Easy oxidation and nitration of human myoglobin by nitrite and hydrogen peroxide

Article abstract of DOI:10.1002/chem.200500361

The modification of human myoglobin (HMb) by reaction with nitrite and hydrogen peroxide has been investigated. This reaction is important because NO₂^- and H₂O₂ are formed in vivo under conditions of oxidative and nitrative stress, where protein derivatization has been often observed. The abundance of HMb in tissues and in the heart makes it a potential source and target of reactive species generated in the body. The oxidant and nitrating species produced by HMb/H ₂O₂/NO₂^- are nitrogen dioxide and peroxynitrite, which can react with exogenous substrates and endogenous protein resi dues. Tandem mass analysis of HMb modified by stoichiometric amounts of H₂O₂ and NO₂^- indicated the presence of two endogenous derivatizations: oxidation of C110 to sulfinic acid (76 %) and nitration of Y103 to 3-nitrotyrosine (44%). When higher concentrations of NO₂^- and H₂O₂ were used, nitration of Y146 and of the heme were also observed. The two-dimensional gel-electrophoretic analysis of the modified HMbs showed spots more acidic than that of wild-type HMb, a result in agreement with the formation of sulfinic acid and nitrotyrosine residues. By contrast, the reaction showed no evidence for the formation of protein homodimers, as observed in the reaction of HMb with H₂O₂ alone. Both HMb and the modified HMb are active in the H₂O₂/NO ₂^--dependent nitration of exogenous phenols. Their catalytic activity is quite similar and the endogenous modifications of HMb therefore have little effect on the reactivity of the protein intermediates.

Full text of DOI:10.1002/chem.200500361

FULL PAPER
DOI: 10.1002/chem.200500361
Easy Oxidation and Nitration of Human Myoglobin by Nitrite and Hydrogen
Peroxide
Stefania Nicolis,[a] Andrea Pennati,[b] Eleonora Perani,[c] Enrico Monzani,[a]
Anna Maria Sanangelantoni,[d] and Luigi Casella*[a]
Abstract: The modification of human
myoglobin (HMb) by reaction with ni-
trite and hydrogen peroxide has been
investigated. This reaction is important
dues. Tandem mass analysis of HMb
modified by stoichiometric amounts of
H2O2 and NO2 indicated the presence
HMbs showed spots more acidic than
that of wild-type HMb, a result in
agreement with the formation of sulfin-
ic acid and nitrotyrosine residues. By
contrast, the reaction showed no evi-
dence for the formation of protein ho-
modimers, as observed in the reaction
of HMb with H2O2 alone. Both HMb
and the modified HMb are active in
ꢀ
of two endogenous derivatizations: oxi-
dation of C110 to sulfinic acid (76%)
and nitration of Y103 to 3-nitrotyrosine
(44%). When higher concentrations of
ꢀ
because NO2 and H2O2 are formed in
vivo under conditions of oxidative and
nitrative stress, where protein derivati-
zation has been often observed. The
abundance of HMb in tissues and in
the heart makes it a potential source
and target of reactive species generated
in the body. The oxidant and nitrating
ꢀ
NO2 and H2O2 were used, nitration of
Y146 and of the heme were also ob-
served. The two-dimensional gel-elec-
trophoretic analysis of the modified
ꢀ
the H2O2/NO2 -dependent nitration of
exogenous phenols. Their catalytic ac-
tivity is quite similar and the endoge-
nous modifications of HMb therefore
have little effect on the reactivity of
the protein intermediates.
ꢀ
species produced by HMb/H2O2/NO2
Keywords: myoglobin · nitration ·
are nitrogen dioxide and peroxynitrite,
which can react with exogenous sub-
strates and endogenous protein resi-
nitrogen oxides
·
oxidation
·
protein modifications
Introduction
ticular interest in the present context is the fact that human
myoglobin (HMb) differs from the other known mammalian
Mbs in the presence of a cysteine residue at position 110.
The abilities of HMb to preserve NO bioactivity through
the formation of an S-nitrosothiol group at this C110 residue
and to regulate NO in vivo either by the formation of an
Although myoglobin (Mb) has been one of the most widely
studied proteins, continuing interest in its study arises from
the new activities and applications that have been reported
through the years. Besides the classical functions of storage
and intracellular transfer of molecular oxygen,[1] several
other activities of this protein have been found.[2–5] Of par-
ꢀ
iron–nitrosyl HMb complex or by oxidation of NO to NO3
are the most important recent findings in the biochemistry
of this protein.[6–8] Other reported activities of Mbs involve
catalytic reactions on exogenous substrates; examples of
these reactions are the peroxide-dependent oxidation[9,10]
and nitration[11] of phenolic substrates, the sulfoxidation of
organic sulfides,[12–16] and the epoxidation of alkenes,[13–16] al-
though the catalytic efficiency of the protein in these pro-
cesses is generally modest.
[a]Dr. S. Nicolis, Dr. E. Monzani, Prof. L. Casella
Dipartimento di Chimica Generale
Università di Pavia, Via Taramelli 12, 27100 Pavia (Italy)
Fax : (+39)0382-528-544
E-mail: bioinorg@unipv.it
[b]Dr. A. Pennati
Dipartimento di Scienze Biomolecolari e Biotecnologie
Università degli Studi di Milano, Via Celoria 26, 20133 Milano (Italy)
Chemical modification of proteins is systematically ob-
served under pathophysiological conditions; the identifica-
tion of specific residues involved and of the nature of the
modification produced is therefore essential for the under-
standing of the mechanisms of development of the patholo-
gies.[17] Reactive oxygen and nitrogen species are typically
responsible for these modifications,[18–20] through the forma-
[c]Dr. E. Perani
Centro Grandi Strumenti
Università di Pavia, 27100 Pavia (Italy)
[d]Prof. A. M. Sanangelantoni
Dipartimento di Scienze Ambientali
Università di Parma, 43100 Parma (Italy)
Chem. Eur. J. 2006, 12, 749 – 757
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
749

tion of protein-centered radicals similar to those involved as
intermediates in the mechanism of many redox en-
zymes.[21–26] For instance, the reaction of ferric human myo-
globin (metHMb) with H2O2 yields protein radicals localized
on tyrosine and/or tryptophan residues.[27,28] Of relevance for
the present investigation is the finding by the group of
Mauk that the reaction of HMb with hydrogen peroxide,
peroxynitrite, or nitric oxide leads to modification of two
specific residues, Y103 and C110, through radical mecha-
nisms.[6,29–31] In particular, addition of H2O2 to HMb produ-
ces a C110 thiyl radical, through an intermolecular electron-
transfer reaction from C110 to a Y103 phenoxy radical,
which results in the formation of an intermolecular disulfide
bond and a protein homodimer.[29,30] The same dimer is also
obtained from the reaction of HMb with peroxynitrite, but
the addition of physiological concentrations of carbonate re-
sults in the nitration of the protein at Y103.[31] The cysteine
residue can also be derivatized to form an S-nitrosothiol
group by the reaction of metHMb and NO.[6] Both protein
and nonprotein thiol groups are potential targets of H2O2
and ONOOꢀ. Besides disulfide bridges, the oxidation of
thiol groups can lead to sulfenic (RSOH), sulfinic (RSO2H),
or sulfonic (RSO3H) acids, depending on the species in-
volved.[32]
Modification of HMb: Modification of HMb under mild
conditions was obtained by reaction of the protein (50 mm),
nitrite, and H2O2 in a molar ratio of 1:2:2. This reaction
yields a protein derivative, m-HMb, with UV-visible spectral
features basically indistinguishable from those of HMb. On
the other hand, when HMb was treated with larger concen-
ꢀ
trations of NO2 (0.8m) and H2O2 (1 mm), the solution
turned from brown to greenish-brown and the reaction
yielded the m’-HMb derivative. The change of color does
not correspond to a shift of the Soret band, with the maxi-
mum remaining always at 408 nm. In a comparison of the
UV-visible spectra of HMb and m’-HMb (Figure 1), only a
slight broadening of the Soret band is observed. The color
change is due to nitration of one of the heme vinyl groups
and tyrosine residues (see below).
