Microperoxidase8catalysednitrogenoxides formation
from oxidation of
N
-hydroxyguanidines byhydrogen peroxide
Re
´
my Ricoux
1
, Jean-Luc Boucher
2
, Dominique Mandon
3
, Yves-Michel Frapart
2
, Yann Henry
4
,
Daniel Mansuy
2
and Jean-Pierre Mahy
1
1
Laboratoire de Chimie Bioorganique et Bioinorganique, Institut de Chimie Mole
´
culaire d’Orsay, Universite
´
Paris-Sud XI,
Orsay, France;
2
Universite
´
R. Descartes, Paris, France;
3
Institut LeBel, Strasbourg, France;
4
Institut Curie-Recherche,
Universite
´
Paris-Sud XI, Orsay, France
Nitric oxide (NO) is a potent intra- and intercellular mes-
senger involved in the control of vascular tone, neuronal
signalling and host response to infection. In mammals, NO is
synthesized byoxidation of
L
-arginine catalysedby heme-
proteins called NO-synthases with intermediate formation
of N
x
-hydroxy-
L
-arginine (NOHA). NOHA and some
hydroxyguanidines have been shown to be able to deliver
nitrogen oxides including NO in the presence of various
oxidative systems. In this study, NOHA and a model com-
pound, N-(4-chlorophenyl)-N¢-hydroxyguanidine, were tes-
ted for their ability to generate NO in the presence of a
haemprotein model, microperoxidase8 (MP8), and hydro-
gen peroxide. Nitrite and nitrate production along with
selective formationof 4-chlorophenylcyanamide was
observed from incubations of N-(4-chlorophenyl)-N¢-
hydroxyguanidine in the presence of MP8 and hydrogen
peroxide. In the case of NOHA, the corresponding cyana-
mide, N
d
-cyano-L-ornithine, was too unstable under the
conditions used and
L
-citrulline was the only product
identified. A NO-specific conversion of 2-(4-carboxyphe-
nyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4-
carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and
formation of MP8–Fe–NO complexes were observed by
EPR spectroscopy and low-temperature UV/visible spectro-
scopy, respectively. These results clearly demonstrate the
formation ofnitrogenoxides including NO from the oxi-
dation of exogenous hydroxyguanidines byhydrogen per-
oxide in the presence of a minienzyme such as MP8. The
importance of the bioactivation of endogenous (NOHA) or
exogenous N-hydroxyguanidinesby peroxidases of physio-
logical interest remains to be established in vivo.
Keywords: N-hydroxyguanidines; nitric oxide synthase; iron
nitrosyl complexes; microperoxidase 8; nitric oxide.
Nitric oxide, NO, is an intra- and intercellular messenger
with important functions in the physiology of mammalian
cardiovascular, immune and nervous systems [1,2]. A major
action of NO is to activate guanylate cyclase and to increase
cyclic GMP levels in target cells. NO is also involved as an
effector of the antiproliferative function exerted by cytotoxic
macrophages against tumour cells or parasites [3,4]. Bio-
synthesis of NO involves the oxidation of
L
-arg by haem-
containing monooxygenases known as NO synthases
(NOSs) that require haem, FAD, FMN and tetrahydro-
biopterine (BH
4
) as cofactors, and NADPH and O
2
as
cosubstrates [1,2,5]. The reaction catalysedby NOSs occurs
in two steps with generation of N
x
-hydroxy-
L
-arginine
(NOHA) as an intermediate [6]. Mechanisms of NO
formation from
L
-arg and NOHA catalysedby NOSs are
the subjects of active studies. NOSs fully saturated with BH
4
specifically catalyse the oxidationof NOHA to
L
-citrulline
and NO, whereas BH
4
-free NOSs oxidize NOHA to a
mixture of
L
-citrulline and N
d
-cyano-
L
-ornithine (CN-orn),
with formationof NO and nitrous oxide (N
2
O) as nitrogen
oxides, N
2
O originating from the initial release of nitroxyl
anion, NO
–
[7,8]. Strong differences in the reactivity of
NOSs’ are also observed with some analogues of NOHA:
mixtures of N-arylureas and N-arylcyanamides are formed
when N-aryl-N¢-hydroxyguanidines are incubated in the
presence of BH
4
-free NOS II, whereas BH
4
-saturated NOS
II selectively forms N-aryl-ureas [9]. In addition to NOSs,
some cytochrome P450s oxidize simple guanidines to the
corresponding N-hydroxyguanidines [10] while distinct
P450 isoforms catalyse the oxidationof N-hydroxyguani-
dines to mixtures of ureas and cyanamides with formation
of nitrogenoxides [10–12]. This reaction of P450s involves
the generation of superoxide anion and is highly sensitive to
the addition of low amounts of superoxide dismutase (SOD)
[11]. In fact, various enzymatic and chemical systems
forming superoxide can generate nitrogenoxides from
Correspondence to J P. Mahy, Laboratoire de Chimie Bioorganique et
Bioinorganique, FRE 2127 CNRS, Institut de Chimie Mole
´
culaire
d’Orsay, Universite
´
Paris-Sud XI, Baˆ timent 420, 91405 Orsay cedex,
France. Fax: + 33 1 69 15 72 81, Tel.: + 33 1 69 15 74 21,
E-mail: jpmahy@icmo.u-psud.fr
Abbreviations: NO, nitric oxide; MP8, microperoxidase 8; MP8-I,
MP8 compound I; MP8-II, MP8 compound II; N-AcMP8,
N-acetylmicroperoxidase 8; NOS, nitric oxide synthase; NOHA,
N
x
-hydroxy-L-arginine; CN-orn, N
d
-cyano-L-ornithine;
BH
4
, (6R)-5,6,7,8-tetrahydrobiopterin; P450, cytochrome P-450;
HRP, horseradish peroxidase; NDA, 2,3-naphthalene-
dicarboxaldehyde; CPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-
imidazoline-1-oxyl 3-oxide; CPTI, 2-(4-carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl.
