Oxidations of Nω-hydroxyarginine analogues and various N-hydroxyguanidines by NO synthase II: Key role of tetrahydrobiopterin in the reaction mechanism and substrate selectivity

Article abstract of DOI:10.1021/tx0001068

Oxidations of L-arginine 2, homo-L-arginine 1, their N^ω-hydroxy derivatives 4 and 3 (NOHA and homo-NOHA, respectively), and four N-hydroxyguanidines, N^ω-hydroxynor-L-arginine 5 (nor-NOHA), N^ω-hydroxydinor-L-arginine 6 (dinor-NOHA), N-(4-chlorophenyl)-N'-hydroxyguanidine (8), and N-hydroxyguanidine (7) itself, by either NOS II or (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄)-free NOS II, have been studied in a comparative manner. Recombinant BH₄-free NOS II catalyzes the oxidation of all N-hydroxyguanidines by NADPH and O₂, with formation of NO₂^- and NO₃^- at rates between 20 and 80 nmol min^-1 (mg of protein)^-1. In the case of compound 8, formation of the corresponding urea and cyanamide was also detected besides that of NO₂^- and NO₃^-. These BH₄-free NOS II-dependent reactions are inhibited by modulators of electron transfer in NOS such as thiocitrulline (TC) or imidazole (ImH), but not by Arg, and are completely suppressed by superoxide dismutase (SOD). They exhibit characteristics very similar to those previously reported for microsomal cytochrome P450-catalyzed oxidation of N-hydroxyguanidines. Both P450 and BH₄-free NOS II reactions appear to be mainly performed by O₂?^- derived from the oxidase function of those heme proteins. In the presence of increasing concentrations of BH₄, these nonselective oxidations progressively disappear while a much more selective monooxygenation takes place only with the N-hydroxyguanidines that are recognized well by NOS II, NOHA, homo-NOHA, and 8. These monooxygenations are much more chemoselective (8 being selectively transformed into the corresponding urea and NO) and are inhibited by Arg but not by SOD, as expected for reactions performed by the NOS Fe^II-O₂ species. Altogether, these results provide a further clear illustration of the key role of BH₄ in regulating the monooxygenase/oxidase ratio in NOS. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics such as N-hydroxyguanidines, not only via their monooxygenase function but also via their oxidase function.

Full text of DOI:10.1021/tx0001068

2
02
Chem. Res. Toxicol. 2001, 14, 202-210
ω
Oxid a tion s of N -Hyd r oxya r gin in e An a logu es a n d
Va r iou s N-Hyd r oxygu a n id in es by NO Syn th a se II: Key
Role of Tetr a h yd r obiop ter in in th e Rea ction Mech a n ism
a n d Su bstr a te Selectivity
†
†
†
Catherine Moali, J ean-Luc Boucher, Axelle Renodon-Corniere,
‡
,†
Dennis J . Stuehr, and Daniel Mansuy*
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS,
Universit e´ Paris V, 45 Rue des Saints-P e` res, 75270 Paris Cedex 06, France, and Department of
Immunology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195
Received May 10, 2000
ω
Oxidations of L-arginine 2, homo-L-arginine 1, their N -hydroxy derivatives 4 and 3 (NOHA
ω
and homo-NOHA, respectively), and four N-hydroxyguanidines, N -hydroxynor-L-arginine 5
ω
(
nor-NOHA), N -hydroxydinor-L-arginine 6 (dinor-NOHA), N-(4-chlorophenyl)-N′-hydroxy-
guanidine (8), and N-hydroxyguanidine (7) itself, by either NOS II or (6R)-5,6,7,8-tetrahydro-
L-biopterin (BH )-free NOS II, have been studied in a comparative manner. Recombinant BH
free NOS II catalyzes the oxidation of all N-hydroxyguanidines by NADPH and O , with
4
4
-
2
-
-
-1
-1
formation of NO
2
and NO
3
at rates between 20 and 80 nmol min (mg of protein) . In the
case of compound 8, formation of the corresponding urea and cyanamide was also detected
-
-
besides that of NO
2
3 4
and NO . These BH -free NOS II-dependent reactions are inhibited by
modulators of electron transfer in NOS such as thiocitrulline (TC) or imidazole (ImH), but not
by Arg, and are completely suppressed by superoxide dismutase (SOD). They exhibit
characteristics very similar to those previously reported for microsomal cytochrome P450-
catalyzed oxidation of N-hydroxyguanidines. Both P450 and BH
4
-free NOS II reactions appear
derived from the oxidase function of those heme proteins. In
the presence of increasing concentrations of BH , these nonselective oxidations progressively
•
-
to be mainly performed by O
2
4
disappear while a much more selective monooxygenation takes place only with the N-
hydroxyguanidines that are recognized well by NOS II, NOHA, homo-NOHA, and 8. These
monooxygenations are much more chemoselective (8 being selectively transformed into the
corresponding urea and NO) and are inhibited by Arg but not by SOD, as expected for reactions
II
performed by the NOS Fe -O
2
species. Altogether, these results provide a further clear
in regulating the monooxygenase/oxidase ratio in NOS. They
illustration of the key role of BH
4
also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of
xenobiotics such as N-hydroxyguanidines, not only via their monooxygenase function but also
via their oxidase function.
In tr od u ction
In the past decade, nitric oxide (NO) has emerged as a
III) are constitutively expressed, and their activity
depends on intracellular Ca2 concentration which can
trigger calmodulin binding and activation of NOS. The
last isoform (NOS II) is regulated at the transcriptional
level by cytokines and other infectious agents and is
+
major biological mediator in mammals (1). It is produced
1
from L-arginine (Arg) by heme thiolate proteins called
NO synthases (NOSs). Three types of NOS have been
characterized in mammals, based on amino acid se-
quence, tissue distribution, and expressional control (2).
