Báo cáo khoa học: Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO pptx

Relationship between the structure of guanidines and
N-hydroxyguanidines, their binding to inducible nitric
oxide synthase (iNOS) and their iNOS-catalysed oxidation
to NO
David Lefe
`
vre-Groboillot
1,2
, Jean-Luc Boucher
1
, Dennis J. Stuehr
2
and Daniel Mansuy
1
1 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite
´
Paris 5, France
2 Department of Immunology, Lerner Research Institute, Cleveland, OH, USA
Keywords
binding kinetic; guanidines
N-hydroxyguanidines; nitric oxide synthase;
UV ⁄ Vis difference spectroscopy
Correspondence
J-L. Boucher, Laboratoire de Chimie et
Biochimie Pharmacologiques et
Toxicologiques, UMR 8601 CNRS,
Universite
´
Paris 5, 45 rue des Saints-Pe
`
res,

75270 Paris Cedex 06, France
Fax: +33 1 42 86 83 87
Tel: +33 1 42 86 21 91
E-mail:
(Received 18 February 2005, revised
20 April 2005, accepted 25 April 2005)
doi:10.1111/j.1742-4658.2005.04736.x
The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines
to the oxygenase domain of inducible NO-synthase (iNOS
oxy
) was studied
by UV ⁄ Vis difference spectroscopy. In a very general manner, monosubsti-
tuted guanidines exhibited affinities for iNOS
oxy
that were very close to
those of the corresponding N-hydroxyguanidines. The highest affinities
were observed for the natural substrates, l-arginine and N
x
-hydroxy-
l-arginine (K
d
at the lm level). The deletion of either the CO
2
H or the
NH
2
function of their amino acid moiety led to dramatic decreases in the
affinity. However, alkylguanidines with a relatively small alkyl chain exhib-
ited interesting affinities, the best being observed for a butyl chain (K
d

¼
20 lm). Arylguanidines also bound to iNOS
oxy
, however, with lower affinit-
ies (K
d
> 250 l m). Many N-alkyl- and N-aryl-N¢-hydroxyguanidines are
oxidized by iNOS with formation of NO, whereas only few alkylguanidines
led to significant production of NO under identical conditions, and all the
arylguanidines tested to date were unable to lead to the production of NO.
The k
cat
values of NO production from the oxidation by iNOS of the stud-
ied N-hydroxyguanidines were found to vary independently of their affinity
for the protein. The k
cat
values determined for the two-step oxidation of
alkylguanidines to NO were not clearly related to the K
d
of these substrates
toward iNOS
oxy
. However, there is a qualitative relationship between these
k
cat
values and the apparent rate constants of dissociation of the complex
between iNOS
oxy
and the corresponding N-alkyl-N¢-hydroxyguanidine
(k

off
app
) that were determined by stopped-flow UV ⁄ Vis spectroscopy. These
data indicate that a key factor for efficient oxidation of a guanidine by
iNOS to NO is the ability of the corresponding N-hydroxyguanidine to
bind to the active site without being too rapidly released before its further
oxidation. This explains why 4,4,4-trifluorobutylguanidine is so far the best
non-a-amino acid guanidine substrate of iNOS with formation of NO,
because the k
off
app
of the corresponding N-hydroxyguanidine is particularly
low. This suggests that the rational design of guanidines as new NO donors
Abbreviations
BH
4
,(6R)-5,6,7,8-tetrahydro-L-biopterin; BuGua, n-butylguanidine; BuNOHG, N-(n-butyl)-N ¢-hydroxyguanidine; BzNOHG, N -benzyl-N ¢-
hydroxyguanidine; ClPhNOHG, N-(4-chlorophenyl)-N ¢-hydroxyguanidine; FPhGua, 4-fluorophenylguanidine; FPhNOHG, N-(4-fluorophenyl)-
N ¢-hydroxyguanidine; HexGua, n-hexylguanidine; HexNOHG, N-(n-hexyl)-N ¢-hydroxyguanidine; homo-
L-Arg, homo-L-arginine; homo-NOHA,
N
x
-hydroxy-homo-L-arginine; HS, high spin; ImH, imidazole; L-Arg, L-arginine; LS, low spin; NOHA, N
x
-hydroxy-L-Arginine; Nor-L-Arg, nor-L-
Arginine; NOHAgma, N
x
-hydroxyagmatine; NOHGPA, N
x
-hydroxyguanidinopentanoic acid; NOS, nitric oxide synthase; NOS