(Received 24 July 2002, revised 23 October 2002,
accepted 7 November 2002)
Eur. J. Biochem. 270, 47–55 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03358.x
NOHA and N-alkyl- (or N-aryl-) N¢-hydroxyguanidines.
For instance, NAD(P)H oxidases are involved in the
formation ofnitrogenoxidesfrom NOHA during oxidative
burst [13]. Chemical systems generating superoxide anion
from KO
2
similarly oxidize N-hydroxyguanidines with
cyanamides as main products with minor amounts of ureas
[14]. Cyanamides and N
2
O are also the main products
identified after the addition of oxidants such as K
3
Fe(CN)
6
,
Ag
2
CO
3
or PbO
2
to N-hydroxyguanidines [15] whereas
mixtures of ureas, cyanamides, NO and N
2
O were identified
after the addition of Pb(OAc)
4
,H
2
O
2
, peracids, hydroper-
oxides or NO itself, to N-hydroxyguanidines [15–17]. It was
previously observed that horseradish peroxidase (HRP)
reacts with NOHA in the presence of H
2
O
2
with formation
of
L
-citrulline and nitrogenoxides [18]. However, the
putative cyanamide derived from NOHA, N
d
-cyano-
L
-
ornithine, is a relatively unstable product [7] and the exact
nature of the organic end-product(s) formed in this HRP-
catalysed oxidationof NOHA remains to be determined.
Furthermore, in addition to the previously identified ureas
and cyanamides, it has been recently reported that a mutant
of cytochrome c peroxidase is able to bind NOHA and to
catalyse its oxidationby H
2
O
2
to a new compound
identified as N
x
-nitroso-
L
-arginine without any significant
accumulation of NO or
L
-citrulline [19]. N-aryl-N¢-hydroxy-
guanidines, N-aryl ureas and N-aryl cyanamides are more
stable compounds than their alkyl-counterparts [9] and we
therefore reinvestigated the reactivity of N-aryl-N¢-hydroxy-
guanidines in the presence of various oxidative systems.
Haem-based mini-enzymes, called microperoxidases,
form a new generation of biocatalysts obtained by hydro-
lytic digestion of cytochrome c. Microperoxidase8 (MP8)
consists of an iron protoporphyrin IX cofactor covalently
bound to an oligopeptide (amino acid residues 14–21 of
horse cytochrome c) which contains His18 acting as the
axial ligand to the haem iron [20–22]. These microperoxi-
dases have been reported to catalyse peroxidase-like reac-
tions involving one-electron oxidations of several typical
peroxidase cosubstrates [21–23]. MP8 can also display
cytochrome P450-type oxygen transfer reactions and cata-
lyses the para-hydroxylation of aniline, the S-oxidation of
thioethers and the N- and O-dealkylation of aromatic
amines and ethers [24,25]. We recently demonstrated that
some N-aryl-N¢-hydroxyguanidines bind to Fe
III
-MP8 with
a high affinity (K
d
in the 10–100 l
M
range) and form new
low-spin complexes [26]. In this paper, we describe the
reactivity of a simple N-hydroxyguanidine, N-(4-chlorophe-
nyl)-N¢-hydroxyguanidine 1 (Scheme 1) and of NOHA
itself, in the presence of MP8 and H
2
O
2
. Our results show
that MP8 selectively catalyses the oxidationof 1 to the
corresponding cyanamide 2 with formationof only minor
amounts of urea 3. Reaction of 1 and NOHA in the
presence of MP8 and H
2
O
2
leads to NO formation as
evidenced by EPR spectroscopy using a specific spin trap for
NO and by low temperature UV/Visible detection of the
MP8–Fe
III
–NO complex. Moreover, MP8 compound II
(MP8-II) reacts with exogenous NOHA to yield the same
MP8–Fe
III
–NO complex. We thus provide experimental
evidence that a peroxidase-like mechanism explains such a
selective cyanamide formation with generation of NO and
NO
–
.