Two isoforms (soluble NOS I and membrane-bound NOS
2+
mostly independent of Ca concentration since it con-
tains calmodulin as a tightly bound prosthetic group. In
the presence of NADPH and dioxygen, all three NOSs
catalyze the two-step oxidation of Arg into nitric oxide
and citrulline (Cit) as follows (3):
*
To whom correspondence should be addressed. Fax: 33 (0)1 42
NOS
8
6 83 87. E-mail: Daniel.Mansuy@biomedicale.univ-paris5.fr.
+
†
Arg + NADPH + H + O
2
8
Universit e´ Paris V.
The Cleveland Clinic Foundation.
‡
+
NOHA + NADP + H O
1
2
Abbreviations: Arg, L-arginine; homo-Arg, homo-L-arginine; NOHA,
ω
ω
N -hydroxy-L-arginine; homo-NOHA, N -hydroxyhomo-L-arginine; nor-
ω
ω
NOS
NOHA, N -hydroxynor-L-arginine; dinor-NOHA, N -hydroxydinor-L-
arginine; OH-gua, N-hydroxyguanidine; Cit, L-citrulline; NMA, N -
methyl-L-arginine; TC, L-thiocitrulline; SEITU, S-ethylisothiourea;
ImH, imidazole; BH
+
ω
NOHA + 0.5NADPH + 0.5H + O
8
2
+
Cit + NO + 0.5NADPH + H O
4
, (6R)-5,6,7,8-tetrahydro-L-biopterin; DTT, D,L-
2
dithiothreitol; NDA, 2,3-naphthalenedicarboxaldehyde; HEPES, N-(2-
hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; NOS, NO synthase;
ω
ω
In the first step, Arg is N -hydroxylated with insertion
P450, cytochrome P450; SOD, superoxide dismutase; NO
nitro-L-arginine.
2
-Arg, N -
of one oxygen atom from dioxygen and consumption of
1
0.1021/tx0001068 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/18/2001

ω
Oxidations of N -Hydroxyarginine Analogues
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 203
two electrons from NADPH to produce the stable inter-
ω
mediate N -hydroxy-L-arginine (NOHA) (4). The second
step which leads to the formation of nitric oxide and Cit
is also a monooxygenation step, but only one electron
from NADPH is necessary (5). The site for NO production
is heme, but other cofactors are required: flavin adenine
dinucleotide and flavin mononucleotide which serve in
the transfer and storage of electrons derived from NAD-
PH and tetrahydrobiopterin (BH
involved at least in the allosteric modulation of NOS (6,
). Even though the presence of the heme thiolate is
4
) which seems to be
7
reminiscent of other monooxygenase enzymes such as
cytochromes P450 (P450), NO synthases are unique
among this family of enzymes in several ways. (1) NO
formation proceeds with consumption of an odd number
of electrons. Whereas the first step seems to involve the
F igu r e 1. Structure of the compounds used in this study.
also been demonstrated in vivo and is suspected to be
involved in several pathological conditions, alone or in
conjunction with peroxynitrite which is the product of its
reaction with nitric oxide (26-28). Thus, the oxidase
activity of NOS may turn NO synthase into a superoxide/
peroxynitrite synthase (29) and induce irreversible tissue
damage.
V
iron-oxo species “Fe dO” as is the case in most P450-
dependent monooxygenations, the catalysis of the second
step is generally attributed to the intermediate complexes
III
•
III
-
(
Fe -OO and Fe -OO ) (8-11). (2) NO synthases are
the only heme proteins involving BH as a supplementary
4
cofactor. Beyond its well-characterized effect on NOS
quaternary structure, stability, and active site structure
Interestingly, it has been recently reported that re-
combinant NOS not containing BH was able to catalyze
4
δ
(12), BH
4
has also been proposed to control the redox
the oxidation of NOHA with formation of Cit, N -cyano-
-
properties of the heme (13), to influence the reactivity of
L-ornithine, and NO2 (30).
II
the Fe -O
2
species (14), and even to deliver electrons to
To better understand the substrate specificity of NOSs,
the heme (15, 16). (3) NOSs are multimeric enzymes
consisting of two identical subunits. Each subunit has a
two-domain structure which allows the association of a
reductase component responsible for electron transfer
between NADPH and flavins and of an oxygenase com-
ponent containing the binding sites for substrate, heme,
the effects of BH on the substrate specificity and reaction
mechanism of NOSs, we have compared the abilities of
4
recombinant NOS II and BH -free NOS II to catalyze the
4
oxidation of a series of N-hydroxyguanidines, including
NOHA 4 and its analogues 3, 5, and 6, N-(4-chlorophe-
nyl)-N′-hydroxyguanidine 8, and N-hydroxyguanidine 7
itself (Figure 1). All these compounds are oxidized with
cleavage of their CdNOH bond, however, with very
different rates and mechanisms when either NOS II or
4
4
and BH which are all located in close proximity (16, 17).
Calmodulin binds at the junction between the two
domains and controls electron transfer between the
flavins and the heme (1-3). Thus, NO synthases are the
most complex heme thiolate proteins known so far, and
mechanistic studies are still needed to explain all the
aspects of NO biosynthesis.
At present, NOSs are known to catalyze two different
kinds of reactions. The first ones are monooxygenations
that appear only to occur on few substrates. Arg, NOHA,
BH -free NOS II is used.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. (6R)-5,6,7,8-Tetrahydro-L-biopterin and L-thio-
citrulline were purchased from Alexis (Coger, Paris, France).
