by nitrite, MS analyses were
performed using the microchip emitter shown in Scheme 1, where a
carbon electrode placed in channel C was used to supply the high-
voltage. For the off-line nitration induced by nitrite, a similar microchip
with a carbon electrode placed in channel A was used as an emitter. The
diluted reaction product was pushed by a syringe pump through the
channel A with a flow rate of 1.1 μL minÀ1. Channels B and C were
blocked during these experiments.
MS analyses of nitration induced by •NO were performed with the
commercial ESI source. The diluted reaction products were pushed by a
syringe pump at a flow rate of 10 μL minÀ1
.
All MS instrumental parameters were kept constant during experi-
ments for the microchip ESIÀMS and commercial source ESIÀMS,
respectively.
Characterization of Hydroxyl Radical Generation from
•
•
Copper(II) and H2O2. OH/(Cu2+À OH) radicals generated from
Cu2+ and H2O2 were characterized using a fluorometric assay.28,29 The
fluorescence spectra of 2-hydroxyterephthalic acid generated by the
•
reaction of terephthalic acid with •OH/(Cu2+À OH) were obtained on
a Perkin-Elmer LS-50B fluorescence spectrometer. An aqueous solution
of terephthalic acid (0.1 mM, saturated), H2O2 (3 mM), and CuCl2
(3 mM) with or without NaNO2 (3 mM) was incubated in ambient
temperature and pressure before the fluorescence detection. The
excitation wavelength for fluorescence detection was 315 nm, and the
signal was recorded at 430 nm.
Nitration Induced by Nitric Oxide. Diethylamine NONOate
sodium salt hydrate (NONOate) was used to generate nitric oxide, •NO.
The nitration was performed off-line by incubating Ang I (0.25 mM,
trifluoroacetate salt) with NONOate (2.5 mM of initial concentration)
in water under persistent shaking at ambient temperature and pressure.
’ RESULTS AND DISCUSSION
On-Line Tyrosine Nitration Induced by Copper(II), Nitrite,
and H2O2. Copper(II) was found to catalyze tyrosine nitration in
the presence of nitrite and H2O2. To efficiently analyze the
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clear that Ang I nitration induced by hydrogen peroxide and
nitrite occurs only in the presence of Cu2+. The weak peak of
OÀAng I observed in both Figure 1a and b can be a result of the
direct oxidation of the peptide during the electrospray process
rather than oxidation induced by Cu2+ and H2O2. Indeed, it was
reported previously that the high voltage applied for ESI could
result in the oxidation of samples.30,31 Besides angiotensin I,
similar tyrosine nitration reactions were observed with leu-enkephalin
or a fragment of human α-synuclein (107À140), as shown in
Supporting Information Figure S-5.
On-Line Tyrosine Nitration under Various Concentrations
of Copper(II), H2O2, and Nitrite. Tyrosine nitration efficiencies
were compared under various experimental conditions. To
calculate quantitatively the nitration levels from the mass spectra,
an external calibration method was employed as illustrated in
Supporting Information S-6. Specifically, M1/(M1 + M2) was
calculated from P1/(P1 + P2) to show the nitration level by using
the calibration curves shown in Figure S-6, where M1 is the
concentration of NO2ÀAng I produced, M2 is the concentration
of unreacted Ang I, P1 is the monoisotopic peak intensity of
three-times protonated NO2ÀAng I (NO2ÀAng I)3+ read from
the mass spectra, and P2 is the monoisotopic peak intensity of
three-times protonated Ang I (Ang I)3+.
As shown in Supporting Information Figure S-7, the nitration
can reach steady-state conditions within 1 min. Therefore, a flow
rate of 0.05 μL minÀ1 for both channels A and B, corresponding
to a reaction time of 1 min, was then used as the standard flow
rate for the following on-line tyrosine nitration studies.
To investigate the effect of NO2À, its concentration was varied
while maintaining the concentrations of Ang I, CuCl2, and H2O2
constant. Flow rates in all three channels were also kept constant.