Materials and methods
Chemicals
MP8 was prepared by sequential peptic and tryptic diges-
tion of horse heart cytochrome c (Sigma) essentially as
previously described [20]. The haem content was determined
spectrophotometrically using e
397
¼ 157 m
M
)1
Æcm
)1
[20].
The purity of the sample was > 97% based on MALDI-
TOF MS analysis. The ferrous form of MP8 was prepared
by reacting MP8 with sodium dithionite in phosphate buffer
under anaerobic conditions. Metal-free MP8 was prepared
by reductive demetallation of Fe
III
–MP8 in the presence of
Fe
II
chloride in glacial acetic acid containing concentrated
HCl [27]. N-Acetylmicroperoxidase 8 (N-AcMP8) was
obtained by treating MP8 with excess of acetic anhydride
in sodium carbonate buffer, as described previously [28].
N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 was prepared
in 65% yield by reaction of cyanamide 2 with hydroxylam-
ine hydrochloride in anhydrous ethanol [29]. 4-chlorophe-
nylurea 3 and 4-chlorophenylcyanamide 2 were obtained,
respectively, by reacting 4-chloroaniline with potassium
cyanate in 0.5
M
HCl and 4-chloroaniline with cyanogen
bromide [11,29]. All compounds were fully identified by
1
Hand
13
C NMR and mass spectra which were identical
to those described previously. N
d
-Cyano-
L
-ornithine was
synthesized from
L
-ornithine following the protocol des-
cribed by Clague et al. [7]. Hydrogenperoxide (30% v/v)
and bovine liver catalase were purchased from Sigma.
2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl
3-oxide (CPTIO) and N
x
-hydroxy-
L
-arginine came from
Alexis. Gaseous NO was from L’Air Liquide. Anaerobic
solutions of NO were prepared by bubbling NO in 0.1
M
phosphate buffer pH 7.4 carefully deoxygenated with
argon. NO concentrations were measured using the con-
version of oxyhemoglobin to methemoglobin following an
Scheme 1. Structure of the studied compounds.
48 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003
established protocol [30]. The concentration of the solution
of hydrogenperoxide was checked spectrophotometrically
using e
240
¼ 39.4
M
)1
Æcm
)1
[21]. All other reagents and
solvents were of the highest commercially available purity.
Incubation conditions and analyses of end-products
Typical incubations contained 1 m
M
N-(4-chlorophenyl)-
N¢-hydroxyguanidine 1 and 1 l
M
MP8 in 250 lL(final
volume) 0.1
M
phosphate buffer pH 7.4. Reactions were
startedbytheadditionof2m
M
H
2
O
2
. Incubations were
shaken at room temperature for the indicated times and
stopped by the addition of 100 lL phosphate buffer
containing 1000 UÆmL
)1
catalase and 400 l
M
4-chloro-
benzamide (internal standard for RP-HPLC analysis). After
10 min at room temperature, aliquots (50 lL) were mixed
with the same volumes of sulfanilamide 1% in HCl 0.5
M
and N-(1-naphthyl)ethylenediamine 0.1% in HCl 0.5
M
for
measurements of NO
2
–
(Griess reaction) [31]. Reduction of
nitrate to nitrite was performed in the presence of nitrate
reductase and a NADPH-regenerating system according to
a previously described protocol [32] and the nitrite formed
was quantified using the Griess reaction. The amounts of
nitrate were thus obtained by difference between the two
measurements. Calibration curves were made under iden-
tical conditions in the presence of known amounts of
NaNO
2
and KNO
3
. Separations of organic products were
routinely carried out on a Thermo-Finnigan system
equipped with a AS3000 auto-sampler, a Focus multiwave-
length detector and a 250 · 4.6 mm Nucleosil ODS column
(5 lm particle size). Flow rate was 1 mLÆmin
)1
and the
mobile phase was a gradient between solvent A (5 m
M
phosphoric acid in water, pH 2.6) and solvent B (acetonit-
rile) using the following program: 0 min, 5% B; 5 min,
linear gradient to 40% B in 10 min; 15 min, linear gradient
to 100% B in 10 min; 30 min, linear gradient to 5% B in
5 min followed by 15 min re-equilibration. The absorbance
was monitored between 200 and 400 nm. The retention
times for 1, 2, 3 and 4-chlorobenzamide (internal standard)
were 7.0, 18.3, 15.2 and 14.2 min, respectively. Calibration
curves were made from identical mixtures containing
various concentrations of 2, 3, and internal standard, but
without MP8. Some incubations were also analysed on a
Thermo-Finnigan LCQ Advantage system (RP-HPLC
system coupled to a mass spectrometer). Separations were
performed on a 150 · 2 mm Chromasil C
18
column
(3.5 lm particle size). Flow rate was 250 lLÆmin
)1
and
the mobile phase was a gradient between solvent A (10 m
M
ammonium acetate) and solvent B (acetonitrile) using the
following program: 0 min, 10% B; 5 min, linear gradient to
40% B in 10 min; 15 min, linear gradient to 100% B in
10 min; 28 min, linear gradient to 10% B in 5 min followed
by 10 min re-equilibration. The absorbance was monitored
between 200 and 400 nm and mass spectra were recorded in
the 100–1000 mass range.