oxy
, oxygenase
domain of NOS; PentylGua, n-pentylguanidine; PentylNOHG, N-(n-pentyl)-N ¢-hydroxyguanidine; ProGua, n-propylguanidine; ProNOHG,
N-(n-propyl)-N ¢-hydroxyguanidine; TFBGua, 4,4,4-trifluorobutylguanidine; TFBNOHG, N-(4,4,4-trifluorobutyl)-N ¢-hydroxyguanidine.
3172 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS
Nitric oxide synthases (NOS) catalyse the oxidation of
l-arginine (l-Arg) into l-citrulline and NO, with the
intermediate formation of N
x
-hydroxy-l-arginine
(NOHA) [1–3]. This reaction ideally consumes 1.5 mol
of NADPH and 2 mol of O
2
. It occurs in the homo-
dimeric N-terminal domain of the protein called NOS
oxygenase domain (NOS
oxy
) that contains two cofac-
tors per monomer, the heme (iron-protoporphyrin IX)
and (6R)-5,6,7,8-tetrahydro-l-biopterin (BH
4
). Elec-
trons from NADPH are provided to heme by flanking
C-terminal reductase domains. NOSs are heme-thiolate
monooxygenases comparable with cytochrome P450.
Whereas proteins of the cytochrome P450 family are
known to be able to bind and oxidize a very large
number of compounds of various structures, until
recently NOSs were only known to be able to oxidize
l-Arg and a very small number of its close a-amino

acid analogues. Recent reports have shown that NOSs
are able to produce NO from the oxidation of many
non-a-amino acid monosubstituted N-hydroxyguani-
dines, including N-alkyl-N¢-hydroxyguanidines and
N-aryl-N¢-hydroxyguanidines, provided that the alkyl
or aryl substituent is neither too small nor too bulky
[4–7]. NOS-catalysed oxidation of some of these com-
pounds showed k
cat
values as high as 80% that
obtained with NOHA, and some proved to be selective
for one of the three isoforms vs. the others [5,7]. More
recently, NO production has also been observed from
the oxidation of several non-a-amino acid alkylguani-
dines by purified iNOS or by activated mouse macro-
phages, opening the way to the design of stable
exogenous NOS substrates of pharmacological interest
[8,9].
Apart from some equilibrium and kinetic constants
related to the binding of l-Arg [10–14] and NOHA
[10], nothing is known about the thermodynamics and
kinetics of the binding of guanidines and N-hydroxy-
guanidines to iNOS. Removal of the a-amino or
a-carboxylate moiety of l-Arg has important effects on
the ability of the resulting compounds to affect the
heme iron spin equilibrium, and to trigger NADPH
consumption and NO production [14–16]. Interestingly,
it has been shown that several binding modes exist for
N-hydroxyguanidines in the heme pocket of NOSs [17–
20]. Also, the fact that isoform-selective substrates for

NOS [5,7,9] were characterized is striking, given the
high level of similarity between the crystal structures of
the oxygenase domains of the three isoforms [18,19,21].
This study was undertaken to determine structural fac-
tors that are important for a guanidine or N-hydroxy-
guanidine to be well recognized by the NOS active site,
and to be efficiently oxidized with NO formation. For
that purpose, the dissociation constants of several com-
plexes of the oxygenase domain of iNOS (iNOS
oxy
) with
various alkyl- and aryl-guanidines and N-hydroxygua-
nidines were determined by UV ⁄ Vis difference spectros-
copy, according to a previously described technique [22].
The kinetics of the binding of some of these substrates
to iNOS
oxy
was also studied by UV ⁄ Vis spectroscopy
using stopped-flow techniques [23,24]. The correspond-
ing thermodynamic and kinetic binding constants were
then compared with the kinetic constants of NO forma-
tion from iNOS-catalysed oxidation of guanidine and
N-hydroxyguanidine substrates. Our results suggest that
a key factor in the efficient oxidation of a guanidine to
NO by iNOS could be the ability of the corresponding
N-hydroxyguanidine to bind to the active site without
being too rapidly released before its further oxidation.
Our results may help in the further rational design of
guanidines as new NO precursors.
Results