NADPH came from Boehringer Mannheim Biochemicals. Arg,
ω
homo-Arg, N-hydroxyguanidine, Cit, N -nitro-L-arginine, bovine
and their homologues with one more CH
2
carbon, homo-
L-arginine (homo-Arg) and N -hydroxyhomo-L-arginine
homo-NOHA), are the only compounds oxidized by NOS
erythrocyte superoxide dismutase (SOD), Aspergillus nitrate
reductase, and bovine serum albumin were purchased from
Sigma. Homo-NOHA, NOHA, nor-NOHA, and dinor-NOHA
were synthesized following previously described procedures (31,
32). N-(4-Chlorophenyl)-N′-hydroxyguanidine 8 was prepared
in 65% yield by reacting cyanamide 8b with hydroxylamine
hydrochloride in anhydrous ethanol according to the procedure
of Schantl and T u¨ rk (33). (4-Chlorophenyl)urea 8a and 4-chlo-
rophenylcyanamide 8b were obtained in 90 and 75% yield by
reacting 4-chloroaniline with potassium cyanate in 0.5 M HCl
and cyanogen bromide in methanol, respectively, according to
ω
(
with formation of NO and the corresponding urea with
a very high catalytic efficiency (18). All the other tested
Arg or NOHA derivatives either failed to produce NO,
as described in the case of nor-Arg or nor-NOHA (18, 19),
or led to small amounts of NO, as in the case of
ω
N -methyl-L-arginine (NMA) (19, 20). However, quite
recently, some N-aryl-N′-hydroxyguanidines have been
reported to be oxidized by NOS II in a manner similar
the method of Schantl and T u¨ rk (33). The compounds used in
this study exhibited 1H NMR and mass spectra in complete
to that seen with NOHA, with V
m
values for NO forma-
tion that are only 4 times lower than those found for
NOHA (21).
agreement with those reported previously (31-33). Other
reagents were purchased from Aldrich.
The second kind of reaction catalyzed by NOSs is the
Recombinant NOS II was isolated and purified from Escheri-
chia coli in the absence of BH4 as described previously (34).
P r otein Qu a n tita tion . Protein concentrations were deter-
mined by the method of Bradford (35) using bovine serum
albumin as a standard and the Bradford reagent from Bio-Rad.
•
-
reduction of O
2
into O
2
2 2
and H O . When NOS isoforms
are saturated with cofactors and Arg, NADPH consump-
tion is fully coupled to NO production, 1.5 equiv of
NADPH being consumed per NO molecule produced (4,
-
-
Assessm en t of NO
2
a n d NO
3
F or m a tion . Nitrite detec-
5
). However, in the absence of Arg or when the enzyme
tion was performed according to a previously described proce-
dure (36). Incubations containing 50 mM N-(2-hydroxyethyl)-
piperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4), 500 µM
substrate, 100 µM NADPH, 100 µM D/L-dithiothreitol (DTT), and
1 µg of NOS II in a final volume of 100 µL were shaken at 37
is not fully loaded with BH , electrons coming from
4
NADPH serve in oxygen reduction and lead to reduced
oxygen species such as superoxide anion (22, 23) and
hydrogen peroxide (24, 25). Superoxide generation has

2
04 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Moali et al.
Ta ble 1. Nitr ite a n d Nitr a te P r od u ction a n d NADP H
°
C for 10 min, the reactions quenched with 25 µL of ethanol,
and the mixtures allowed to stay at room temperature for at
least 1 h before addition of the Griess reagent (125 µL of 1%
sulfanilamide in 0.5 N HCl and 125 µL of 0.1% N-(1-naphthyl)-
ethylenediamine in 0.5 N HCl) (36). Absorbances were measured
at 550 nm in a 96-well microplate, and standard curves with
Con su m p tion by BH4-F r ee NOS II in th e P r esen ce of
Com p ou n d s 1-8
activity [nmol min-1 (mg of protein)-1]a
nitrite
nitrate
NADPH
production
production
oxidation
NaNO
2
in the presence of 100 µM NADPH were used to
determine the amount of nitrite in the incubations. When nitrate
quantification was necessary, incubations were stopped by
heating for 3 min at 95 °C, and reduction of nitrate to nitrite
was performed in the presence of nitrate reductase and an
NADPH-regenerating system, following a previously described
protocol (18, 36).
no substrate
0 µM BH4
homo-Arg 1
Arg 2
e1
e1
e1
e1
e5
nd
e5
e5
45 ( 8
b
1
112 ( 11
b
nd
b
nd
homo-NOHA 3
NOHA 4
nor-NOHA 5
dinor-NOHA 6
OH-gua 7
28 ( 10
49 ( 9
26 ( 11
33 ( 15
7 ( 5
30 ( 6
22 ( 10
31 ( 10
12 ( 4
13 ( 8
10 ( 5
18 ( 10
55 ( 7
92 ( 8
50 ( 11
47 ( 7
NADP H Con su m p tion . NADPH consumption was followed
at 37 °C by monitoring the decrease in absorbance at 340 nm
and quantitated using an extinction coefficient of 6.2 mM
55 ( 2
-
1
4-Cl-Ph-NH-
C(dN-OH)-NH2
8
150 ( 30
-
1
cm . Unless otherwise indicated, cuvettes containing 100 µM
NADPH, 100 µM DTT, and 500 µM substrate in 150 µL of
HEPES buffer (50 mM, pH 7.4) were allowed to stay at 37 °C
for 2 min prior to the addition of 1.5 µg of NOS II to the sample
cuvette.
a
Nitrite and nitrate production were quantitated from incuba-
tions containing 100 µM NADPH, 100 µM DTT, 1 µg of BH4-free
NOS II, and 500 µM potential substrate (when present) in a total
volume of 100 µL. Nitrite and nitrate formation was linear over
time for at least 10 min. After 10 min at 37 °C, incubations were
stopped by heating for 3 min at 90 °C and submitted to the Griess
assay as described in Materials and Methods. The rate of NADPH
oxidation was measured spectrophotometrically at 340 nm with
reference and sample cuvettes having the same composition (100
µM NADPH and 100 µM DTT) except that BH4-free NOS II (1.5
HP LC Id en tifica tion a n d Qu a n tita tion of Meta bolites
fr om NOS II-Dep en d en t Oxid a tion of NOHA a n d 8. Amino
acid products were derivatized with 2,3-naphthalenedicarbox-
aldehyde (NDA) in the presence of NaCN and separated by
reverse-phase HPLC according to the procedure recently de-
scribed by Clague et al. (37).