Incubations of 1 m
M
NOHA in the presence of 1 l
M
MP8 and 2 m
M
H
2
O
2
were performed as previously
described for N-hydroxyguanidine 1, except that stop buffer
contained 1000 UÆmL
)1
catalase only. Amino acid products
were derivatized and separated by RP-HPLC according to
the procedure described by Clague et al.[7].Thus,50lLof
the reaction mixtures were mixed in HPLC vials containing
20 l
M
phenylalanine (internal standard), 100 lL0.1
M
potassium borate (pH 9.5), 10 lL0.5
M
NaCN in the same
borate buffer and 50 lL10m
M
2,3-naphthalenedicarbox-
aldehyde (NDA) in methanol. The mixtures were allowed to
stay at room temperature for 15 min and applied to a Nova-
Pak C
18
column (150 · 3.9 mm, 4 lm particle size, Waters)
which was maintained at 40 °C and equilibrated with 60%
solvent A (5 m
M
ammonium acetate buffer, pH 6.0) and
40% methanol (solvent B). Elution conditions were as
follows: 2 min at 40% solvent B, a linear increase from 40 to
60% solvent B over 20 min, and from 60 to 100% solvent B
over the next 5 min, followed by 3 min of 100% solvent B,
and a return to initial conditions in 3 min. The flow rate was
0.5 mLÆmin
)1
and detection was performed at 420 nm.
Retention time for NDA derivatives of NOHA, CN-orn,
L
-citrulline and phenylalanine were 10.0, 6.2, 5.0 and
18.5 min, respectively.
EPR Spectroscopy
Room temperature EPR measurements were performed on
mixtures containing 1 l
M
MP8, 1 m
M
N-(4-chlorophenyl)-
N¢-hydroxyguanidine 1 or NOHA, 200 l
M
H
2
O
2
and 20 l
M
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl
3-oxide (CPTIO) in 0.1
M
phosphate buffer pH 7.4. After
1 min, the reaction mixture was transferred via a Teflon
capillary tube to a Bruker Aqua-X cell inserted in a shq 0011
cavity (Bruker). EPR spectra were recorded on a Elexsys E
500 EPR spectrometer using the following instrument
settings: microwave frequency, 9.82 GHz; field modulation
frequency, 100 kHz; sampling time, 0.04 s; field sweep, 6
mTÆmin
)1
, 0.05 mT modulation amplitude and microwave
power, 10 mW.
UV/visible spectroscopy
UV/visible spectra were recorded on a Varian Cary 05E
spectrophotometer equipped with a variable temperature
Dewar using a home-made quartz cuvette with a 0.1-cm
path length. Solutions of MP8 (2 l
M
in phosphate buffer
containing 50% methanol as antifreeze adduct) were
introduced into the quartz cuvette which was set up in the
beam and used as reaction vessel. After equilibration at
)15 °C, 2 m
M
N-hydroxyguanidine 1 or NOHA, and
10 m
M
H
2
O
2
, all in phosphate buffer containing 50%
methanol, were introduced directly in the cuvette via a steel
needle and the medium was homogenized by cold nitrogen
bubbling prior to data collection.
Results
Reaction of
N
-(4-chlorophenyl)-
N
¢-hydroxyguanidine 1
in the presence of H
2
O
2
and MP8
In preliminary experiments, mixtures containing 1 m
M
NOHA, 1 l
M
MP8 and 2 m
M
H
2
O
2
were incubated for
2 min at room temperature before the addition of catalase
to stop the reaction. Amino acids were derivatized in the
presence of NDA and NaCN [7], and analysis of the
reaction mixtures by RP-HPLC showed the disappearance
of NOHA with formationof citrulline as the only identified
product (data not shown). Incubation of authentic CN-orn
Ó FEBS 2003 Microperoxidase8 and nitrogenoxidesformation (Eur. J. Biochem. 270)49
followed by derivatization under the same conditions also
resulted in the detection of citrulline (data not shown).
These results indicated that the low stability of CN-orn
under those conditions prevents any clear indentification of
the end products of the reaction.
Similar incubations containing 1 m
M
N-(4-chlorophe-
nyl)-N¢-hydroxyguanidine 1,1l
M
MP8 and 2 m
M
H
2
O
2
were thus performed and stopped after 2 min by the
addition of catalase. Analysis of the reaction mixtures by
RP-HPLC using UV-detection revealed a significant
decrease of the amount of starting N-hydroxyguanidine 1
and the formationof three new organic compounds (Fig. 1).
The very predominant one was identified to be 4-chloro-
phenylcyanamide 2 by comparison of its retention time and
UV spectrum to those of the authentic compound and by
coinjection with the authentic compound. A second, minor,
metabolite was similarly found to be 4-chlorophenylurea 3.