Zoomed mass spectra of the three-times protonated NO2ÀAng I
generated under different experimental conditions are shown in
Figure 2a. Each mass spectrum shown is an average of ∼500
spectra collected continually during 3 min. The mass spectra
were normalized by always taking the intensity of the mono-
isotopic peak of (Ang I)3+ equal to 100 for comparison. The
calculated nitration levels were plotted against the nitrite con-
centrations, as shown in the inset. The data illustrate that tyrosine
Figure 1. (a) Mass spectrum of Ang I on-line nitration products. Flow
rates in channels A and B = 0.05 μL minÀ1 and in channel C =
1 μL minÀ1. Channel A, 0.5 mM of Ang I, 1 mM of H2O2, and 2 mM of
NaNO2 in H2O; channel B, 0.5 mM of CuCl2 in H2O; channel C, ESI
buffer. (b) Mass spectrum obtained under the same experimental
conditions as in (a) except that the solution in channel B was changed
to deionized water.
nitration products, a three-channel microchip was fabricated
working both as a reactor for tyrosine nitration and as an emitter
for electrospray ionization, as shown in Scheme 1. Peptide
solutions were pumped into channel A together with H2O2
and sodium nitrite (NaNO2). Copper(II) chloride (CuCl2)
solutions were brought by channel B to react with nitrite,
H2O2, and peptide in the 2 cm long reaction flow channel A.
At the end of the reaction channel, a large amount of ESI buffer
was introduced by channel C to help sample ionization and to
maintain steady ionization conditions so as to obtain a good
reproducibility required for quantitative analysis. The reaction
products were directly ionized and detected by a linear ion trap
mass spectrometer. Angiotensin I (Ang I) was used as a test
sample for the nitration study. As shown in Figure 1a, both
nitrated and unmodified Ang I were detected by mass spectro-
metry. With tandem MS experiments, it was further demon-
strated that the nitration happens always on the single tyrosine
residue of Ang I (see Supporting Information S-3). Oxidized Ang
I (OÀAng I) and copper(II)-binding Ang I (Cu(II)ÀAng I)
were also observed.
À
nitration strongly relies on the presence of NO2 with an
observed optimal concentration of between 1 and 2.5 mM.
Interestingly, the nitration leÀvel was attenuated greatly with
higher concentrations of NO2
.
Similarly, the influences of H2O2 and copper(II) on the
tyrosine nitration yield were also investigated. As was observed
in Figure 2b, the nitration level increases quickly when the H2O2
concentration increases from 0.125 to 0.5 mM, reaching a
maximum for H2O2 concentrations above 1 mM. This indicates
that nitration is then limited by other reactants. A strong
copper(II)-dependent nitration is observed when the concentra-
tion of CuCl2 increases from 12.5 μM to 0.5 mM, while the
nitration yield does not vary much when the concentration of
CuCl2 increases from 0.5 to 5 mM; see Figure 2c. The highest
observed nitration level of Ang I with optimized reactant concen-
trations is ∼10%. The nitration of Ang I induced by copper(II),
nitrite, and H2O2 happened always under weak acidic conditions
(pH = 4.0 ( 0.3) due to the acidity of angiotensin I trifluoro-
acetate salt itself.
When the CuCl2 solution in channel B was changed to pure
water, the nitrated Ang I (NO2ÀAng I) and Cu(II)ÀAng I were
no longer observed, while peaks of the bare and oxidized Ang I
remained, as shown in Figure 1b. The details of the observed
peaks are listed in Supporting Information Table S-4. It is then
•
Off-Line Tyrosine Nitration Induced by NO/O2 with the
Catalysis of Copper(II)/H2O2. It was found that a trace amount
•
of copper(II) can also catalyze nitration induced by NO and
O2 in the presence of H2O2. Diethylamine NONOate sodium
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Figure 3. NONOate (2.5 mM)/O2-induced Ang I (0.25 mM) nitration
in water (a) with or (b) without H2O2 (1 mM) and CuCl2 (12.5 μM)
after 4 h of reaction. The products were diluted 11 times in the ESI buffer
and then analyzed by ESIÀMS with a flow rate of 10 μL minÀ1
.