Herein we report on the modifications undergone by
HMb in the reaction with nitrite and hydrogen peroxide in
conditions of low and high reagent concentrations, which
produce modified protein derivatives labeled as m-HMb
and m’-HMb, respectively. In both cases, the major protein
modification occurs at the cysteine residue, which is trans-
formed into sulfinic acid, in addition to the expected tyro-
sine nitration. This extremely facile reaction is of some in-
terest in view of the emerging role of Mb in the heart,
where the protein is particularly abundant.[17,33] Cardiovascu-
lar pathologies like heart failure and ischemia-reperfusion
injury are typically characterized by sudden formation of
oxygen radicals and NO overexpression, which lead to ex-
tensive nitration of protein tyrosine residues through mecha-
nisms that are still not completely understood.[17] While tyro-
sine nitration is the commonly accepted marker of these
events, we show here that modification of the cysteine resi-
due of Mb occurs to a much larger extent. Comparative
studies show that the modified Mb derivative maintains the
same ability to catalyze nitrative reactions on exogenous
substrates as wild-type Mb.
Figure 1. UV/Vis spectrum of m’-HMb (thin upper trace) compared with
the spectrum of HMb (bold lower trace), both in 0.2m phosphate buffer
(pH 7.5) at 258C. The concentration of the proteins was 7.5 mm.
Tandem mass analysis of m’-HMb: The characterization of
the modifications introduced into HMb by reaction with
ꢀ
NO2 and H2O2 was initially carried out on m’-HMb, where
the extent of the modifications was larger. To this end, the
polypeptide fragments resulting from tryptic digestion of
apoHMb and apo-m’-HMb were analyzed by HPLC–ESI-
MS/MS. Comparison of the data showed the presence of
two modified peptide fragments as bicharged ions with
m/z=973 (mass of 1944 amu) and m/z=995.5 (mass of
1989 amu). These both correspond to modification of the
103–118 peptide (containing Y103 and C110, with a mass of
1912 amu). The peptide with a mass of 1944 amu contains
two oxygen atoms more than the parent peptide, while the
one with a mass of 1989 amu shows the further replacement
of a proton with NO2. Both modified peptides were ana-
lyzed by collision-induced dissociation (CID). The MS/MS
spectrum of the latter is shown in Figure 2, where the y and
b ion series are reported. A mass difference of 135 amu was
detected between b8 and b7, a difference indicative of the
presence of a cysteinyl residue modified by two oxygen
atoms. In the y ion series, the mass difference of 117 amu
between y09 and y8 corresponds to the same cysteine modifi-
Results
Cloning and expression of HMb: The fully successful ex-
pression of HMb in bacteria is reported here for the first
time. All previous attempts gave the protein in inclusion
bodies,[34,35] while we obtained the holoprotein in soluble
form. In our hands, complete maturation of the apoprotein
into its holo form, with the heme bound to the polypeptide,
was observed only when bacterial growth was carried out
under slow mechanical stirring.
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Chem. Eur. J. 2006, 12, 749 – 757

Oxidation and Nitration of Human Myoglobin
FULL PAPER
trum of m’-HMb, three peptides containing the Y146 residue
were found that are absent in the LC–MS spectrum of HMb
and that had a mass difference of 45 amu with respect to the
corresponding unmodified peptides. These peptides were
140–153 (mass difference from 1587 to 1632 amu), 141–153
(mass difference from 1459 to 1504 amu), and 141–147
(mass difference from 827 to 872 amu). These data indicate
the presence of another endogenous nitration at Y146; this
modification occurs to a lower extent with respect to the ni-
tration at Y103 and the modified protein represents ꢁ10–
20% of the initial HMb, as judged by relative peak areas in
the EIC chromatograms.
The nitration of one heme vinyl group was reported to
occur upon treatment of Mb at pH 5.5 with a large excess of
nitrite; in this case, the reaction was also accompanied by a
change of color of the protein solution to greenish-brown.[36]
At pH 7.5 this reaction is much slower. We previously found
the same heme modification in horse heart Mb after treat-
ꢀ
ment with NO2 and H2O2 at pH 7.5.[11] To assess the occur-
rence of the modification of the heme prosthetic group in
m’-HMb, an HPLC–ESI-MS/MS analysis was performed on
the protein solution. The injected m’-HMb sample was dena-
tured in the HPLC column and the released hemin was sep-
arated from the apoprotein; ions at m/z=616 and 661, cor-
responding to hemin and to a modified hemin where a
proton of the prosthetic group was replaced by a NO2 group
in the porphyrin, respectively, were detected in MS/MS
mode. Integration of the peaks in the MS/MS chromato-
grams indicated that the nitration occurred with 8% conver-
sion.
Figure 2. MS/MS spectrum of the m/z=995.5 peak (mass of 1989 amu)
assigned to the 103–118 peptide in a double-charged state and with two
modifications: the oxidation of C110 to the corresponding sulfinic acid,
C110-SO2H, and the nitration of Y103 to 3-nitrotyrosine, Y103-NO2. The
assignment of the y and b ion series is shown. Above the spectrum, the
sequence of the 103–118 peptide, with the two modified amino acids in
bold and with the summary of the y and b ions found in the spectrum, is
reported.
cation (with consideration of the loss of a water molecule in
the y0 series). These results allowed unequivocal assignment
of the oxidation of C110 to the corresponding sulfinic acid.
The mass of the peptide, together with the b ion series, is in-
dicative of the presence of another modification, corre-
sponding to a mass increment of 45 amu; the known reactiv-
ity of tyrosine residues under these conditions[11] allowed the
assignment of the modification as the nitration of Y103 to 3-
nitrotyrosine.
A quantitative analysis of the modified 103–118 peptide
was obtained by comparing, in the extracted ion current
(EIC) chromatograms, the areas of the peaks corresponding
to the derivatized peptides (m/z=973 and 995.5 for the bi-
charged ions, with masses of 1944 and 1989 amu, respective-
ly) with that of the starting peptide (m/z=957 for the bi-
charged ion, with a mass of 1912 amu). The relative amounts
of the three peptides were as follows: unmodified peptide
5%, peptide containing only the C110-SO2H modification
41%, and the peptide containing both the Y103-NO2 and
C110-SO2H modifications 54%. The peptide nitrated only at
Y103 was not found in the HPLC–MS/MS analysis, although
it might be present in a very small amount. This result indi-
cates that the cysteine residue is more reactive than tyro-
sine.