The amounts of N-hydroxyguanidine that had disappeared
were almost identical to the amounts of cyanamide 2 and
urea 3 formed. An unknown product exhibiting a retention
time of 13.2 min and a UV peak at 240 nm was also
detected by RP-HPLC (Fig. 1). Further studies of the
reaction mixtures using RP-HPLC coupled to MS analysis
completely confirmed the identification of 4-chlorophenyl-
cyanamide 2 as the main end product of the reaction and of
4-chlorophenylurea 3 in minor amounts (data not shown).
However, we were unable to identify unambiguously the
unknown product using our RP-HPLC system coupled to a
mass spectrometer, operating either in the positive or in the
negative mode in the 100–1000 mass range (data not
shown). Aliquots of the reaction mixtures were simulta-
neously studied for NO
2
–
and NO
3
–
formation using a
classical colorimetric assay. Nitrate ion was the predomin-
ant nitrogen product with only low amounts of NO
2
–
(Table 1). Total amounts of measured nitrogen oxides
(NO
2
–
+NO
3
–
) were about one-third of the sum of the
organic products (2 + 3,Table1).Incontrolexperiments,
4-chlorophenylurea 3 remained unchanged when incubated
10 min at room temperature in the presence of 1 l
M
MP8
and 2 m
M
H
2
O
2
whereas 4-chlorophenylcyanamide 2 gave
low amounts of unidentified products, but without any
formation of urea 3 (data not shown).
Formations of cyanamide 2,urea3 and (NO
2
–
+NO
3
–
)
upon MP8- and H
2
O
2
-dependent oxidationof 1 were
linearly dependent upon the amounts of added MP8 (in the
0.1–2 l
M
range). Accumulation of 2 and 3 reached a
maximum 3–4 min after the addition of MP8 at room
temperature, indicating that degradation of MP8 had
occured under those oxidative conditions (data not shown).
The effect of changing the pH of the reaction medium on
the efficiency and selectivity of the reaction of 1 in the
presence of MP8 and H
2
O
2
was also investigated. Increasing
the pH from 5.0 to 8.5 resulted in a fourfold increase in the
rate offormationof cyanamide 2 and urea 3, but no change
in the ratio 2/3 (35 ± 5) was measured (data not shown).
This increase in activity is consistant with the fact that in this
pH range, the substrate is HO
2
–
rather than H
2
O
2
[21].
The reaction of N-(4-chlorophenyl)-N¢-hydroxyguanidine
1 in the presence of MP8 and H
2
O
2
was clearly dependent
upon the presence of all components as formation of
4-chlorophenylcyanamide 2 and NO
3
–
was not observed in
incubations performed in the absence of either MP8 or
H
2
O
2
. Complementary experiments performed under
anaerobic conditions resulted in almost similar yields,
without significant changes in the cyanamide/urea, and
NO
2
–
/NO
3
–
ratios. This indicated that dioxygen was not
involved in catalysis (Table 1).
The reaction was strongly inhibited by the addition to the
incubation mixture of ascorbic acid and N-acetyl-
L
-cysteine,
two classical reductants (Table 1). Mannitol and dimethyl-
sulfoxide, two scavengers of OH
Æ
, and EDTA, an iron
chelator, were without effect on the reaction efficiency and
selectivity, indicating that neither OH
Æ
nor free iron were
directly involved in the formationof cyanamide. Further-
more, implication of the iron atom of MP8 in the catalysis
was indicated by the almost complete inhibition of the
reaction performed in the presence of 1 l
M
metal-free
MP8 (Table 1). Using N-AcMP8 in place of MP8 led to a
significant increase in the efficiency of the reaction without
significant modification of the cyanamide/urea ratio
(Table 1). This higher activity could result from less
degradation, or aggregation, of N-AcMP8 by comparison
to MP8 [28].
Selective formationof cyanamide 2 was also observed
when haemin was used instead of MP8 but the reaction with
haemin was much less efficient (Table 1). As reported
previously [15], a simple oxidant proceeding through
monoelectronic transfers, Fe(CN)
6
K
3
,alsoledtothe
selective formationof cyanamide 2 (data not shown). This
constitutes another argument in favour of a monoelectronic
process in the reactions catalysedby MP8.
Detection of nitric oxide by EPR spectroscopy
To detect the possible generation of NO from the oxidation
of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H
2
O
2
in the presence of MP8, we used a NO-specific reactant,
the nitronyl-nitroxide CPTIO and EPR spectroscopy.
Nitric oxide reacts with CPTIO to generate an imino-
nitroxide, CPTI, and this NO-dependent conversion is easily
Fig. 1. RP-HPLC profile obtained after reaction of 1 m
M
N-(4-chlo-
rophenyl)-N¢-hydroxyguanidine 1 with 2 m
M
H
2
O
2
, in the presence of
1 l
M
MP8in0.1
M
phosphate buffer pH 7.4. The reaction was started
by the addition of H
2
O
2
at room temperature and was stopped after
2 min by addition of catalase and analysed by RP-HPLC as described
in Materials and methods. IS: Internal standard.