As is shown in Figure 3b, strong nitration of Ang I can be
observed. However, a more efficient nitration was obtained when
H2O2 and a trace amount of Cu2+ (12.5 μM) were added into the
above reaction system, Figure 3a.
A quantitative analysis was performed with the external calibra-
tion method given in Supporting Information S-6. The nitration
levels of Ang I achieved under various reactant conditions were
systematically compared in Figure 4. As shown in Figure 4b, oxygen
is necessary for •NO-induced nitration. Only background levels of
nitration were observed when the reaction was performed under a
nitrogen-protected environment. The nitration induced by •NO/O2
can be significantly catalyzed only in the presence of H2O2 and trace
amounts of copper(II), while high concentrations of copper(II)
would inhibit the •NO/O2-induced nitration.
À
Figure 2. Ang I nitration under different concentrations of (a) NO2
,
(b) H2O2, and (c) CuCl2. Flow rates in channels A and B = 0.05 μL
minÀ1 and in channel C = 1 μL minÀ1. Channel A, Ang I (0.5 mM),
H2O2, and NaNO2 in H2O; channel B, CuCl2 in H2O; channel C, ESI
buffer. All of the concentrations shown in the figure are those of the
reactants in the microreactor just after mixing solutions from channels A
and B. (a) CuCl2 = 0.25 mM, H2O2 = 0.5 mM. (b) CuCl2 = 0.25 mM,
NO2À = 1 mM. (c) H2O2 = 0.5 mM, NO2À = 1 mM. Mass spectra were
normalized by setting the monoisotopic peak intensities of (Ang I)3+
always as 100%. Insets: Calculated nitration levels versus concentrations.
The error bars show the standard deviations of the calculated nitration
levels.
Figure 4a compares the nitration levels that were obtained
under trace and large amounts of copper(II) for various reaction
times. The nitration catalyzed by 12.5 μM of copper(II) is very
fast and reaches a steady state within 1 h, while in the presence
of 2.5 mM of copper(II) it reaches steady state after 7 h of
incubation. Nitration under various concentrations of copper(II)
was further investigated. As shown in Figure 4c, the nitration
level decreases with the increase of copper(II) concentration.
When the Cu2+ concentration is higher than 0.5 mM, nitration
salt hydrate (NONOate) was used as an NO generator32 to
•
incubate with Ang I in water. Because •NO was generated during
the decomposition of NONOate, batch reactions were carried
•
induced by NO/O2 without copper(II) is even more efficient
•
out instead of the on-line microchip strategy to study NO-
induced nitration. After 4 h of reaction in water, the nitration
products were analyzed by MS using the commercial ESI source.
than that with copper(II). The influence of H2O2 on •NO/O2-
induced nitration catalyzed by a trace amount of Cu2+ was
also studied. As shown in Figure 4d, the nitration level always
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Figure 4. (a) NONOate (2.5 mM)/O2-induced Ang I nitration in water with H2O2 (1 mM) and CuCl2 (12.5 μM or 2.5 mM) under various reaction
times. (b) NONOate (2.5 mM)/O2-induced Ang I nitration in water under various reactant conditions after 4 h of reaction. The concentration of H2O2
was always 1 mM. (c) NONOate (2.5 mM)/O2 induced Ang I nitration in water with H2O2 (1 mM) and various amounts of CuCl2 after 4 h of reaction.