µg) was only present in the sample cuvette. Results are means
Incubations containing 500 µM N-(4-chlorophenyl)-N′-hy-
droxyguanidine 8, 1 mM NADPH, and 2.5 µg of NOS II in 50
µL (final volume) of 50 mM HEPES (pH 7.4) were shaken at 37
((SD) of four separate experiments. b Not determined.
guanidines 3-8, we have studied the influence of some
°
C for 5 min. The reactions were quenched with 50 µL of a 1:1
mixture of acetonitrile and 5 mM H PO containing 40 µM
-chlorobenzamide as an internal standard. Separation of the
-
classical effectors of NOSs on NO
2
formation from
3
4
NOHA itself and from compound 8.
4
(
1) Ch a r a cter istics of th e Oxid a tion of NOHA
metabolites was performed as previously described (38) on a 250
mm × 4.6 mm Nucleosil ODS 5 µm column (SFCC-Shandon).
The mobile phase was a gradient between solvent A [water
containing 5 mM phosphoric acid (pH 2.6)] and solvent B
4
w ith BH -F r ee NOS II. In the absence of NOHA, nitrite
production was indistinguishable from background noise,
showing that the observed nitrite production was directly
linked to NOHA oxidation (Figure 2A).
Oxidation of NOHA was fully NOS- and NADPH-
dependent since removal of either the enzyme or the
cosubstrate led to a 90% decrease in the level of nitrite
production (Figure 2A). Moreover, the level of NOHA
(acetonitrile) using the following program: at 0 min, 10% B; at
5
min, linear gradient to 40% B over the course of 15 min; at 20
min, linear gradient to 100% B over the course of 10 min; at 30
min, linear gradient to 10% B over the course of 5 min followed
by reequilibration for 15 min. The flow rate was 1 mL/min, and
the absorbance was monitored at 240 nm (λmax for 8, 8a , and
4
oxidation increased linearly with BH -free NOS II con-
8
b are 232, 241, and 236 nm, respectively). The retention times
for 8, 8a , 8b, and 4-chlorobenzamide were 6.7, 19.5, 24.7, and
8.1 min, respectively. Calibration curves were made from
centration up to 0.1 mg/mL and with time up to 10 min
at 37 °C (data not shown). More surprisingly, Arg, which
is not a substrate under the conditions studied here but
should compete with NOHA for binding in the active site,
1
identical mixtures containing various concentrations of 8, 8a ,
and 8b but without NOS II.
did not exhibit any inhibitory effect on the reaction
ω
(
Figure 2A). A similar result was obtained with N -
Resu lts
methyl-L-arginine (NMA), a classical inhibitor of NOS,
which even led to a slight increase in activity. Other NOS
inhibitors such as L-thiocitrulline (TC), S-ethylisothiourea
4
Oxid a tion of Com p ou n d s 1-8 w ith BH -F r ee NOS
II. Whereas guanidines (homo-Arg and Arg) could not
evoke any significant nitrite and nitrate production in
4
the presence of BH -free NOS II and NADPH under our
conditions (see Materials and Methods), all six N-hy-
droxyguanidines 3-8 produced variable amounts of
nitrite and nitrate ions (Table 1). NOHA seems to be the
most efficient substrate in the series, while nitrite and
nitrate production from the unsubstituted N-hydroxy-
guanidine 7 was hardly detectable. Intermediate amounts
of nitrite and nitrate were obtained with NOHA ana-
logues 3, 5, and 6, and with N-aryl-N′-hydroxyguanidine
(
SEITU), and imidazole (ImH), which act as substrate
competitors but also as inhibitors of the electron transfer
between flavins and heme (40), inhibit nitrite production
by 60-70% when used at 1 mM. Thus, whereas electron
transfer appears to be important for BH -free NOS II-
4
dependent oxidation of NOHA, binding of NOHA in the
active site does not seem to be required.
Finally, addition of very small amounts of SOD (10
units/mL) led to an almost complete inhibition of nitrite
production (Figure 2A). This result is the most striking
8
. However, it is noteworthy that experiments using the
4
one since, in the presence of saturating amounts of BH ,
hemoglobin assay for NO formation (39) showed that not
one of compounds 1-8 was able to produce detectable
amounts of NO in the presence of BH
NADPH (data not shown).
SOD failed to inhibit nitrite production from NOHA and
NOS II at concentrations up to 100 units/mL (Figure 3A).
Figure 3A also demonstrates that the concentration of
4
-free NOS II and
4
SOD which allows the half-maximum effect in BH -free
To gain further insight into the characteristics of the
BH -free NOS II-dependent oxidation of N-hydroxy-
NOS II-catalyzed oxidation of NOHA is very low (0.6 unit/
mL). Such spectacular inhibitory effects of SOD were
4

ω
Oxidations of N -Hydroxyarginine Analogues
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 205
F igu r e 2. Influence of cofactors, inhibitors, and SOD on nitrite
production (A) and NADPH oxidation (B) in oxidation of NOHA
by BH -free NOS II. The control incubation (complete system,
4
CS) was obtained with 500 µM NOHA under the conditions
described in Table 1. In experiments regarding NADPH con-
sumption, 100 µM NADPH was used. When mentioned, NOHA,
NADPH, or NOS was removed and Arg, NMA, TC, SEITU, or
ImH was added at a final concentration of 1 mM. The SOD
concentration was 10 units/mL. Means ((SD) of four separate
experiments.