50 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003
monitored by EPR at room temperature [33]. Fig. 2, trace a
shows the EPR spectrum of 20 l
M
CPTIO in the presence
of 1 m
M
N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 and
2m
M
H
2
O
2
. The spectrum consists of five lines with an
intensityratioof1:2:3:2:1(Fig.2,tracea).Thesame
kind of EPR spectrum was also obtained when N-hydroxy-
guanidine 1,H
2
O
2
, or both reagents were omitted from the
reaction mixture (data not shown). Moreover, the addition
of 1 l
M
MP8 to this mixture led to a gradual disappearance
ofthefive-lineEPRspectrumofCPTIOandtothe
appearance of a seven-line spectrum (Fig. 2, trace b). After
about 8 min, the only observable spectrum (Fig. 2, trace c)
was identical to the spectrum of CPTI obtained after the
addition of a solution of NO to CPTIO, without detection
of any other transient radical (data not shown). The
intensity of this spectrum slowly decreased and was barely
detectable 20 min after the addition of MP8 to the mixture
of CPTIO, 1 and H
2
O
2
. The simultaneous presence of all
components of the reaction mixture was required for the
formation of the CPTI spectrum. This disappearance of the
EPR signal of CPTI could result from further oxidation of
CPTI to EPR-silent products under those conditions or
from the coupling of CPTI(O) to intermediate radicals
derived from N-hydroxyguanidine 1.Incontrolexperi-
ments: (a) CPTI formed from the addition of NO solutions
to CPTIO was stable in the presence of 2 m
M
H
2
O
2
, but
further addition of 1 l
M
MP8 led to the slow disappearance
of any observable EPR spectra after 10 min; (b) the
incubation of 20 l
M
CPTIO with 1 l
M
MP8 and 2 m
M
H
2
O
2
but without N-hydroxyguanidine 1 did not lead to
any change in the five-line signal of CPTIO for at least
15 min. Very similar results were obtained using NOHA in
place of N-hydroxyguanidine 1 (data not shown). These
results show that oxidationof N-hydroxyguanidine 1 (or
NOHA) in the presence of H
2
O
2
and MP8 leads to the
transient formationof NO and that further reaction of NO
either with O
2
inthepresenceofFe
III
–MP8, or with MP8-I/
MP8-II leads to its stable end-products, NO
2
–
and NO
3
–
.In
order to get more information on the nature of nitrogen
oxides involved during these oxidations, low temperature
UV/visible studies were performed.
Low temperature UV/visible spectroscopy
In the absence of substrate, the reaction of MP8 with H
2
O
2
yields the oxo-ferryl derivative MP8 compound II (MP8-II)
[34]. Monitoring the spectroscopic changes following the
addition of H
2
O
2
to MP8 at )15 °C in phosphate buffer
containing 50% methanol, showed absorption shifts from
395 to 408 nm for the Soret band and from 535 nm to 522
and 551 nm for the Q bands (Fig. 3A, traces a and b). MP8-
II is known to be relatively unstable and to undergo
complete bleaching within minutes [34]. However, when
10 m
M
H
2
O
2
and 2 m
M
NOHA were simultaneously added
Table 1. Effects of incubation conditions on the oxidationof N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H
2
O
2
catalysed by MP8. Reactions were
performed in 0.1
M
phosphate buffer pH 7.4 containing 1 m
M
N-(4-chlorophenyl)-N¢-hydroxyguanidine 1, 2 m
M
H
2
O
2
and 1 l
M
MP8. After 2 min
at room temperature, the reactions were stopped by the addition of catalase and aliquots were analysed as described in Materials and methods for
measurements of cyanamide 2, urea 3, NO
2
–
and NO
3
–
. Results are expressed in l
M
of products formed under the conditions used and are means
± SD from four to eight experiments (three experiments in the cases of anaerobic conditions, metal-free MP8 and N-AcMP8).
Products
Conditions Cyanamide 2 Urea 3 NO
2
–
NO
3
–
Complete system 175 ± 45 6 ± 2 5 ± 2 60 ± 15
–MP8 2 ± 1 2 ± 1 1 ± 1 2 ± 1
–H
2
O
2
2±1 2±1 1±1 2±1
–O
2
130±12 3±1 8±3 40±8
+10m
M
ascorbic acid 2 ± 1 3 ± 1 1 ± 1 2 ± 1
+10m
M
N-acetylcysteine 12 ± 4 3 ± 1 3 ± 1 12 ± 3
+ 100 m
M
mannitol 165 ± 35 8 ± 3 5 ± 2 80 ± 10
+ 100 m
M
dimethylsulfoxide 166 ± 20 12 ± 3 5 ± 2 85 ± 20
+ 0.1 m
M
EDTA 155 ± 25 15 ± 5 5 ± 1 90 ± 20
Metal-free MP8 14 ± 5 2 ± 1 5 ± 3 6 ± 3
N-AcMP8 200 ± 15 3 ± 1 15 ± 3 75 ± 10
1 l
M
Heme + 2 m
M
H
2
O
2
58±10 2±1 5±1 38±10
Fig. 2. EPR detection of NO formation. Spectra obtained at room
temperature from 20 l
M
CPTIO in 0.1
M
phosphate buffer containing
1m
M
N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 and 2 m
M
H
2
O
2
without MP8 (a). (b) Same as in (a) but 2 min after the addition of
1 l
M
MP8. (c) Same as (b) 10 min after the addition of MP8.