(d) NONOate (2.5 mM)/O2-induced Ang I nitration in water with CuCl2 (12.5 μM) and various amounts of H2O2 after 4 h of reaction. The error bars
show the standard deviations of the calculated nitration levels.
increases with the increase of H2O2 concentration. The acidity of
angiotensin I trifluoroacetate salt was neutralized by the basic
NONOate. As a result, the nitration of Ang I induced by •NO/O2
happened always under neutral conditions (pH = 7.5 ( 0.2).
Role of Copper(II) in Nitrating Tyrosine. Tyrosine nitration
can occur via several mechanisms originating either from nitric
oxide (•NO) or from nitrite (NO2À).
copper is less known in protein nitration. According to previous
studies, copper(II) is able to catalyze the decomposition of
peroxynitrite to oxidize special substrates, such as ascorbic acid,
or to induce nitration.43À46 Alternatively, peroxynitrite coordi-
nated copper(II), which is generated in the presence of copper(I)
•
complexes, NO, and O2, can undergo further decomposition
inducing tyrosine nitration.47À49 The nitrite related tyrosine
nitration was reported to be induced by copperÀzinc superoxide
dismutase (SOD) via Fenton reactions14 in the presence of
hydrogen peroxide.21À24 Indeed, Cu2+ in SOD is assumed to be
reduced to copper(I) by H2O2 that in turn can react with H2O2
•
The NO pathways involve its oxidation by oxygen to form
ultimately nitrogen dioxide (•NO2),12 which is the key nitrating
agent. •NO can also react with superoxide anions (O2À) to form
peroxynitrite (ONOOÀ) that may decompose, depending on
•
+ 33À35
via a Fenton-like reaction to generate copper(II)-bound OH
the biological conditions, to form nitronium ion (NO2 )
(Cu2+À OH) that oxidizes nitrite to nitrogen dioxide.
•
•
À
or NO2.7,11,13 In the presence of CO2, ONOOCO2 can be
generated from ONOOÀ, and further decomposes into carbon-
ate radicals and •NO2 to induce tyrosine nitration.7,11
The nitrite (NO2À) pathways are usually considered to be
enzymatically driven where NO2À is oxidized in the presence of
H2O2 and peroxidase to form nitrogen dioxide (•NO2).14,36
Alternatively, enzymeÀONOOÀ complÀexes can also be gener-
ated from peroxidases, H2O2, and NO2 to react directly with
tyrosine and induce nitration.37
On the basis of the enzymatic redox mechanisms,21À24
a
pathway for the present copper(II)-induced nitration by nitrite
is proposed as shown in Scheme 2. In the presence of H2O2/
HO2À, copper(II) can be coordinated by hydroperoxide anion
(HO2À) to form a key complex of copper(II)Àhydroperoxo.
The complex can further decompose to generate copper(I) to
react with H2O2 via a Fenton-like reaction to produce •OH and/
•
or Cu2+À OH, as highlighted to be the Fenton reaction route
•
shown in Scheme 2. The •OH/Cu2+À OH radicals contribute to
Beside peroxidases, heme, heme-containing proteins, such as
myoglobin and cytochrome c,andFe2+/Fe3+ can also catalyze nitration
through mechanisms similar to those involving peroxidases.14,38,39 The
hemeÀprotein/H2O2/NO2À facilitated nitration and the chemical
biology of ONOOÀ have been extensively reviewed in recent
publications.9,36,40À42
•
both the generation of NO2 in the presence of nitrite and the
production of Tyr• from tyrosine, leading to the nitration of
tyrosine. With this mechanism, the on-line nitrite-induced nitra-
tion should happen mainly in aqueous channel A before the
addition of the ESI buffer. This is because methanol in this buffer
is a scavenger of hydroxyl radicals thereby terminating the radical
reactions.50 Considering the pH conditions used in the experiments,
In comparison to the well-established mechanisms for perox-
idase, heme, and iron-induced tyrosine nitration, the role of
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Scheme 2. Proposed Mechanism for Copper(II) and
PeptideÀCopper(II)-Induced Tyrosine Nitrationa
Figure 5. Characterization of •OH radical production from H2O2
(3 mM) and copper(II) (3 mM) with or without nitrite (3 mM):
fluorescence intensity (430 nm) versus reaction time plot. Baselines
were subtracted from the spectra to calculate the fluorescence intensities.