F igu r e 3. Effects of increasing amounts of SOD on nitrite
production from NOHA and NOS II (A) and on the oxidation of
8
4
by NOS II (B). Incubations in the absence of BH (white
symbols) were carried out as described in Table 1 except that
increasing concentrations of SOD were added. For the incuba-
tions in the presence of BH
120 µM DTT were also added. Activities in panel A are
expressed in nanomoles of NO
4 4
(black symbols), 10 µM BH and
previously observed in the oxidation of several com-
pounds containing a CdN-OH function, including N-
hydroxyguanidines, with microsomal cytochromes P450
-
2
per minute per milligram of
protein; in panel B, they are expressed in nanomoles of 8a
(
circles) or 8b (squares) per minute per milligram of protein
means ((SD) of four experiments]. Ordinate values indicated
(
38). In those reactions, small amounts of SOD (1-10
[
units/mL) were also sufficient to lead to almost complete
4
on the left are for BH -containing NOS II, whereas those on
-
the right are for BH -free NOS II.
inhibition of P450-dependent formation of NO
Consequently, we suggest that superoxide ion may also
be directly involved in NOHA oxidation by BH -free NOS
II. Figure 2B shows the effects of the various compounds
used in Figure 2A on NADPH consumption by BH -free
NOS II in the presence of NOHA. As previously reported
39), the level of NADPH consumption is decreased in
2
.
4
the N-hydroxyguanidines (3, 5, 6, or 7) was added (Table
4
1
). These compounds do not seem to affect electron
transfer from NADPH when used at concentrations as
high as 500 µM. On the contrary, addition of NOHA or 8
led to a significant increase in the rate of NADPH
4
(
-1
-1
consumption [around 100 nmol min (mg of protein) ].
Interestingly, NADPH consumption and nitrite and
nitrate formation seem to be relatively well correlated,
approximately 1.2 mol of NADPH being consumed to
the absence of NOHA. Addition of Arg and NMA had
weak effects on both NADPH consumption (Figure 2B)
-
and NO
2
formation (Figure 2A), whereas imidazole had
a clear inhibitory effect on both NADPH consumption and
-
-
produce 1 mol of NO
2
3
and NO , except in the case of
-
NO
sumption and NO
of NOHA by BH
2
formation. This parallel variation of electron con-
N-hydroxyguanidine 7 itself, and of 8, for which about 3
-
2
formation in SOD-inhibited oxidation
-free NOS II is in agreement with the
mol of NADPH was consumed for the formation of 1 mol
4
-
and NO
-
(Table 1).
of NO
2
3
•
-
involvement of O
2
in this oxidation. Other NOS inhibi-
(
3) Or ga n ic P r od u cts F or m ed in th e BH -F r ee
4
tors, TC and SEITU, had intermediate effects (Figure 2);
NOS II-Dep en d en t Oxid a t ion of N-H yd r oxy-
gu a n id in es. Quantitative measurements of the organic
products derived from NOHA and its R-amino acid
analogues were found to be very difficult after derivati-
zation of their R-amino acid function (18, 30, 37). This
originated from the relatively low enzymatic activities
-
they inhibited NO
2
formation slightly more than NAD-
PH consumption. This could be due to direct chemical
•
-
reactions of these compounds with O
2
, in competition
-dependent oxidation of NOHA.
2) NADP H Con su m p tion d u r in g BH
II-Dep en d en t Oxid a tion of N-Hyd r oxygu a n id in es
-8. Basal NADPH oxidation by BH -free NOS II in the
absence of either the cofactor or substrate was found to
•
-
with O
2
(
4
-F r ee NOS
4
of BH -free NOS II (Table 1) and the great importance
3
4
of small amounts of products containing a urea function
that were formed from hydrolysis of the corresponding
cyanamides under the derivatization conditions (37).
However, quantitative detection of substrate 8 and of
its metabolites was easily performed after HPLC separa-
tion and detection with UV spectroscopy at the wave-
-
1
occur at a relatively low rate of 45 ( 8 nmol min (mg
of protein) at 37 °C (Table 1), in agreement with the
-
1
value obtained by Presta et al. under similar conditions
(39). This rate was only slightly modified when one of

2
06 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Moali et al.
F igu r e 4. Influence of cofactors, inhibitors, and SOD on urea
a and cyanamide 8b production (A) and NADPH oxidation (B)
in oxidation of 8 by BH -free NOS II. The control incubation
complete system, CS) was carried out with 500 µM 8, 1 mM
NADPH, and 2.5 µg of NOS II in a total volume of 50 µL. After
min at 37 °C, incubations were quenched and submitted to
HPLC analysis as described in Materials and Methods. In
experiments regarding NADPH consumption, 100 µM NADPH
was used. When mentioned, 8, NADPH, or NOS was omitted
8
4
F igu r e 5. Effect of increasing concentrations of BH on the
4
reaction between NOS II and N-hydroxyguanidines 3-7 (A) in
the absence of SOD and (B) in the presence of 50 units/mL SOD.
Incubations were carried out as described in Table 1 except for
(
5
the presence of BH
prepared just before the experiment in 50 mM HEPES (pH
.4): (b) homo-NOHA, (O) NOHA, ([) nor-NOHA, (]) dinor-
NOHA, and (×) OH-gua. Means of three separate experiments
SD < 15%).
4
which was added from a 250 µM solution
7
and Arg, NMA, NO
2
-Arg (1 mM final concentration), or ImH
(
(10 mM final concentration) was added. The SOD concentration
was 10 units/mL. Activities for urea 8a (white bars) and
cyanamide 8b formation (black bars) are means ( SD of four
separate experiments.
II in the presence of 8 exhibited similar variations upon
addition of modulators of electron transfer. Arg and NMA
had little effect or led to an increase in the level of product
formation (8a and 8b) and of NADPH consumption
length corresponding to the absorption of their aryl
chromophore. Formation of two products, 4-chlorophe-
nylurea (8a ) and 4-chlorophenylcyanamide (8b), was
demonstrated from comparison of their retention times
and UV spectra with those of authentic samples.