Ó FEBS 2003 Microperoxidase8 and nitrogenoxidesformation (Eur. J. Biochem. 270)51
to a 2-l
M
solution of MP8 at )15 °C, MP8-II formed first
and smoothly turned into a new intermediate with maxima
at 412, 528 and 563 nm (Fig. 3A, trace c). Contrary to
MP8-II, the new intermediate slowly turned back to ferric
MP8 (Fig. 3A, trace d). This intermediate was obtained as a
more stable species upon careful addition of NOHA to a
previously prepared solution of MP8-II maintained at
)15 °C (data not shown). Therefore, this new intermediate
was a reaction product of MP8-II with NOHA and was
tentatively identified by comparison to the UV/visible
spectra of the MP8–Fe
III
–NO and MP8–Fe
II
–NO com-
plexes formed, respectively, by the addition of a NO-solu-
tion to MP8, either in the ferric or ferrous form. These two
Fe–NO complexes showed characteristic spectra with peaks
at 414, 526 and 560 nm (MP8–Fe
III
–NO, Fig. 3B, trace a)
and 411, 537 and 563 nm (MP8–Fe
II
–NO, Fig. 3B, trace b).
It appears that the spectrum of the intermediate formed
during the reaction was very close to that of the MP8–Fe
III
–
NO complex but significantly differed from that of the
MP8–Fe
II
–NO complex (compare Fig. 3A, trace c, and
Fig. 3B, trace a). These data suggest that MP8–Fe
III
–NO
was the main species formed during the oxidationof NOHA
by H
2
O
2
in the presence of MP8. Similar results were
observed using N-(4-chlorophenyl)-N¢-hydroxyguanidine 1
instead of NOHA (data not shown).
Discussion
Our results show that MP8 efficiently catalyses the oxida-
tion of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 in the
presence of H
2
O
2
with predominant formationof 4-chlo-
rophenylcyanamide 2 and only minor amounts of urea 3
(5–10% of cyanamide 2, Eqn 1). An unidentified product
was formed in minor amounts (Fig. 1) but could not be fully
characterized under our RP-HPLC conditions with MS
operating in the positive or negative modes. This product
did not display the typical absorption at 400 nm described
for nitrosoguanidines [19] but could be a dimeric com-
pound. Stable end-products of NO, NO
2
–
and NO
3
–
,were
detected in lower amounts than the organic compounds
(Table 1), indicating that nitrogenoxides distinct from NO
are formed but were not detected by our methods. The most
plausible candidate is nitroxyl anion, NO
–
, which upon
dimerization, protonation and dehydration would release
gaseous N
2
O [7,15]. Using NOHA instead of hydroxygu-
anidine 1 led to the detection of citrulline as the only stable
identified derivative, in agreement with the known instabi-
lity of the corresponding cyanamide, CN-orn [7].
NO
x
¼ NO
À
; NO
Á
; NO
À
2
; NO
À
3
ð1Þ
The oxidationof N-hydroxyguanidine 1 and of NOHA by
H
2
O
2
catalysed by MP8 clearly involves an active species
derived from the haem of MP8 as metal-free MP8 was
inactive. It also involves high oxidation states of the haem of
MP8 as it is strongly inhibited by reductants such as
ascorbate and N-acetylcysteine. Such a selective oxidation
of an N-hydroxyguanidine to the corresponding cyanamide
has been reported previously in the case of monoelectronic
oxidants such as K
3
Fe(CN)
6
and Ag
2
CO
3
[15]. This new
MP8-catalysed reaction thus displays strong analogies with
chemical systems known to proceed by mono-electronic
transfers and with peroxidase-type reactions catalysed
by MP8. It probably involves initial oxidationof MP8-
Fe
III
by H
2
O
2
to MP8 compound I (MP8
+
–Fe
IV
¼ O,
MP8-I). Successive monoelectronic reductions of MP8-I
by N-hydroxyguanidine could give MP8-compound-II
(MP8-II) and restore MP8–Fe
III
(Scheme 2). One-electron
oxidations of a N-hydroxyguanidine should result in the
formation of the iminoxy-radical 4 and one can propose the
mechanism shown in Scheme 2 for the oxidation of
N-hydroxyguanidine 1 by H
2
O
2
in the presence of MP8.
Further one-electron oxidations of the intermediate imin-
oxy-radical 4 (or its mesomeric form, the nitroso-C-centered
radical 5), by MP8-I (or MP8-II) can lead to the interme-
diate nitroso-imine 6 and cyanamide 2 (Scheme 2, paths a
or b). Such mechanisms can generate nitric oxide NO and
nitroxyl anion, NO
–
. Formationof N
2
O could explain the
marked differences observed between the amounts of
nitrogenous compounds derived from NO (NO
2
–
+NO
3
–
)
Fig. 3. Low temperature UV/visible spectroscopy. (A) To a mixture of
2m
M
NOHA and 2 l
M
MP8 in 0.1
M
phosphate buffer containing
50% methanol was added 10 m
M
H
2
O
2
at )15 °C and spectra were
recorded at various time intervals: 0 min (a), 1 min (b), 2 min (c) and
5 min (d). (B) Spectra of authentic MP8–Fe
III
–NO (a) and MP8–
Fe
II
-NO (b) obtained after the addition of a solution of NO to 2 l
M
MP8 followed by the addition of sodium dithionite.