Unmodified fluorescence spectra are shown in Figure S-8.
•
characterized OH by terephthalic acid can be a sum of both
diffusible and copper(II)-bound hydroxyl radicals.
According to the fluorometric results in Figure 5, •OH and/or
•
Cu2+À OH can be generated from H2O2 and copper(II) and
a L: ligand.
•
further scavenged by nitrite to form possibly NO2. This se-
H2O2 (pKa = 11.6) should be the main species to react with
cupric ions in the case of copper(II), nitrite, and H2O2-induced
Ang I nitration (pH = 4.0 ( 0.3). In theory, more HO2À could
participate in the reaction in the case of •NO/O2 induced Ang I
nitration (pH = 7.5 ( 0.2), leading to an increased reaction rate
to produce •OH.
quence is in accordance with the proposed Fenton reaction route
in Scheme 2. Another important reactant that can mediate
tyrosine nitration is peroxynitrite. Although it was reported that
enzyme-bound peroxynitrite can be formed from nitrite and
H2O2 in the presence of peroxidases or heme containing
proteins,37À39 copper(II) was reported to catalyze peroxynitrite
decomposition.44 Therefore, the peroxynitrite is not likely to be
generated from nitrite and H2O2 under our experimental condi-
tions, and the •NO2-mediated nitration is more likely to be the
main reaction route of the nitrite-induced nitration.
To validate the proposed mechanism, fluorometric experi-
ments were carried out using terephthalic acid to characterize the
generation of •OH radicals.28,29 Terephthalic acid has been used
in the past as a highly specific and sensitive hydroxyl radical
dosimeter in radiation chemistry and sonochemistry.51,52 In the
presence of •OH, 2-hydroxyterephthalic acid is generated, yield-
ing a strong fluorescence signal at 430 nm under UV light
irradiation at 315 nm. It was proven that other radicals, such as
•OOH and O2À, cannot directly induce a similar fluorescence.53
As shown in Figure 5 and Supporting Information Figure S-8,
fluorescence was observed from the reaction products of H2O2,
CuCl2, and terephthalic acid, rising linearly with the reaction
time, indicating the abundant generation of •OH from H2O2 and
CuCl2. When NaNO2 was added, the production of 2-hydroxy-
terephthalic acid decreased, illustrating that NO2À competed for
The results of nitratioÀn occurring under various concentra-
tions of H2O2 and NO2 shown in Figure 2 also support the
proposed mechanism in Scheme 2. A large amount of •OH can be
generated with concentrated H2O2, which would favor the
nitration of tyrosine, in accordance with Figure 2b. The ratio
between •OH and NO2À in the reaction system is an important
factor. With an excess amount of NO2À, all •OH radicals would
be scavenged by nitrite. Therefore, Tyr• can only be produced
from the reaction between •NO2 and tyrosine, leading to limited
nitration, in agreement with Figure 2a.