(
Figure 4A,B); imidazole led to a decrease in these two
activities, and NO -Arg did not exhibit any significant
effect. The strong inhibitory effects of SOD on formation
of 8a and 8b from oxidation of 8 by BH -free NOS II and
2
4
the parallel variation of 8a and 8b formation and
NADPH consumption in the presence of NOS regulators
•
-
are in agreement with an oxidation of 8 by O
from the oxidase function of BH -free NOS II.
E ffect s of BH on t h e Oxid a t ion of N-Hyd r oxy-
gu a n id in es 3-8 w ith NOS II. Preincubation of recom-
binant BH -free NOS II with increasing concentrations
of BH led to a variation of the rates of NO
upon oxidation of N-hydroxyguanidines that is greatly
dependent on the substrate structure. In fact, two very
different kinds of behavior were observed (Figure 5). With
2
derived
4
4
8
a and 8b were formed with relatively low rates [34
4
-1
-1
-
and 16 nmol min (mg of protein) , respectively, for 500
4
2
formation
-
µM 8] and with a stoichiometry relative to NO
2
and
of ∼1 (Table 1). Both products were hardly detect-
able after incubation in the absence of either NADPH or
-
NO
3
-
-
BH
in BH
4
-free NOS II. As previously found for NO
2
formation
NOHA and homo-NOHA as substrates, the rate of NO
2
-
4
-free NOS II-dependent oxidation of NOHA, ad-
(and NO
increased with increasing BH
with BH concentrations of >10 µM, a maximum level
6-10-fold higher than that observed in the absence of
BH (Figure 5A). In the presence of such concentrations
of BH , SOD did not show any inhibitory effects (Figure
3A). Thus, nitrite formation from BH -free NOS II-
catalyzed oxidation of NOHA and homo-NOHA is due to
3
, data not shown) formation dramatically
dition of Arg or NMA did not significantly inhibit the
formation of 8a and 8b. Addition of N -nitro-L-arginine
NO
formation, while imidazole inhibited 60% of this activity
Figure 4A). Moreover, even low levels of SOD (1 unit/
mL) almost completely inhibited formation of 8a and 8b
Figure 3B) as well as formation of NO
shown), whereas oxidation of 8 to 8a by BH
NOS II was not inhibited by SOD (Figure 3B), as
previously reported (21).
4
concentration and reaches,
ω
4
(
2
-Arg) resulted in only 40% inhibition of 8a and 8b
4
(
4
4
-
(
2
(data not
-sufficient
•
-
4
oxidation of these compounds by O
oxidase function of the enzyme, while in the presence of
BH , it is due to the classical monooxygenase function of
2
derived from the
4
As in the case of NOHA (Figure 2A,B), 8a and 8b
formation and NADPH consumption by BH -free NOS
4
NOS II, which occurs in the active site and is not
inhibited by SOD. This conclusion was completely con-

ω
Oxidations of N -Hydroxyarginine Analogues
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 207
N-hydroxyguanidine 7 itself (Table 1). The main char-
acteristics of these reactions, i.e., (i) their almost complete
inhibition by very low concentrations of SOD, (ii) their
-
1
-1
relatively low rates [20-80 nmol min (mg of protein)
at 500 µM substrate], (iii) their broad substrate specific-
ity, (iv) their poor chemoselectivity, as shown by the
formation of amounts of urea 8a and cyanamide 8b in a
2
/1 ratio in the case of 8, are very similar to those
previously reported for the oxidations of N-hydroxy-
guanidines with microsomal cytochromes P450 (38).
These results show that BH
chromes P450, oxidizes N-hydroxyguanidines via O
4
-free NOS II, like cyto-
•
-
2
derived from the oxidase function of these two classes of
heme proteins. Accordingly, the aforementioned charac-
teristics of these reactions are very similar to those
4
F igu r e 6. Effect of increasing concentrations of BH on the
reaction between NOS II and 8. Incubations were carried out
as described in the legend of Figure 4, except for the addition
reported for the chemical oxidation of N-hydroxy-
of BH
4
which was preincubated for 10 min at 4 °C in the
•-
guanidines by O
(
2
itself in aqueous or organic solvents
presence of NOS II at the indicated concentrations. Reactions
were started by the addition of 500 µM 8 and 1 mM NADPH
and carried out for 5 min at 37 °C. They were quenched and
analyzed by HPLC as described in Materials and Methods to
quantify urea 8a (white bars) and cyanamide 8b (black bars)
formation. Means ( SD of three separate experiments.
•
-
38, 41-44). Thus, the ability of O
2
to perform the
oxidative cleavage of the CdNOH function of various
N-hydroxyguanidines, whereas it seems to be inert
toward guanidines (43), allows one to understand the
4
broad substrate specificity of BH -free NOS II toward
N-hydroxyguanidine and its inertness toward Arg (ref 30
and this work) and homo-Arg (Table 1).
As mentioned above, Arg and NMA, which were
expected to compete with the N-hydroxyguanidine sub-
strate for binding to the active site, failed to inhibit the
firmed by experiments similar to those described in the
legend of Figure 5A, except for the presence of 50 units/
mL SOD in the incubations (Figure 5B). In the presence
of SOD, very similar curves were obtained for the
-
variation of the extent of NO
of BH
detectable in the absence of BH
2
formation as a function
oxidation of NOHA and 8 with BH
4
-free NOS II (Figures
4
concentration, except that nitrite was hardly
2
and 4). This result suggests that the reaction between
4
.
•
-
these N-hydroxyguanidines and O
2
does not occur in
In the case of the other N-hydroxyguanidines, nor-
NOHA (5), dinor-NOHA (6), and hydroxyguanidine itself
the active site but rather in the surrounding buffer.