52 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003
and organic products (2 and 3) in our experiments (Table 1).
It seems likely that, in the presence of H
2
O
2
and Fe
III
,NO
–
could be oxidized to NO with formationof MP8–Fe
III
–NO
[35,36]. Alternatively, NO
–
could add to MP8–Fe
III
,form-
ing MP8–Fe
II
–NO that can be reoxidized to MP8–Fe
III
–
NO under those conditions (Scheme 2, path c). MP8-I and
MP8-II could also react with NO and oxidize it either into
NO
+
or into NO
2
.
, leading after hydrolysis to NO
2
–
and
NO
3
–
(Scheme 2). As control experiments showed that
cyanamide 2 did not lead to any formationof urea and that
dioxygen is probably not involved in the reaction, the
formation of urea 3 could result from the hydrolysis of one
of the intermediates indicated on Scheme 2, the most
plausible candidate being nitroso-imine 6.
These mechanisms are in complete agreement with those
previously indicated for the oxidationof N-hydroxyguan-
idines by one-electron oxidants and for the oxidation of
benzamidoximes by H
2
O
2
in the presence of HRP
[15,37,38]. In addition, the formationof intermediate
nitroso-imines has been clearly established in the oxidation
of disubstituted amidoximes by Pb(OAc)
4
[39].
This new reaction of MP8 is in full agreement with a
peroxidase-like activity of MP8 as it involves high-valent
compounds I and II of MP8 and proceeds through mono-
electronic transfers that lead to the selective formationof a
cyanamide and a mixture ofnitrogen oxides, NO and N
2
O. It
clearly differs from the oxidationof NOHA and N-hydroxy-
guanidine 1 catalysedby hepatic cytochromes P450 and
BH
4
-free NOS II that give 1 : 1 mixtures of the correspond-
ing cyanamide and urea [9,11]. This is easily understandable
as the active species in the later reactions has been shown to
be O
2
•)
derived from the oxidase function of these haem
proteins [9,13]. This MP8-catalysed reaction also differs from
the oxidations of NOHA and 1 by BH
4
-containing NOS II,
Scheme 2. Possible mechanisms for the oxidationof N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H
2
O
2
in the presence of MP8.
Ó FEBS 2003 Microperoxidase8 and nitrogenoxidesformation (Eur. J. Biochem. 270)53
which led almost exclusively to citrulline and the corres-
ponding urea 3 [6,9,40]. It has been proposed that the active
species responsible for these NOS-catalysed reactions is
either a Fe
II
–O
2
or a Fe
III
–O–O
–
complex [1,2]. The very
different behaviour of the MP8–H
2
O
2
system, that should
involve a high-valent iron-oxo species for cyanamide forma-
tion (see above), seems to confirm that the active species
responsible for urea formation in NOS-catalysed oxidations
is not a high-valent iron-oxo complex which could have
derived from the NOS–Fe
III
–O–O
–
intermediate.
According to the proposed mechanism, many other
one-electron oxidants could generate NO from N-hydroxy-
guanidines through reactions that do not require the
participation of BH
4
-containing NOS. The formation of
NO in a model system for peroxidase may rationalize the
observations that some N-hydroxyguanidines are potent
vasodilators in endothelium-denuded vasculature [41,42].
The antihypertensive effects ofN-hydroxyguanidines have
been related to their ability to form NO under oxidative
conditions [10,12] and a recent study has shown that
hydroxyguanidine-induced toxicity toward leukaemia
HL60 cells parallels the intracellular production of NO
[43]. The mechanisms of NO generation from such
compounds should differ from those observed from NOHA
itself as most N-hydroxyguanidines are not substrates for
NOS and they can form nitrogenoxides (NO and NO
–
)by
clearly distinct enzymatic processes. These results using
haem-octapeptide MP8 as a model open the way to a better
understanding of the mechanisms of NO formation from
N-hydroxyguanidines, or more generally from molecules
bearingaC¼NOH function, in the presence of peroxidases
of physiological relevance.
Acknowledgements
The authors thank R. Azerad for his help in performing RP-HPLC
coupled to mass spectra analysis.
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. Microperoxidase 8 catalysed nitrogen oxides formation
from oxidation of
N
-hydroxyguanidines by hydrogen peroxide
Re
´
my Ricoux
1
,. NO
À
3
ð1Þ
The oxidation of N-hydroxyguanidine 1 and of NOHA by
H
2
O
2
catalysed by MP8 clearly involves an active species
derived from the haem of MP8 as metal-free