Tandem mass analysis of m-HMb: To assess whether the
modifications taking place in HMb in the presence of high
ꢀ
concentrations of NO2 and H2O2 could also be obtained
under milder conditions, the polypeptide fragments resulting
from tryptic digestion of apo-m-HMb were analyzed by
HPLC–ESI-MS/MS. The modified 103–118 peptides corre-
sponding to the oxidation of C110 to sulfinic acid (mass of
1944 amu) and to both the oxidation of C110 and the nitra-
tion of Y103 (mass of 1989 amu) were found. The latter
peak was analyzed by CID obtaining an MS/MS spectrum
analogous to that previously reported for m’-HMb
(Figure 2). The relative amounts of the starting and modi-
fied 103–118 peptides resulting from quantitative analysis
were as follows: unmodified peptide 24%, peptide contain-
ing only the C110-SO2H modification 32%, and peptide
containing both the Y103-NO2 and the C110-SO2H modifi-
cations 44%.
As in the case of m’-HMb, the peptide nitrated only at
Y103 was also absent in the tryptic fragments resulting from
the analysis of m-HMb. Furthermore, unlike m’-HMb, pep-
tide fragments containing neither nitration at residue Y146
nor nitration at the heme prosthetic group (analyzed by
HPLC–ESI-MS/MS on the protein solution) could be de-
tected, a result indicating that both types of modification
were at best present in very small, undetectable amounts. It
should be noted that in the conditions employed here, that
The occurrence of nitration at the other tyrosine residue
of HMb, Y146, was also investigated. In the LC–MS spec-
Chem. Eur. J. 2006, 12, 749 – 757
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751

L. Casella et al.
is, with only a slight excess H2O2 with respect to HMb,
almost all of the peroxide was consumed for the modifica-
tion of Y103 and C110, so only a small fraction was actually
available for nitration of Y146 and the heme.
Gel electrophoresis analysis of modified HMb: The two-di-
mensional polyacrylamide gel of m’-HMb showed that the
modified protein is not homogeneous: two bands with differ-
ent isoelectric points and similar intensity could be observed
in the gel. The comparison of the two-dimensional poly-
acrylamide gel of m’-HMb with that of HMb indicated that
both the spots of m’-HMb where more acidic than the
native protein. By considering the linear pH gradient from
6.5–8.5 that was used, it was possible to estimate pI shifts of
0.1 and 0.2 units for the two spots of m’-HMb from the
7.2 value for HMb (as reported by Varadarajan et al.).[34] We
can assign the spot with pI 7.1 to the C110-SO2H protein de-
rivative (41% from MS/MS analysis) and the spot with
pI 7.0 to the Y103-NO2/C110-SO2H protein derivative
(54%). The increased acidity of the modified protein can be
related both to tyrosine nitration and to sulfinic acid forma-
tion. In fact, the oxidation of one cysteine residue to sulfinic
acid in peroxiredoxin I (an enzyme catalyzing the destruc-
tion of peroxides under oxidative stress conditions) was re-
ported to cause a decrease in the isoelectric points of 0.5 pH
units.[37]
Scheme 1. Structures of the phenols employed in the kinetic studies.
reactivity of the Y103-NO2/C110-SO2H HMb derivative,
while the contribution of the protein fraction containing par-
tial nitration at Y146 and at the heme can be neglected.
Therefore, m’-HMb could be a good probe for the analysis
of the effect of Y103 and C110 modification on the reactivi-
ty of the protein in the presence of nitrite and hydrogen per-
oxide.
In the phenol nitration, each reactant affects the reaction
rate in a way dependent on the concentration of the others.
In this study, the concentration of the Mb derivatives was
kept constant at 1 mm, because under these conditions the
noncatalytic reaction could always be neglected.[11] The con-
centration of the phenols and nitrite were instead varied; in
order to simplify the analysis, it was found convenient to
vary the concentration of one reagent while maintaining
that of the others constant at the value that maximizes the
rate. The plots of rate versus [phenol]showed saturation be-
havior, a result suggesting the need for an interaction with
Homodimers of wild-type HMb were formed through di-
sulfide linkage involving C110 residues following the reac-
[30]
tion with H2O2
or peroxynitrite.[31] However, no dimer
ꢀ
was detected in the sodium dodecylsulfate (SDS) polyacryl-
amide gel of m’-HMb performed in nonreducing conditions:
this gel was, in fact, equivalent to the gel of wild-type HMb.
the protein. The rate versus [NO2 ]exhibited a slightly
more complicated dependence; in this case, an initial sig-
ꢀ
moidal phase, at low [NO2 ], is followed by nitrite satura-
ꢀ
Thus, the HMb/NO2 /H2O2 system performs the endogenous
tion. This behavior can be explained by considering for
HMb and m’-HMb a mechanism similar to that observed
with the protein from horse heart: two competing pathways
are in fact operative in the nitration of the phenols, depend-
ing on nitrite concentration.[11] In the first one, which domi-
modifications identified by the MS analysis without forma-
tion of protein homodimers (either by dicysteine or dityro-
sine linkages).
ꢀ
Binding of nitrite to HMb: The iron(iii) center of HMb
nates at low [NO2 ], the protein reacts through a perox-
binds nitrite to form the low-spin HMbFeIII–NO2 complex;
idase-like cycle involving two active intermediates, which
can give one-electron oxidation of the substrates, thereby
producing a phenoxy radical from the phenol and NO2C from
ꢀ
the reaction can be followed through the shift of the Soret
band of the protein from 408 to 410 nm. From spectrophoto-
metric monitoring of the changes of the Soret band as a
function of nitrite concentration at pH 7.5, the binding con-
stant KB =(76ꢂ2)mꢀ1 was obtained. This value is compara-
ble to that obtained with horse heart Mb (KB =51mꢀ1)
under the same conditions.[11]
ꢀ
nitrite. The other mechanism predominates at high [NO2 ]
and does not involve peroxidase-like intermediates; hydro-
gen peroxide reacts with the iron-bound nitrite to produce a
nitrating active species that we assumed to be a protein-
bound peroxynitrite, MbFeIII–N(O)OO.[11] With changes in
ꢀ
[NO2 ], the fraction of protein–nitrite complex changes and
Phenol nitration catalyzed by HMb and m’-HMb: Like the
met form of horse heart Mb,[11,38–40] HMb and m’-HMb also
catalyze the nitration of phenols to ortho-nitrophenols in
the presence of hydrogen peroxide and nitrite. The reactions
studied here are the nitration of representative phenolic
substrates 1–3 (Scheme 1). By considering that in the case of
m’-HMb the extent of the modifications at residues Y103
and C110 was in excess of 50% and very close to 100%, re-
spectively, the reactivity of the modified HMb deduced from
the kinetic studies can be considered as an indication of the
this necessarily affects the relative importance of the two
ꢀ
paths (via NO2C or via ONOOꢀ) operating in the Mb/NO2
/
H2O2-dependent nitration.[11] A simplified kinetic study of
the nitration process can be carried out by assuming that the
peroxynitrite path dominates at relatively high nitrite con-
centration.[11] The rate dependence on the phenol concentra-
tion at constant (high) nitrite level is then given by Equa-
tion (1). The rate dependence on nitrite concentration
should instead consider that the NO2C path can be observed
only at low nitrite concentration [Eq. (2)].[11]
752
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Chem. Eur. J. 2006, 12, 749 – 757

Oxidation and Nitration of Human Myoglobin
FULL PAPER
kcat½MbŠ½PhOHŠ
rate ¼
to the fact that the binding of the phenolic substrate near
the heme site during catalysis hinders the interaction of
NO2 at the iron center. Furthermore, it should be taken
ð1Þ
ð2Þ
ðKPMhOHþ½PhOHŠÞ
ꢀ
ꢀ
into consideration that the change in the mechanism of the
kcat½MbŠð½NO2 ŠꢀbÞ
rate ¼
ꢀ
ðKMnitriteþ½NO2 Šꢀ2bÞ
ꢀ
reaction with changing [NO2 ]prevents the precise determi-
nation of KnMitrite. In general, however, the reactivity of the
modified HMb in the nitration of phenolic substrates is
quite similar to that of wild-type HMb. By comparing the
parameters kcat and kcat/KPMhOH (obtained at saturating nitrite
concentration) and kcat and kcat/KnMitrite (obtained at saturating
phenol concentration) for HMb and m’-HMb, a general
slight decrease in the rate constants can be noted for the
modified protein.