Study of the binding of guanidines and
N-hydroxyguanidines to iNOS
oxy
by UV ⁄ Vis
difference spectroscopy
Purified recombinant iNOS
oxy
showed a wide Soret
band with a maximum absorption wavelength around
400 nm, indicating that the heme-iron existed in equi-
librium between a hexacoordinated low-spin (LS) state
and a pentacoordinated high-spin (HS) state, the major
fraction being in the HS state. As previously described,
addition of l-Arg leads to conversion of the minor
population of heme centres being in the LS state into
the HS state and to the appearance of a difference
spectrum [22,25–27]. However, the intensity of this dif-
ference spectrum is small, because the spin state of the
major fraction of the protein is not affected. Imidazole
(ImH) was thus used to completely convert iNOS
oxy
into a LS state iNOS
oxy
–Fe(III)–ImH complex allow-
ing one to more easily follow the binding of guanidines
or N-hydroxyguanidines to the iNOS
oxy
substrate
binding site [12,22,28]. iNOS
oxy

(1 lm) in the presence
of 400 lm ImH was first titrated with l-Arg. A differ-
ence spectrum displaying a peak at 392 nm and a
upon in situ oxidation by NOSs should take into account both thermody-
namic and kinetic characteristics of the interaction of the protein not only
with the guanidine but also with the corresponding N-hydroxyguanidine.
D. Lefe
`
vre-Groboillot et al. Substrate specificity of iNOS
FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS 3173
trough at 430 nm (Fig. 1) resulting from the conver-
sion of the LS NOS–Fe(III)–ImH complex to the
HS NOS–Fe(III)–l-Arg complex was observed. Inhibi-
tion of the binding of l-Arg to iNOS
oxy
by ImH, and of
the iNOS-catalysed conversion of l-Arg into l-citrulline
has previously been shown to be competitive [11]. Equa-
tion (1) was thus used to calculate corrected equi-
librium constants, K
d
, for the iNOS
oxy
–substrate
complexes from apparent constants K
app
[12–14,23,24].
K
app
=K

d
¼ 1 þ½ImH=K
ImH
ð1Þ
With the ImH concentration used in this study
(400 lm), Eqn (1) became
K
app
¼ 8:7K
d
ð1
0
Þ
Variations in the amplitudes of the difference spectra
with the concentrations of l-Arg were in agreement
with a single binding site model (see Experimental pro-
cedures) and K
app
value of 26 ± 2 lm was found for
the apparent equilibrium constant for the dissociation
of the iNOS
oxy
–l-Arg complex, in good agreement
with a previously reported value (28 ± 4 lm) obtained
with the same ImH concentration [25]. At the end of
the titration, the absolute spectrum of the iNOS
oxy
solution containing 400 lm ImH and 1 mml-Arg
showed a maximum absorption wavelength at
395 ± 3 nm (not shown).

Similar titrations of iNOS
oxy
in the presence of
400 lm ImH were then performed with a large number
of guanidines and N-hydroxyguanidines previously
evaluated as iNOS substrates [4–9,29]. The positions of
the peaks and troughs of the difference spectra
observed during these titrations were similar to those
observed when l-Arg was used (Fig. 1). Variation
in the amplitude of the observed difference spectra
with the concentration of the studied guanidines or
N-hydroxyguanidines was always in reasonable agree-
ment with a single binding site model. The apparent
equilibrium constants derived from these experiments
are shown in Table 1.
NOHA was found to bind to iNOS with a K
app
value slightly lower than that of l-Arg (18 ± 7 lm,
Table 1). Homo-L-Arg and homo-NOHA, the l-Arg
and NOHA analogues bearing one extra methylene
group in the alkyl side-chain, were found to bind to
iNOS
oxy
with higher K
app
values than l-Arg (80 ± 13
and 150 ± 40 lm, respectively). Finally, a much
higher K
app
value (2.4 mm) was found for nor-l-Arg,