•
In the presence of NO, several pathways are possible to
•
•OH quenching to form NO2. All of the fluorometric experi-
induce tyrosine nitration by considering the coÀpper(II) redox
mechanisms shown in Scheme 2. Because O2 is produced
during the decomposition of H2O2 catalyzed by copper(II),
ONOOÀ may be generated from the combinatioÀn of •NO and
ments were performed under weak acidic conditions (pH = 4.2 (
0.1) due to the acidity of terephthalic acid, in accordance with the
nitration induced by copper(II), nitrite, and H2O2. It is calculated
that ∼70% of •OH generated is scavenged by NO2À under the
experimental conditions in the presence of nitrite, and that the
À
O2 to induce tyrosine nitration. However, O2 can also be
removed by the excess of copper(II)/copper(I) efficiently,54,55
•
rate constant for OH scavenging by nitrite is 8% that of
resulting in the ONOOÀ route being less feasible. Alternatively,
•
terephthalic acid. Thus, this shows that nitrite is a rather efficient
•OH scavenger but is not as efficient as terephthalic acid; see
Supporting Information S-8. According to the mechanism pro-
posed for nitration induced by SOD, an oxidant of SODÀ
•NO2 can be generated by oxidizing NO with O2 to induce
nitration.12 Considering the observation that •NO-induced tyro-
sine nitration occurs only in the presence of oxygen and that
•
copper(II) is not essential for the NO induced nitration, the
•
•
Cu2+À OH is generated instead of diffusible OH.22 Similarly,
the copper(II)-bound hydroxyl radical may also be generated in
this mechanism, perhaps exhibiting chemical properties similar to
those of diffusible •OH to induce the fluorescence. Therefore, the
•NO oxidation route highlighted in Scheme 2 is the most likely
under the present conditions. In the presence of Cu2+/H2O2,
production of Tyr• is promoted by the production of •OH/Cu2+À
•OH radicals, and therefore more efficient nitration was observed
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Scheme 3. Structures of the TCÀCu2+, EDTAÀCu2+, and
Ang IÀCu2+ Complexes
Figure 6. Ang I on-line nitration catalyzed by different metal ions. Flow
rates in channels A and B = 0.05 μL minÀ1 and in channel C =
1 μL minÀ1. Channel A, 0.5 mM of Ang I, 1 mM of H2O2, and 2 mM of
NaNO2 in H2O; channel B, 25 μM of Aβ-16ÀCu2+, Cu2+, TCÀCu2+,
EDTAÀCu2+, heme, Fe2+, or Fe3+ in H2O; channel C, ESI buffer. The
error bars show the standard deviations of the calculated nitration levels.
The concentrations of different metal ions were all 25 μM because the
maximum concentration of heme in water under ambient conditions is
25 μM.
as shown in Figures 3 and 4. In addition, it is possible to generate
peroxynitrite or copper(II) bound peroxynitrite from •NO, O2,
and copper(I) complexes to induce nitration.47À49 However,
abundant copper(II) inhibits nitration induced by •NO, possibly
because a large amount of copper(II) can scavenge the •NO by
forming a NOÀCu2+(L) complex, where L is any ligand.56
Coordinated Copper(II) as Fenton Catalyst. Copper can be
easily coordinated by various ligands. The copper(II)/copper(I)
complexes were also reported to catalyze peroxynitrite decom-
position to induce ascorbic acid oxidation and nitration, and to
catalyze the formation of peroxynitrite coordinated copper(II)
from •NO and O2.43,47,57 Copper complexes that hold SOD-like
properties, peroxidase properties, and antioxidant properties
have already been synthesized.58,59
In Figure 1a, the peaks of Ang I monocoordinated copper(II)
are observed, demonstrating the presence of Ang IÀCu2+ during
the Ang I nitration. According to the tandem MS results shown in
Supporting Information S-9, Cu2+ should bind to the carbonyl
group on the backbone between valine and tyrosine, in accor-
dance with the literature.60 The structure of Ang IÀCu2+ is
shown in Scheme 3. Because it has been reported that amino acid
monocoordinated Cu2+ shows enhanced activity for the decom-
position of H2O2,61 it can be proposed that both peptide-
coordinated and noncoordinated Cu2+ may play a role in the
copper-catalyzed tyrosine nitration.
As shown in Scheme 3, the hydroperoxide anion should therefore
coordinate TCÀCu2+ as an axial ligand to form the key complex
of copper(II)Àhydroperoxo. The complex can further decom-
pose to generate TCÀcopper(I) complex. When EDTAÀCu2+
was used, only a background level of nitrite-induced tyrosine
nitration was observed. This phenomenon supports the pro-
posed mechanism shown in Scheme 2. In the very stable
structure of EDTAÀCu2+, Cu2+ is 6-fold coordinated, and
therefore the key copper(II)Àhydroperoxo complex necessary
for inducing copper(II) reduction cannot be generated, as shown
in Scheme 3.