In these equations, [Mb]is the total HMb or m ’-HMb con-
centration, kcat represents the maximum turnover number of
the proteins, and KnMitrite and KPMhOH give an indication of the
ꢀ
dissociation constant of NO2 and PhOH, respectively, from
the complexes with the proteins in the presence of the other
reagent. The parameter b had to be introduced into Equa-
tion (2) to account for the change in the mechanism at in-
creasing nitrite concentration; furthermore, the data at very
ꢀ
low [NO2 ]were not considered in the interpolation of the
Discussion
experimental data. The values of kcat, KPMhOH, and kcat/KPMhOH
obtained from the rate dependence on [PhOH]and those of
kcat, KMnitrite, and kcat/KnMitrite obtained from the rate dependence
In analogy with horse heart Mb,[11] HMb is able to use ni-
trite and hydrogen peroxide to generate the oxidant and ni-
trating species nitrogen dioxide and peroxynitrite. Both
these species perform nitration
ꢀ
on [NO2 ]for the nitration of substrates 1–3 are reported in
Table 1.
of exogenously supplied phe-
ꢀ
Table 1. Steady-state kinetic parameters for the HMb- and m’-HMb-dependent phenol nitration by NO2
/
nols and the protein is thus an
efficient catalyst in the nitration
of substrates 1–3. However,
NO2C and ONOOꢀ can also oxi-
dize and nitrate protein resi-
dues. The tandem mass analysis
of HMb modified with stoichio-
metric amounts of the reagents
indicates the presence of two
endogenous derivatizations: ni-
tration of Y103 to 3-nitrotyro-
sine (at 44%) and/or oxidation
of C110 to the corresponding
sulfinic acid C110-SO2H (at
76%). Upon reaction of HMb
with high concentrations of
H2O2 in 0.2m phosphate buffer (pH 7.5) at 258C. For comparison purposes, corresponding kinetic data for the
lactoperoxidase (LPO) catalyzed reactions under the same conditions are included (from ref. [54]).
Phenol
KPMhOH
[mm]
kcat
kcat/KPMhOH
[mꢀ1 sꢀ1
KnMitrite
[m]
kcat
kcat/KnMitrite
[mꢀ1 sꢀ1
[sꢀ1
]
]
[sꢀ1
]
]
HMb
1
2
3
0.35ꢂ0.07
0.12ꢂ0.02
0.47ꢂ0.04
2.1ꢂ0.1
0.67ꢂ0.03
2.4ꢂ0.1
6000ꢂ1000
5600ꢂ900
5200ꢂ200
0.30ꢂ0.04
0.24ꢂ0.04
0.25ꢂ0.04
2.1ꢂ0.1
1.03ꢂ0.08
1.7ꢂ0.1
6.9ꢂ0.6
4.3ꢂ0.5
6.9ꢂ0.8
m’-HMb
1
2
3
0.40ꢂ0.07
0.16ꢂ0.02
0.5ꢂ0.1
1.26ꢂ0.06
0.62ꢂ0.03
1.3ꢂ0.2
3100ꢂ400
3800ꢂ400
2400ꢂ200
0.24ꢂ0.04
0.23ꢂ0.04
0.20ꢂ0.04
1.31ꢂ0.09
0.68ꢂ0.05
1.0ꢂ0.1
5.5ꢂ0.6
3.0ꢂ0.4
5.1ꢂ0.7
LPO
1
2
3
0.12ꢂ0.01
0.14ꢂ0.02
0.11ꢂ0.01
380ꢂ10
130ꢂ5
75ꢂ2
3.2”106
9.4”105
6.8”105
48ꢂ5
30ꢂ3
16ꢂ1
380ꢂ15
135ꢂ5
80ꢂ2
7.9”103
4.5”103
5.0”103
ꢀ
NO2 and H2O2, the extent of
The data show that the catalytic activity of HMb and m’-
HMb in the phenol nitration is comparable to that of horse
heart Mb, with HMb being slightly more efficient in the pro-
duction of 3-nitrotyramine and 3-nitrotyrosine.[11] The rela-
tively small differences observed in the kcat parameters for
the different substrates with different charges at pH 7.5 are
probably connected to the high efficiency of the nitrating
oxidation of C110 (at 95%) and nitration of Y103 (at 54%)
increased and nitration of Y146 (at ꢁ10–20%) and the
heme (at ꢁ8%) was also observed. Although the ferric
form studied here is not the physiological form of the pro-
tein, the presence of hydrogen peroxide and nitrite would
rapidly convert ferrous or oxy Mb into the oxidized form.
Both NO2C and ONOOꢀ may be responsible for the oxida-
tion of the cysteine residue. A free-radical pathway can be
proposed for the formation of sulfinic acid by nitrogen diox-
ide, according to the Equations (3)–(5), in analogy with the
mechanism proposed for the oxidation of human serum al-
bumin by peroxynitrite in the presence of carbon dioxide.[41]
ꢀ
agent generated by the Mb/H2O2/NO2 systems. As ob-
served with horse heart Mb,[11] the KPMhOH values are below
the millimolar range; this is compatible with a disposition of
the substrate at the protein surface. It is interesting to note
that the values of KnMitrite are quite similar for the different
substrates. The reciprocal of KnMitrite value should give an in-
dication, extrapolated from kinetic studies, of the nitrite
binding constant to the protein (KB =1/KnMitrite ꢁ4mꢀ1). This
value differs significantly from the binding constant ob-
HMb-SHþNO2 ! HMb-SCþHNO2
ð3Þ
ð4Þ
ð5Þ
C
HMb-SCþO2 ! HMb-SOOC
ꢀ
tained independently for the HMbFeIII–NO2 complex
eꢀ
HMb-SOOC
HMbꢀSO H
(KB =76mꢀ1). The smaller value of 1/KnMitrite can be ascribed
ƒ!