the analogue bearing one methylene fewer than l-Arg.
Removal of either the a-COOH or the a-NH
2
group
of NOHA led to a dramatic decrease in the affinity of
the resulting compounds, the K
app
values measured for
N
x
-hydroxyagmatine (NOHAgma), and N
x
-hydroxy-
guanidino-pentanoic acid (NOHGPA), being > 1 mm
(2 and > 4 mm, respectively; Table 1). However, the
simultaneous removal of both the a-NH
2
and a-COOH
functions of NOHA led to N-(n-butyl)-N¢-hydroxygu-
anidine (BuNOHG), which showed a much lower K
app
value of 160 ± 40 lm (Table 1). Replacement of the
terminal CH
3
group of the n-butyl chain by a CF
3
group, leading to N-(4,4,4-trifluorobutyl)-N¢-hydroxy-
guanidine (TFBNOHG), resulted in a sixfold increase
in the K
app

value. Shorter nonfunctionalized analogues
N-(n-propyl)-N¢-hydroxyguanidine (ProNOHG) and
longer ones N-(n-pentyl)-N¢-hydroxyguanidine (Pentyl-
NOHG) and N-(n-hexyl)-N¢-hydroxyguanidine (Hex-
NOHG) showed higher K
app
values than the
N-(n-butyl) compound (270, 900 and >1000 lm,
respectively). Finally, N-benzyl-N¢-hydroxyguanidine
(BzNOHG) and the three para-substituted aryl-deriva-
tives N-(4-fluoro-, 4-methyl- and 4-chlorophenyl)-N¢-
hydroxyguanidines (FPhNOHG, TolNOHG and
ClPhNOHG), showed K
app
values > 2 mm.
A study of the binding of the corresponding non-
functionalized alkylguanidines to iNOS
oxy
led to very
similar conclusions. In the studied series, the alkylgu-
anidine exhibiting the highest affinity for iNOS was
n-butylguanidine (BuGua), with a K
app
value of
140 ± 20 lm (Table 1). Trifluorination of the terminal
methyl group of the n-butyl chain, leading to 4,4,4-tri-
fluorobutylguanidine (TFBGua), increased the K
app
value by 10-fold. The longer nonsubstituted n-pentyl-
and n-hexylguanidines (PentylGua and HexGua) also

Fig. 1. Difference spectrum obtained upon addition of increasing
concentrations of
L-Arg to iNOS
oxy
in the presence of ImH. iNOS
oxy
and ImH concentrations were 1 and 400 lM, respectively. (Inset)
Plot of 1 ⁄DA vs. 1 ⁄ [
L-Arg].
Substrate specificity of iNOS D. Lefe
`
vre-Groboillot et al.
3174 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS
showed higher K
app
values (600 lm and > 4 mm for
the n-pentyl and n-hexyl derivatives, respectively). The
shorter n-propylguanidine (ProGua) showed a K
app
value similar to that found for BuGua. Finally, the
arylguanidines 4-fluorophenyl- and 4-methylphenyl-
guanidines (FPhGua and TolGua) interacted with
iNOS with K
app
values>2 mm.
Relationship between the equilibrium constants
measured for the binding of guanidines and
N-hydroxyguanidines to iNOS
oxy
and the kinetic