A β-16ÀCu2+ was also employed instead of free copper(II)
to catalyze the Ang I nitration in the presence of H2O2 and nitrite.
A β-16 has been reported to have a strong affinity to coordinate
copper(II),62À64 and to further control the redox activity of
Cu2+/Cu+.65,66 As shown in Figure 6, A β-16ÀCu2+ is a better
nitration catalyst than free Cu2+ under the same experimental
conditions, illustrating that A β-16ÀCu2+ is a stronger Fenton
catalyst.
Additionally, because proteins/peptides are able to bind
various free metal ions, the abilities of different metal ions to
catalyze nitration with H2O2 and NO2À were examined. Speci-
fically, Fe2+, Fe3+, and heme were used for comparison. As shown
in Figure 6, the Cu2+-induced nitration is almost as efficient as
that mediated by Fe3+. An increase in efficiency of the A
β-16ÀCu2+-induced nitration, in comparison to that catalyzed
by heme under the same experimental conditions, implies that
Cu2+ or peptide-coordinated Cu2+ may play an important role in
many neurodegenerative disorders related to tyrosine nitration.
To study if Cu2+ can catalyze nitration without peptides,
4-aminophenol (AP) was used as a substrate for a nitrite-induced
nitration. In the presence of Cu2+, nitrite, and H2O2, nitrated AP
was generated together with the oxidized AP and detected by
mass spectrometry. This illustrates that copper(II) itself is a good
Fenton catalyst, Supporting Information S-10.
To evaluate the performance of coordinated copper(II) in tyrosine
nitration, different copper(II) complexes were prepared, including
β-amyloid-16 peptide coordinated copper(II) (A β-16ÀCu2+),
1,4,8,11-tetraazacyclotetradecane coordinated copper(II)
(TCÀCu2+), and ethylenediaminetetraacetic acid coordinated
copper(II) (EDTAÀCu2+). When TCÀCu2+ was used instead
of uncoordinated Cu2+ to catalyze the nitration of Ang I, nitrite-
induced tyrosine nitration was still realized. The nitration level
observed was similar to that when uncoordinated Cu2+ was
employed; see Figure 6. The affinity between TC and Cu2+ is
very strong; thus, no free Cu2+ or peptide coordinated Cu2+ can
take part in the nitration of tyrosine when an excess of TC is used.
’ CONCLUSION
A mass spectrometry-based methodology has been developed
to study the on-line nitration of tyrosine by nitrite and hy-
drogen peroxide. We have demonstrated that cupric ions and
cupricÀpeptides complexes act as efficient Fenton catalysts able
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Journal of the American Chemical Society
ARTICLE
to catalyze this reaction even better than heme. This chemical
pathway is similar to the enzymatic pathways classically consid-
ered. Using an off-line approach, we have shown that cupric
ions in the presence of hydrogen peroxide can promote tyrosine
nitration through promotion of the production of tyrosine
radicals by nitric oxide. This study shows that cupric ions play
a polyvalent role as Fenton catalysts to efficiently catalyze the
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Supporting Information. Chemicals employed, micro-
b
chip fabrication, tandem MS characterization of various peptides,
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’ AUTHOR INFORMATION
Corresponding Author
hubert.girault@epfl.ch
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’ ACKNOWLEDGMENT
We thank the Swiss National Science Foundation for support-
ing the project “Analytical tools for proteome analysis and
redoxomics (200020-127142)” and NSFC 20925517. B.L. is
grateful to EPFL for a visiting professor fellowship. We thank
Laboratoire de neurobiologie molꢀeculaire et neuroprotꢀeomique
of Ecole Polytechnique Fꢀedꢀerale de Lausanne for the synthesis of
human α-synuclein (107-140) peptide.
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