2
Hþ
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753

L. Casella et al.
The thiol group of HMb is oxidized by nitrogen dioxide to
the thiyl radical [Eq. (3)]; this radical can react with dioxy-
gen to form a peroxy radical [Eq. (4)], which in turn produ-
ces the sulfinic acid after hydrogen-atom abstraction from a
reductant, according to Equation (5). Peroxynitrite can also
promote the oxidation of cysteinyl residues beyond sulfenic
acid; this has been observed in the oxidation of bovine
serum albumin, which has only one sterically isolated thiol
group.[32] Both the oxidation of C110 and the nitration of
Y103 were apparent in the two-dimensional polyacrylamide
gel of m’-HMb, where the two spots that were more acidic
than HMb and have similar intensity correspond to the
modified proteins with both sulfinic acid at C110 and 3-ni-
trotyrosine at Y103 and with sulfinic acid at C110, respec-
tively. Sulfinylation is not a rare event, given that 1–2% of
the cysteine residues of soluble proteins from rat liver were
detected as cysteine sulfinic acid.[42] Formation of sulfinic
acid is a reversible reaction, as is the case for the oxidation
of the active-site cysteine of peroxiredoxins.[43] Nitration of
tyrosine residues (Y103 and Y146) can also be promoted by
both nitrogen dioxide and peroxynitrite, according to the
mechanisms previously described.[11]
K45R/C110A mutant of HMb (Figure 3).[44] In this structure,
Y103 is the tyrosine residue nearest to the heme group and
is exposed to solvent, while Y146 is an internal residue, as is
Witting et al. reported that the reactions between HMb
and H2O2 or ONOOꢀ result in the formation of a protein
homodimer through an intermolecular disulfide bond.[30,31]
The first step of this process is the oxidation of the Y103
residue to the Y103 phenoxy radical (as a component of the
compound I like species). In our case, where NO2C is also
produced, the protein radical is intercepted in a fast process
that leads to the formation of nitrotyrosine, according to
Equation (6).
Figure 3. Structure of the K45R/C110A mutant of HMb.[44] The disposi-
tion of the side chains of Y103, A110, and Y146 is shown; HMb contains
a cysteine residue at position 110.
also the case for horse heart Mb. Our previous studies
showed that in the presence of nitrite and hydrogen perox-
ide the latter protein undergoes endogenous nitration only
at the Y146 residue, not at Y103.[11] This result was of some
importance because a controversy exists concerning the po-
sition of the tyrosine residue (Y103 versus Y146) in horse
heart Mb that is nitrated by peroxynitrite.[31,45,46] The reac-
tion of the latter species and HMb in the presence of car-
bonate results in nitration at Y103; in the presence of hy-
drogen peroxide, or peroxynitrite without carbonate, both
residues Y103 and C110 of HMb undergo radical reactions
which eventually lead to the formation of protein homodim-
ers.[30,31] A different modification at the cysteinyl residue
was only observed in the reaction of HMb with nitric oxide,
which yielded an S-nitrosated protein derivative.[6]
HMb-Y103CþNO2 ! HMb-Y103-NO2
ð6Þ
C
In this way, the electron transfer from C110 to Y103C to pro-
duce a thiyl radical and hence the dicysteine linkage is pre-
vented. In fact, in the SDS polyacrylamide gel of m’-HMb,
the band corresponding to the HMb homodimer is absent or
below the detection limit.
The MS analysis of m’-HMb (but not that of m-HMb)
also revealed the presence of a modification on the heme
prosthetic group that corresponds to the replacement of a
proton with an NO2 group; the modification probably
occurs at one of the heme vinyl groups, in analogy to the
heme nitration process by a large excess of nitrite reported
for Mb at pH 5.5.[36] We previously found similar heme nitra-
In our system, once the oxidizing and nitrating species
(that is, NO2C or ONOOꢀ) is produced near the heme, it per-
forms derivatization at the nearest protein residues, Y103
and C110. According to the mechanism previously reported
ꢀ
tion in horse heart Mb upon treatment with NO2 and
H2O2.[11] However, nitration of HMb occurs only to the
extent of 8%, while for horse heart Mb conversion into the
nitrated heme accounted for 50% of the starting protein
under the same conditions. This difference clearly depends
on the presence in HMb of the reactive cysteine residue
that can capture the oxidant species and thereby protect the
prosthetic group from nitration.
ꢀ
for the nitration catalyzed by horse heart Mb, at low [NO2 ]
(such as that employed for the production of m-HMb) the
ꢀ
oxidant and nitrating species is NO2C, while at high [NO2 ]
(employed for the production of m’-HMb) the reactive spe-
cies is mainly a protein-bound peroxynitrite species. As dis-
cussed before, both NO2C and ONOOꢀ may be responsible
for oxidation of C110 and nitration of Y103 and the two
modifications are actually observed in both the m-HMb and
m’-HMb derivatives. The cysteine residue is always more re-
The three-dimensional structure of wild-type HMb is not
available. A reference structure for localizing the residues
subjected to modifications is the crystal structure of the
754
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Chem. Eur. J. 2006, 12, 749 – 757

Oxidation and Nitration of Human Myoglobin
FULL PAPER
Cloning, expression, and protein purification: The open reading frame of
the human myoglobin gene was amplified from the fagmide pDNR-LIB,
clone IRALp962G1819Q2 (genome bank RZPD, Deutsches Ressourcen-
zentrum für Genomforschung GmbH, Heubnerweg 6, 14059 Berlin), by
using the polymerase chain reaction (PCR) with the nucleotides 5’-
CGCGGATCCTAGCCCTGGAAGCCCAG-3’ and 5’-GGGAATTCCA-
TATGGGGCTCAGCGAC-3’ which contain the BamHI and NdeI re-
striction sites (italicized). The BamHI–NdeI fragment containing hmb
was cloned in the BamHI–NdeI sites of the expression vector pET-11a,
thereby yielding pEThmb. The presence of the wild-type hmb gene was
confirmed by DNA sequence analysis prior to protein expression in bac-
teria. Escherichia coli BL21(DE3) (E. coli) cells transformed with
pEThmb were grown in flasks under shaking at 378C in Luria–Bertani
medium supplemented with ampicillin (100 mgmLꢀ1) for the purpose of
plasmid selection and maintenance. Cells were grown to the midexponen-
tial growth phase before induction with 1 mm isopropyl-b-d-thiogalacto-
side. Bacterial growth was carried out under slow stirring conditions.
Cells were harvested after 24–28 h and analyzed for the presence of solu-
ble HMb by SDS polyacrylamide gel electrophoresis (SDS PAGE) analy-
sis.
active than tyrosine: it is oxidized to a larger extent both in
m-HMb (by NO2C) and in m’-HMb (by ONOOꢀ).
In the presence of high nitrite concentration, the peroxy-
nitrite species can also react, albeit with lower efficiency,
with the prosthetic group and diffuse to the Xe1 cavity of
HMb, where it performs the nitration of the Y146 residue,
which is located in the proximity of this cavity.[3,35,47] As sug-
gested by a comparison of the data on the catalytic activity
of wild-type HMb and the m’-HMb derivative (Table 1), it is
apparent that the endogenous modifications have little
effect on the reactivity of the protein intermediates.
Conclusion
Myoglobin is abundant in tissues and particularly in the
heart, where it can be present at concentrations up to
0.2 mm.[7] Experiments with transgenic mice deficient in
HMb have recently shown that the protein exhibits a cardio-
protective role against nitrosative[33,48] and oxidative
stress.[49] However, HMb is itself a potential source and, at
the same time, a potential target of the oxidant and nitrating
species generated in the body in the presence of hydrogen
peroxide and nitrite. This situation can occur during inflam-
matory processes in tissues and in cardiovascular patholo-
gies, where H2O2 and nitrite are continuously produced.[17,50]
The oxidant and nitrating species produced by the HMb/
All purification steps were performed at 48C except for HPLC runs. The
E. coli cell paste was resuspended in 20 mm tris(hydroxymethyl)amino-
methane–HCl (Tris-HCl)/1 mm ethylenediaminetetraacetate (EDTA)
buffer (pH 8.0, 2 mLgꢀ1) containing 1 mm b-mercaptoethanol and 1 mm
phenylmethylsulfonyl fluoride (PMSF) and disrupted by sonication.