Therefore, the only ways to prevent substrate oxidation
(7), the presence of increasing concentrations of BH
4
-
with NO
2
formation are inhibition of superoxide syn-
failed to produce any increase in the activity. It even led
-
thesis with inhibitors of the electron transfer to the heme
such as imidazole, NO -Arg, and TC (Figures 2A and 4A)
and scavenging of the freshly produced superoxide by
to a slight decrease in the level of NO
2
formation, which
2
became hardly detectable at BH concentrations of >20
4
µM (Figure 5A). In experiments performed in the pres-
ence of 50 units/mL SOD (Figure 5B), the level of nitrite
formation remained close to zero, indicating that the
oxidase activity of NOS II is the only activity responsible
for the oxidation of 5-7, whatever the concentration of
•
-
SOD. Diffusion of O
2
toward the solvent buffer also
explains why total inhibition of its reaction with the
studied N-hydroxyguanidines can be achieved with very
small quantities of SOD.
Thus, oxidation of N-hydroxyguanidines by BH -free
4
NOS II probably decomposes as follows:
4
BH (compare panels A and B of Figure 5).
Compound 8, which has been recently described to act
as an exogenous substrate of NOS II with formation of
stoichiometric amounts of NO and urea 8a (21), exhibited
a behavior similar to those of NOHA and homo-NOHA.
For instance, Figure 6 shows that, in the presence of
4
increasing concentrations of BH , the rate of formation
of urea 8a was dramatically increased, by a factor of at
least 10, whereas that of cyanamide 8b was only slightly
modified. As in the case of NOHA and homo-NOHA,
The first step is totally NOS-dependent; however, some
N-hydroxyguanidines may exert a subtle control on this
step as shown in Table 1 in the case of NOHA. On the
contrary, the yield and rate of the second step will only
depend on the reactivity of the N-hydroxyguanidine
oxidation of 8 with BH
pressed in the presence of SOD (Figure 3B), whereas its
oxidation with BH -containing NOS II was not affected
4
-free NOS II was almost sup-
4
by the presence of SOD (Figure 3B).
•
-
toward O
2
, which is based on the structural and
electronic characteristics of the CdNOH function, as was
demonstrated in the case of P450-dependent oxidation
of N-hydroxyguanidines (38).
Discu ssion
4
In the absence of BH , NOS II is not able to catalyze
the oxidation of compounds containing a guanidine
function, such as Arg and homo-Arg, as shown by the
4 4
Addition of BH to recombinant BH -free NOS II
dramatically changes the behavior of this enzyme toward
N-hydroxyguanidines, by playing two key roles as shown
in Figures 5A and 6. The first role is to decrease the
-
-
lack of any formation of NO
conditions used here (Table 1). However, BH
2
or NO
3
under the
-free NOS
4
•-
II does catalyze the oxidation of a wide range of com-
importance of the O
2
-dependent oxidation of N-hydroxy-
pounds involving an N-hydroxyguanidine function, 3-8,
guanidines. This is shown by the progressive decrease
-
-
-
with similar rates of NO
2
and NO
3
formation, if one
in the level of and final complete loss of NO
2
formation
excepts a markedly lower rate in the particular case of
from the oxidation of N-hydroxyguanidines 5-7 that are

2
08 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Moali et al.
II
T FeIII-OO ] in the presence of N -hydroxyguanidines.
•
ω
F igu r e 7. Possible evolutions of the NOS complex [Fe -O
2
not substrates of BH
4
-containing NOS II (18) (Figure 5A).
to strongly decrease the level of
derived from the NOS oxidase function
has been reported for the three main NOS isozymes (23,
5-47). The detailed molecular origin of this effect
remains to be determined.
The second role of BH in NOS II-dependent oxidation
tioned close to it in the NOS active site. Such a fast
II
This aptitude of BH
4
reaction of the NOS Fe -O
2
intermediate with the
•
-
formation of O
2
chemically reactive CdNOH function of N-hydroxy-
guanidines appears to occur only with compounds well
recognized and bound close to the heme, such as NOHA,
4
homo-NOHA, and 8, when BH
situations, i.e., with the other N-hydroxyguanidines 5-7
in the presence or in the absence of BH , and with all
the N-hydroxyguanidines in the absence of BH , the NOS
species does not react with the CdNOH function.
4
is present. In all the other
4
of N-hydroxyguanidines is more spectacular (Figures 5A
and 6). It is to start a new monooxygenase activity which
4
4
II
only occurs with substrates of BH
able to produce NO, such as NOHA, homo-NOHA, and
. The characteristics of this reaction are completely
different from those of the BH -free NOS II reactions,
4
-containing NOS II
Fe -O
It thus dissociates its Fe -O
that have been observed come from reactions of O
the N-hydroxyguanidines. In that respect, it is notewor-
2
II
2
bond, and all the products
•-
8
2
with
4
•
-
and correspond well to those previously described for
NOS II-dependent monooxygenation of Arg, NOHA,
homo-NOHA, and 8 (3, 18, 21). They include (i) a weak
effect of SOD on the activity, (ii) a clear substrate
selectivity as compounds 5-7 are not transformed, (iii)
much higher activity levels [between 100 and 320 nmol
thy that O
2
might come not only from dissociation of
II
the NOS Fe -O
at the level of the NOS reductase domain (23, 46, 49-
51). However, the 80% inhibition of BH -free NOS
2 2
complex but also from reduction of O
4
II-dependent oxidation of 8 by 10 mM imidazole, a known
NOS-iron ligand (Figure 4A), indicates that only a small
-1
-1
•-
min (mg of protein) ], and (iv) a high chemoselectivity
part of O
2
directly comes from the reductase domain
with the almost exclusive formation of urea 8a from 8
under the conditions that were used.