constants measured for their iNOS-catalysed
oxidation to NO
In previous studies, we have identified some N-alkyl-
and N-aryl-N¢-hydroxyguanidines, and alkylguanidines
as NO donors following their oxidation catalysed by
iNOS containing all its cofactors [4,5,8,9]. Table 2 gives
the K
m
and k
cat
values measured for the oxidation of
seven N-hydroxyguanidines leading to the highest pro-
duction of NO in the presence of iNOS, together with
K
m
and k
cat
values for the oxidation of the correspond-
ing guanidines [4,5,8,9]. The seven N-hydroxyguani-
dines NOHA, homo-NOHA, BuNOHG, TFBNOHG,
PentylNOHG, FPhNOHG and TolNOHG were oxid-
ized with formation of NO with similar high k
cat
values
ranging from 58 to 100% of that found for NOHA.
They showed widespread K
m ⁄
K
d
ratios, generally >1

and that varied from  1to 20 (Table 2). In that ser-
ies, the k
cat
value for the production of NO from the
oxidation of the N-hydroxyguanidines varied by less
than a factor 2, whereas the k
cat
value for the produc-
tion of NO from the oxidation of the guanidines varied
a great deal from 0 to 100% of the k
cat
value obtained
Table 2. Kinetic constants for the formation of NO from the oxidation of guanidines and N-hydroxyguanidines by recombinant iNOS. See
Table 1 for the structure of compounds. K
m
and k
cat
values are taken from previous publications [4,5,8,9,29]. k
cat
values are expressed per
NOS dimer. The corrected dissociation equilibrium constants (K
d
) for the binding of guanidines and N-hydroxyguanidines to iNOS
oxy
were
obtained by dividing K
app
values (taken from Table 1) by 8.7.
Compounds
K

m
(lM)
N-Hydroxy
guanidine Guanidine
k
cat
(min
)1
)
N-Hydroxy
guanidine Guanidine
K
m
⁄ K
d
N-Hydroxy
guanidine Ref.
NOHA ⁄
L-Arg 40 ± 10 5 ± 1 480 ± 60 400 ± 50 19.3 [4,8,9]
Homo-NOHA ⁄ Homo-
L-Arg 146 ± 21 33 ± 8 410 ± 50 215 ± 50 8.5 [29]
BuNOHG ⁄ BuGua 55 ± 10 45 ± 10 320 ± 50 23 ± 5 3.0 [4,8]
TFBNOHG ⁄ TFBGua 840 ± 100 275 ± 50 780 ± 100 220 ± 50 8.1 [8,9]
PentylNOHG ⁄ PentylGua 310 ± 50 250 ± 100 280 ± 50 60 ± 15 3.8 [4,9]
FPhNOHG ⁄ FPhGua 300 ± 40 – 350 ± 80 < 2
a
1.1 [5,8,9]
TolNOHG ⁄ TolGua 1100 ± 300 – 295 ± 50 < 2
a
< 2.0 [5,9]

a
The rates of the production of NO from the oxidation by iNOS of 1 mM FPhGua or TolGua were lower than 2 min
)1
.
Table 1. Apparent equilibrium constants (K
app
) for the binding of N-hydroxyguanidines R-NH-C(¼ NOH)-NH
2
and guanidines R-NH-C(¼ NH
2
)-
NH
2
to iNOS
oxy
. Titrations were performed by UV ⁄ Vis difference spectroscopy in the presence of 25 lM BH
4
,1mM dithiothreitol and
400 l
M ImH. K
app
values were calculated as described in Experimental procedures. Values ± SD from three different experiments. n.d., not
determined.
R
N-Hydroxyguanidines
Compound K
app
Guanidines
Compound K
app

HO
2
C-CH(NH
2
)-(CH
2
)
3
- NOHA 18 ± 7 lML-Arg 26 ± 2 lM
HO
2
C-CH(NH
2
)-(CH
2
)
4
- Homo-NOHA 150 ± 40 lM Homo-L-Arg 80 ± 13 lM
HO
2
C-CH(NH
2
)-(CH
2
)
2
- – n.d. Nor-L-Arg 2.4 ± 1.0 mM
HO
2
C-(CH

2
)
4
- NOHGPA > 4 mM – n.d.
H
2
N-(CH
2
)
4
- NOHGAgma 2.0 ± 0.7 mM – n.d.
CH
3
(CH
2
)
2
ProNOHG 270 ± 50 lM ProGua 140 ± 20 lM
CH
3
(CH
2
)
3
BuNOHG 160 ± 40 lM BuGua 140 ± 20 lM
CF
3
(CH
2
)