After removal of cell debris by centrifugation at 20000 g for 1 h, the
dark-brown solution was brought to 60% ammonium sulfate saturation,
the precipitate was discarded, and the soluble fraction was precipitated
with 95% ammonium sulfate saturation. The solution was stirred over-
night at 48C and then centrifuged (at 48C, 43000 g, for 1 h); the pellet
was resuspended in 20 mm Tris-HCl/1 mm EDTA buffer (pH 8.0) contain-
ing 1 mm b-mercaptoethanol. The solution was dialyzed overnight (with
several buffer changes) against buffer and then it was loaded onto a di-
ethylaminoethyl (DEAE) CL-6B anion-exchange column, which was
eluted with 20 mm Tris-HCl/1 mm EDTA buffer (pH 6.5) at a flow rate of
12 mLminꢀ1. The fractions containing the protein were collected and the
purity was checked by monitoring the absorbance ratio ASoret/A280 (which
was above 2.5). The protein solution was loaded onto a Superdex G75
ꢀ
H2O2/NO2 system can react both with exogenous substrates
and endogenous protein residues. In particular, we have
found here that modification of the Y103 and C110 residues
is a remarkably efficient process which occurs extensively
on HMb in the presence of stoichiometric amounts of nitrite
and hydrogen peroxide. Nitrite actually appears to potenti-
ate the harmful effect of the peroxide on the protein and
can completely depress the formation of myoglobin dimers,
which are the most typical modification product of the pro-
tein with H2O2.[29,30] The present results show that the pat-
tern of covalent modifications that HMb can undergo in
pathological conditions is more complex than was previously
thought.
gel-filtration column (Amersham Biosciences) connected to
a Jasco
HPLC instrument with a MD-1510 diode array detector and it was eluted
with 20 mm Tris-HCl/150 mm NaCl buffer (pH 8.0) at a flow rate of
0.5 mLminꢀ1. The fractions containing the protein were collected and the
purity was checked as described above, with an ASoret/A280 value >4.0
being achieved.
The expression of HMb yields the protein in the FeII–O2 form; the met
(Fe3+) species was obtained by treating the protein with diluted H3PO4
until pH 3.5 was reached and then adding diluted NaOH until pH 7 was
reached. In all the experiments described below, the proteins (wild-type
HMb and modified proteins) are utilized in their met form.
ꢀ
Modification of HMb in the presence of NO2 and H2O2: The solution of
metHMb (ꢁ50 mm) was dialyzed against 20 mm phosphate buffer
(pH 7.5). The samples of modified HMbs, m-HMb and m’-HMb, were
prepared by adding sodium nitrite (final concentrations of 0.1 and
800 mm, respectively) and hydrogen peroxide (final concentrations of 0.1
and 1 mm, respectively) to the metHMb solution. The proteins were al-
lowed to react at 208C for 10 min. While the color of the solution of m-
HMb remained unchanged, the solution turned from brown to greenish-
brown in the case of m’-HMb. Excess nitrite and oxidant were removed
by overnight dialysis against 20 mm phosphate buffer (pH 7.5).
Experimental Section
Materials: Hydrogen peroxide (30% solution), tyramine (1), 3-(4-hy-
droxyphenyl)propionic acid (2), and l-tyrosine (3) were obtained from
Aldrich. Ampicillin was from Shelton Scientific, trypsin from Sigma, and
BamHI and NdeI from Amersham Biosciences. The other reagents were
obtained at the best grade available. All the buffer solutions were pre-
pared by using deionized, double-distilled water. The concentration of
hydrogen peroxide solutions was controlled by monitoring the formation
of the 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radi-
cal cation according to a standard method that we followed previously.[51]
The concentration of HMb solutions was determined from the extinction
coefficient of metHMb e=1.60”105 mꢀ1 cmꢀ1 at 408 nm;[34] the same
value was assumed for the extinction coefficient of the modified proteins
m-HMb and m’-HMb. UV/Vis spectra were recorded on a Hewlett Pack-
ard HP 8452 A diode array spectrophotometer.
Analysis of protein fragments and heme modification: For the analysis of
protein fragments, a portion of each sample (HMb, m-HMb, and m’-
HMb, about 0.5 mg) was transformed into the apoprotein by the standard
hydrochloric acid/2-butanone method[1] and subsequently hydrolyzed by
trypsin. Digestion was performed with 1:50 (w/w) trypsin at 378C for 3 h
in 20 mm phosphate buffer (pH 7.5). The samples were then analyzed by
HPLC–MS/MS. The heme modification was studied by direct HPLC–MS/
MS analysis on solutions of m-HMb and m’-HMb in 20 mm phosphate
buffer (pH 7.5).
Chem. Eur. J. 2006, 12, 749 – 757
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755

L. Casella et al.
LC–MS and LC–MS/MS data were obtained by using an LCQ DECA
ion-trap mass spectrometer equipped with ESI ion source and controlled
by Xcalibur 1.3 software (Thermo-Finnigan, San Jose, CA). ESI experi-
ments were carried out in positive-ion mode under the following constant
instrumental conditions: source voltage 4.5 kV, capillary voltage 15 V, ca-
pillary temperature 2508C, and tube lens voltage 15 V. The system was
run in automated LC–MS/MS mode and by using a Surveyor HPLC
system (Thermo Finnigan, San Jose, CA, USA) equipped with a Simme-
try300 C18 column (3.5 mm, 2.1”100 mm, Waters, Milford, MA). The elu-
tion was performed with a 0–55% linear gradient over 65 min with 0.1%
trifluoroacetic acid (TFA) in water as solvent A and 0.1% TFA in aceto-
nitrile as solvent B. MS/MS spectra obtained by CID were performed
with an isolation width of 3 Th (m/z). The activation amplitude was
around 35% of the ejection RF amplitude that corresponds to 1.58 V.
efficient of the nitrophenols at 450 nm obtained from their absorbance
spectra (in phosphate buffer at pH 7.5):[54] for 3-nitrotyramine e450 =2300,
for 3-(4-hydroxy-3-nitrophenyl)propionic acid e450 =3350, and for 3-nitro-
l-tyrosine e450 =3100mꢀ1 cmꢀ1. The kinetic parameters were obtained
from fitting the plots of experimental rates at different substrate/nitrite
concentrations to the appropriate equations.
For each substrate, the rate dependence on the various reactant concen-
trations was studied through three series of steps: 1) finding a suitable
[H2O2]maximizing the rate but avoiding unwanted excess of the oxidant,
and then using this [H2O2]2) to study the dependence of the rate versus
[substrate], and 3) to study the dependence of the rate versus [NO2ꢀ] , by
following the iterative procedure described previously in detail.[11] The
protein concentration (HMb or m’-HMb) was kept at 1 mm in all the reac-
tions, while the concentrations of the other reactants used in the kinetic
studies were as follows:
For the analysis of protein fragments derived from m’-HMb, the mass
spectrometer was set such that one full MS scan was followed by a zoom-
scan and MS/MS scan on the most intense ion from the MS spectrum.