(Figure 6). Moreover, this activity is strongly inhibited
One easily understands the requirement of a strict
by compounds known to bind at the active site of NOS,
such as Arg (16, 17). Therefore, this monooxygenase
proximity between the substrate CdNOH function and
II
the NOS Fe -O intermediate for a fast enough reaction
2
II
reaction is performed inside the active site presumably
of the poorly reactive Fe -O species, which explains why
2
II
by the NOS Fe -O
2
species (8-12).
only some N-hydroxyguanidines undergo efficient mo-
Another important difference between the oxidations
catalyzed either by BH -containing NOS II or by BH
free NOS II is the clear formation of NO, in a NO/urea
ratio close to 1, in the former reactions (4, 5, 18, 21)
contrary to the very low level of formation of NO in the
latter reactions. In fact, as mentioned above in Results,
4
nooxygenation. The absolute requirement of BH for this
reaction could be explained, at least in part, by its role
4
4
-
in favoring a good positioning of the CdNOH function of
II
NOHA and related substrates in proximity of the Fe -
2 4
O species. Accordingly, BH is known to play a key role
in the formation of active NOS II dimers (12), and recent
oxidation of compounds 1-8 by BH
lead to any significant detection of NO by the oxyhemo-
globin assay. On the other hand, oxidation of NOHA,
4
-free NOS II did not
X-ray data about NOSs have shown the presence of a
network of hydrogen bonds between BH
4
, heme, and Arg
(16, 17) that should be crucial to controlling the proximity
II
homo-NOHA, and 8 by BH
4
-containing NOS II has been
between the substrate and heme Fe -O
2 4
species. BH
reported to produce NO in stoichiometric amounts rela-
tive to the corresponding urea (18, 21).
The different behavior exhibited by BH -free and BH -
4 4
containing NOS II toward N-hydroxyguanidines could be
could also favor the reaction between the substrate Cd
II
2
NOH function and heme Fe -O species by increasing
II
the intrinsic reactivity of the Fe -O
transferring electron density to the heme (15, 16).
Moreover, evidence for electron transfer from BH to the
2
species (14), or by
interpreted with the different possible fates of the NOS
4
II
II
heme of the NOS Fe -O species has been provided very
Fe -O
2
intermediate (Figure 7). This species may either
2
recently (52, 53).
directly react with the CdNOH function of N-hydroxy-
guanidine substrates such as NOHA or dissociate its
In conclusion, the aforementioned results show that
NOS II and BH -free NOS II are able to catalyze the
4
oxidation of many N-hydroxyguanidines with formation
II
III
•-
Fe -O
the relatively weak oxidizing properties of heme Fe -
complexes (48), it is likely that it should rapidly react
only with especially reactive substrates very well posi-
2
bond, leading to NOS Fe and O
2
. Because of
II
O
2
of the corresponding ureas and cyanamides and nitrogen
•
-
oxides. Most of these reactions are performed by O
2

ω
Oxidations of N -Hydroxyarginine Analogues
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 209
derived from the oxidase function of NOS II. Only few of
(15) Bec, N., Gorren, A. C. F., Voelker, C., Mayer, B., and Lange, R.
(1998) Reaction of neuronal nitric-oxide synthase with oxygen at
them are selective monooxygenations performed by the
low temperature. Evidence for reductive activation of the oxy-
ferrous complex by tetrahydrobiopterin. J . Biol. Chem. 273,
13502-13508.
II
NOS Fe -O
2
species, which exclusively occur with
is present. These
NOHA, homo-NOHA, and 8, when BH
4
results suggest a possible implication of NOSs in the
oxidative metabolism of certain classes of xenobiotics not
only via their monooxygenase function, as previously
mentioned (case of 8; 21) but also via their oxidase
function. In fact, oxidation of exogenous N-hydroxy-
guanidines, which are not substrates of the NOS mo-
nooxygenase function and which lead to easily detected
ureas or cyanamides because of the NOS oxidase func-
(16) Raman, C. S., Li, H., Martasek, P., Kral, V., Masters, B. S. S.,
and Poulos, T. L. (1998) Crystal structure of constitutive endot-
helial nitric oxide synthase: A paradigm for pterin function
involving a novel metal center. Cell 95, 939-950.
(
17) Crane, B. R., Arvai, A. S., Ghosh, D. K., Wu, C. Q., Getzoff, E.
D., Stuehr, D. J ., and Tainer, J . A. (1998) Structure of nitric oxide
synthase oxygenase dimer with pterin and substrate. Science 279,
2
121-2126.
(
18) Moali, C., Boucher, J . L., Sari, M. A., Stuehr, D. J ., and Mansuy,
D. (1998) Substrate specificity of NO synthases: Detailed com-
•
-
ω
tion, could be used to evaluate O
II. Finally, our results provide a further clear illustration
of the key role of BH in regulating the monooxygenase/
2
formation by NOS
parison of L-arginine, homo-L-arginine, their N -hydroxy deriva-
ω
tives, and N -hydroxynor-L-arginine. Biochemistry 37, 10453-
1
0460.
4
(
(
(
19) Grant, S. K., Green, B. G., Stiffey-Wilusz, J ., Durette, P. L., Shah,
S. K., and Kozarich, J . W. (1998) Structural requirements for
human inducible nitric oxide synthase substrates and substrate
analogue inhibitors. Biochemistry 37, 4174-4180.
oxidase ratio in NOS (23, 29, 39, 45-47).
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health (Grant CA
20) Olken, N. M., Osawa, Y., and Marletta, M. A. (1994) Character-
G
ization of the inactivation of nitric oxide synthase by N -methyl-
5
3914 to D.J .S.) and from the Minist e` re de l’′Enseigne-
L-arginine: Evidence for heme loss. Biochemistry 33, 14784-
ment Sup e´ rieur et de la Recherche (to C.M. and A.R.-
C.). We thank Qian Wang, Christine Curran, and Abby
Meade for excellent technical assistance.
1
4791.
21) Renodon-Corni e` re, A., Boucher, J . L., Dijols, S., Stuehr, D. J ., and
Mansuy, D. (1999) Efficient formation of nitric oxide from selective
oxidation of N-aryl N′-hydroxyguanidines by inducible nitric oxide
synthase. Biochemistry 38, 4663-4668.
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