The acquired MS/MS spectra were automatically searched against a pro-
tein database for human proteins (human.fasta) by using the SEQUEST
algorithm to identify the modified residues. This algorithm has been in-
corporated into the Bioworks 3.0 software (ThermoFinnigan, San Jose,
CA). For the analysis of protein fragments of m-HMb, the mass spec-
trometer was set in MS/MS mode for the parent bicharged ions at m/z=
957 (mass of 1912 amu), 995.5 (mass of 1989 amu), and 973 (mass of
1944 amu). To compare the amount of modified heme, the mass spec-
trometer was set in MS/MS mode for the parent ions at 616 and 661 amu
For the phenol nitration catalyzed by HMb: 1) optimization of the perox-
ide concentration: for substrate
1
(2.0 mm): [NO2ꢀ]=1.3m, [H2O2]=
0.071–0.71m; for substrate 2 (0.40 mm): [NO2ꢀ]=0.30m, [H2O2]=0.053–
0.36m; for substrate 3 (0.3 mm): [NO2ꢀ]=0.8m, [H2O2]=0.071–0.71m;
2) dependence of the rate on phenol concentration: for substrate 1:
[H2O2]=0.36m, [NO2ꢀ]=1.3m, [phenol]=0.31–3.7 mm; for substrate 2:
[H2O2]=0.36m, [NO2ꢀ]=0.3m, [phenol]=0.062–1.6 mm; for substrate 3:
[H2O2]=0.53m, [NO2ꢀ]=0.8m, [phenol]=0.013–0.71 mm; 3) dependence
of the rate on nitrite concentration: for substrate 1 (3.1 mm): [H2O2]=
0.36m, [NO2ꢀ]=0.075–2.0m; for substrate 2 (0.62 mm): [H2O2]=0.36m,
[NO2ꢀ]=0.062–0.92m; for substrate
[NO2ꢀ]=0.050–1.1m.
3
(0.71 mm): [H2O2]=0.53m,
Two-dimensional and SDS PAGE analysis: Two-dimensional polyacryl-
amide gel electrophoresis was performed by using the immobilized pH
gradient system.[52] The first dimension, isoelectric focusing, was per-
formed on laboratory-made gels, cast on Gel-Bond (Amersham Biosci-
ences) with a 6.5–8.5 linear immobilized pH gradient obtained with
Acrylamido buffer solutions (Fluka), and the separation was run in the
Multiphor II horizontal system (Amersham Biosciences). The protein so-
lutions (HMb and m’-HMb) were diluted 1:2 with a solution containing
8m urea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-
sulfonate (CHAPS), and 40 mm Tris. After centrifugation, 125 mL of each
sample containing 30 mg protein were loaded. The gel strips were then
equilibrated with SDS and placed on top of vertical 17% gels, before the
second dimension was performed by using a Mini PROTEAN II cell
(BioRad). SDS PAGE (17% gel) was performed by the method of
Laemmli[53] and the gel was stained with Coomassie Blue.
For the phenol nitration catalyzed by m’-HMb: 1) optimization of the per-
oxide concentration: for substrate 1 (2.0 mm): [NO2ꢀ]=1.0m, [H2O2]=
0.071–0.71m; for substrate 2 (0.40 mm): [NO2ꢀ]=0.80m, [H2O2]=0.071–
0.71m; for substrate 3 (0.30 mm): [NO2ꢀ]=0.80m, [H2O2]=0.071–0.71m;
2) dependence of the rate on phenol concentration: for substrate 1:
[H2O2]=0.36m, [NO2ꢀ]=1.0m, [phenol]=0.062–3.1 mm; for substrate 2:
[H2O2]=0.36m, [NO2ꢀ]=0.80m, [phenol]=0.026–1.3 mm; for substrate 3:
[H2O2]=0.36m, [NO2ꢀ]=0.8m, [phenol]=0.031–0.68 mm; 3) dependence
of the rate on nitrite concentration: for substrate 1 (3.1 mm): [H2O2]=
0.36m, [NO2ꢀ]=0.075–1.7m; for substrate 2 (0.5 mm): [H2O2]=0.36m,
[NO2ꢀ]=0.050–1.1m; for substrate 3 (0.68 mm): [H2O2]=0.36m, [NO2ꢀ]=
0.050–0.80m.
Binding of nitrite: Increasing quantities of a concentrated nitrite solution
in 200 mm phosphate buffer (pH 7.5, final concentration of 0.0019–0.53m)
were added to a solution of HMb (3.8 mm, 1600 mL) in the same buffer in
a quartz cuvette with a pathlength of 1 cm thermostated at 25.0ꢂ0.18C;
UV/Vis spectra were recorded after each addition. Blank spectra were
recorded in the same way but in the absence of protein. After subtracting
the corresponding blank from each spectrum, the resulting spectra were
corrected for dilution and then transformed into difference spectra by
subtracting the native protein spectrum. A plot was constructed with the
difference between the absorbance changes at 424 and at 406 nm, the
wavelengths at which the difference spectra exhibit the maximum varia-
tions, versus the ligand concentration. The binding constant, KB, was ob-
Acknowledgements
This work was supported by funds from the Progetto di Ricerca di Inter-
esse Nazionale (PRIN) of the Italian Ministero dellꢁIstruzione dellꢁUni-
versità e della Ricerca (MIUR). The University of Pavia and the Europe-
an COST D21 Action are also gratefully acknowledged for support. We
thank Prof. Monica Galliano and Dr. Monica Campagnoli for useful sup-
port in the electrophoretic characterization of the proteins.
tained by interpolation of the absorbance data with the binding isotherm
[1]E. Antonini, M. Brunori, Hemoglobin and Myoglobin in Their Reac-
tions with Ligands, North Holland, Amsterdam, 1971.
[2]J. B. Wittenberg, B. A. Wittenberg, Annu. Rev. Biophys. Biophys.
Chem. 1990, 19, 217–241.
ꢀ
for low-affinity binding of
a
]
single ligand: DA=DA1KB[NO2
represents the total nitrite concentra-
]
/
total
ꢀ
ꢀ
total
(1+KB[NO2
]
total), where [NO2
tion.[11]
Kinetic studies of phenol nitration catalyzed by HMb and m’-HMb: The
kinetic experiments were carried out in 200 mm phosphate buffer
(pH 7.5) by using a quartz cuvette with a pathlength of 1 cm thermostat-
ed at 25.0ꢂ0.18C and equipped with a magnetic stirrer. The initial solu-
tion containing protein (HMb or m’-HMb, 1 mm) and variable substrate
and nitrite concentrations (final volume 1600 mL) was obtained by mixing
solutions of appropriate concentration of the reagents in the buffer. The
reaction was started by addition of the H2O2 solution and was followed
by monitoring the absorbance change at 450 nm during the initial 10–
15 s. The transformation of the rate data from absorbance per second
into [nitrophenol]produced per second was done by using the extinction co-
[3]H. Frauenfelder, B. H. McMahon, R. H. Austin, K. Chu, J. T.
Groves, Proc. Natl. Acad. Sci. USA 2001, 98, 2370–2374.
[4]D. J. Garry, S. B. Kanatous, P. P. A. Mammen, Trends Cardiovasc.
Med. 2003, 13, 111–116.
[5]U. Flçgel, A. Gçdecke, L.-O. Klotz, J. Schrader, FASEB J. 2004, 18,
1156–1158.
[6]P. K. Witting, D. J. Douglas, A. G. Mauk, J. Biol. Chem. 2001, 276,
3991–3998.
[7]M. Brunori, Trends Biochem. Sci. 2001, 26, 21–23.
[8]M. Brunori, Trends Biochem. Sci. 2001, 26, 209–210.
[9]Y. Watanabe, T. Ueno, Bull. Chem. Soc. Jpn. 2003, 76, 1309–1322.
756
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 749 – 757

Oxidation and Nitration of Human Myoglobin
FULL PAPER
[10]R. Roncone, E. Monzani, S. Nicolis, L. Casella, Eur. J. Inorg. Chem.
2004, 2203–2213.
[34]R. Varadarajan, D. G. Lambright, S. G. Boxer, Biochemistry 1989,
28, 3771–3781.
[11]S. Nicolis, E. Monzani, R. Roncone, L. Gianelli, L. Casella, Chem.
Eur. J. 2004, 10, 2281–2290.
[35]R. Varadarajan, A. Szabo, S. G. Boxer, Proc. Natl. Acad. Sci. USA
1985, 82, 5681–5684.
[12]V. Pironti, S. Nicolis, E. Monzani, S. Colonna, L. Casella, Tetrahe-
dron 2004, 60, 8153–8160.
[13]S.-I. Ozaki, H. Yang, T. Matsui, Y. Goto, Y. Watanabe, Tetrahedron:
Asymmetry 1999, 10, 183–192.
[14]S. Ozaki, T. Matsui, Y. Watanabe, J. Am. Chem. Soc. 1996, 118,
9784–9785.
[15]S. Ozaki, T. Matsui, Y. Watanabe, J. Am. Chem. Soc. 1997, 119,
6666–6667.
[36]L. L. Bondoc, R. Timkovic, J. Biol. Chem. 1989, 264, 6134–6145.
[37]K.-S. Yang, S. W. Kang, H. A. Woo, S. C. Hwang, H. Z. Chae, K.
Kim, S. G. Rhee, J. Biol. Chem. 2002, 277, 38029–38036.
[38]K. Kilinc, A. Kilinc, R. E. Wolf, M. B. Grisham, Biochem. Biophys.
Res. Commun. 2001, 285, 273–276.
[39]S. Herold, F.-J. K. Rehmann, J. Biol. Inorg. Chem. 2001, 6, 543–555.
[40]L. Casella, E. Monzani, R. Roncone, S. Nicolis, A. Sala, A. De Riso,
Environ. Health Perspect. 2002, 110, 709–711.
[16]S. Kato, H. Yang, T. Ueno, S. Ozaki, G. N. Philips, S. M. Fukuzumi,
Y. Watanabe, J. Am. Chem. Soc. 2002, 124, 8506–8507.
[17]I. V. Turko, F. Murad, Pharmacol. Rev. 2002, 54, 619–634.
[18]J. Hunt, R. E. Byrns, L. J. Ignarro, B. Gaston, Lancet 1995, 346,
1235–1236.
[19]D. Torre, G. Ferrario, F. Speranza, A. Orani, G. P. Fiori, C. Zeroli, J.
Clin. Pathol. 1996, 49, 574–576.
[20]W. A. Pryor, G. L. Squadrito, Am. J. Physiol. 1995, 268, L699–L722.
[21]M. A. Halcrow, Angew. Chem. 2001, 113, 358–362; Angew. Chem.
Int. Ed. 2001, 40, 346–349.
[41]S. Carballal, R. Radi, M. C. Kirk, S. Barnes, B. A. Freeman, B. Al-
varez, Biochemistry 2003, 42, 9906–9914.
[42]M. Hamann, T. Zhang, S. Hendrich, J. A. Thomas, Methods Enzy-
mol. 2002, 348, 146–156.
[43]H. A. Woo, S. W. Kang, H. K. Kim, K.-S. Yang, H. Z. Chae, S. G.
Rhee, J. Biol. Chem. 2003, 278, 47361–47364.
[44]S. R. Hubbard, W. A. Hendrickson, D. G. Lambright, S. G. Boxer, J.
Mol. Biol. 1990, 213, 215–218.
[45]J. L. Bourassa, E. P. Ives, A. L. Marqueling, R. Shimanovich, J. T.
Groves, J. Am. Chem. Soc. 2001, 123, 5142–5143.
[46]S. Herold, K. Shivashankar, M. Mehl, Biochemistry 2002, 41, 13460–
13472.
[22]O. M. Lardinois, K. F. Medzihradszky, P. R. Ortiz De Montellano, J.
Biol. Chem. 1999, 274, 35441–35448.
[23]O. M. Lardinois, P. R. Ortiz De Montellano, Biochem. Biophys. Res.
Commun. 2000, 270, 199–202.
[24]H. Zhang, S. He, A. G. Mauk, Biochemistry 2002, 41, 13507–13513.
[25]A. Wilks, P. R. Ortiz de Montellano, J. Biol. Chem. 1992, 267, 8827–
8833.
[47]M. Brunori, Q. H. Gibson, EMBO Rep. 2001, 2, 674–679.
[48]A. Gçdecke, A. Molojavyi, J. Heger, U. Flçgel, Z. Ding, C. Jacoby,
J. Schrader, J. Biol. Chem. 2003, 278, 21761–21766.
[49]U. Flçgel, A. Gçdecke, L. O. Klotz, J. Schrader, FASEB J. 2004, 18,
1156–1158.
[26]S. I. Rao, A. Wilks, P. R. Ortiz de Montellano, J. Biol. Chem. 1993,
268, 803–809.
[50]A. Dejam, C. J. Hunter, A. N. Schechter, M. T. Gladwin, Blood Cells
Mol. Dis. 2004, 32, 423–429.
[27]M. R. Gunther, Free Radical Biol. Med. 2004, 36, 1345–1354.
[28]P. K. Witting, A. G. Mauk, P. A. Lay, Biochemistry 2002, 41, 11495–
11503.
[51]L. Casella, L. De Gioia, G. Frontoso Silvestri, E. Monzani, C. Re-
daelli, R. Roncone, L. Santagostini, J. Inorg. Biochem. 2000, 79, 31–
39.
[29]P. K. Witting, D. J. Douglas, A. G. Mauk, J. Biol. Chem. 2000, 275,
20391–20398.
[52]E. Gianazza, S. Astrua-Testori, P. Giacon, P. G. Righetti, Electropho-
resis 1985, 6, 332–339.
[30]P. K. Witting, A. G. Mauk, J. Biol. Chem. 2001, 276, 16540–16547.
[31]P. K. Witting, A. G. Mauk, D. J. Douglas, R. Stocker, Biochem. Bio-
phys. Res. Commun. 2001, 286, 352–356.
[53]U. K. Laemmli, Nature 1970, 227, 680–685.
[54]E. Monzani, R. Roncone, L. Casella, M. Galliano, W. H. Koppenol,
Eur. J. Biochem. 2004, 271, 895–906.
[32]R. Radi, J. S. Beckman, K. M. Bush, B. A. Freeman, J. Biol. Chem.
1991, 266, 4244–4250.
[33]D. J. Garry, S. B. Kanatous, P. P. A. Mammen, Trends Cardiovasc.
Med. 2003, 13, 111–116.
Received: March 30, 2005
Revised: July 12, 2005
Published online: October 10, 2005
Chem. Eur. J. 2006, 12, 749 – 757
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