3
- TFBNOHG 0.9 ± 0.2 mM TFBGua 1.5 ± 0.4 mM
CH
3
(CH
2
)
4
PentylNOHG 0.7 ± 0.3 mM PentylGua 0.6 ± 0.1 mM
CH
3
(CH
2
)
5
HexNOHG > 1 mM HexGua > 4 mM
4-F-C
6
H
4
- FPhNOHG 2.4 ± 0.1 mM FPhGua 2.0 ± 0.1 mM
4-CH
3
-C
6
H
4
- TolNOHG > 4 mM TolGua 6.0 ± 1.5 mM
4-Cl-C
6

H
4
- ClPhNOHG 3.0 ± 0.5 mM – n.d.
C
6
H
4
-CH
2
- BzNOHG > 4 mM – n.d.
D. Lefe
`
vre-Groboillot et al. Substrate specificity of iNOS
FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS 3175
with l-Arg, with the order l-Arg > homo-l-
Arg  TFBGua>PentylGua>BuGua>>FPhGua
 TolGua (Table 2).
The K
m
and k
cat
values calculated for NO formation
from iNOS-catalysed oxidation of guanidines are com-
plex parameters as they correspond to a two-step
reaction with intermediate formation of N-hydroxygu-
anidines. The data are difficult to correlate with kinetic
or thermodynamic constants clearly describing individ-
ual reactions, such as K
d
(or K

app
). The situation
should be less complex for K
m
and k
cat
for NO forma-
tion from N-hydroxyguanidines that are more closely
related to a one-step enzymatic reaction.
It is actually well known that, for enzymes having
high k
cat
values, the K
m
values can be markedly higher
than the K
d
values, as indicated by the classical rela-
tion given here [30].
K
m
¼ K
d
þ k
cat
=k
on
or
K
m

=K
d
¼ 1 þ k
cat
=k
off
This equation implies that K
m
⁄ K
d
will increase as k
cat
increases and k
off
decreases. Because the k
cat
value
found for these seven N-hydroxyguanidines varied by
less than a factor 2, it was tempting to investigate a
possible relationship between K
m
⁄ K
d
and k
off
. The fol-
lowing experiments were performed as a first approach
to find the variation in k
off
as a function of the iNOS

substrate structure.
Kinetics of the binding of guanidines and
N-hydroxyguanidines to iNOS
oxy
measured by
stopped-flow UV ⁄ Vis spectroscopy
An iNOS
oxy
solution containing 400 lm ImH was rap-
idly mixed with a solution of the studied ligand contain-
ing the same concentration of ImH. Postmixing ligand
concentrations corresponded to pseudo-first-order con-
ditions. Absorption variations were monitored at 430
and 392 nm (Fig. 2), allowing one to follow, respect-
ively, the disappearance of the NOS–Fe(III)–ImH com-
plex and the appearance of the high-spin NOS–Fe(III)
species. The calculated kinetic constants k
obs
were plot-
ted against the ligand concentration and satisfactorily
fitted with a linear function
k
obs
¼ k
off
app
þ k
off
app
½L

where L is the guanidine or N-hydroxyguanidine used
(Fig. 3), in agreement with a competitive model for the
interaction between ImH and the studied guanidine or
N-hydroxyguanidine [23,24,28]. It has previously been
shown that displacement of ImH from the NOS heme-
iron by l-Arg or its analogues is a two-step process
[23,28] and might involve an intermediate and transient
ternary complex between the protein, ImH and the
l-Arg analogue [23]. The k
on
app
and k
off
app
values are
thus apparent association and dissociation rate con-
stants of the guanidine or N-hydroxyguanidine with
the protein in the presence of 400 lm ImH.
Three guanidines and the corresponding N-hydroxy-
guanidines were studied. l-Arg and NOHA were used
0.33
0.32
430 nm
392 nm
0.31
0.29
0.28
0.27
0.26
0.25

00.511.5
Time (s)
Absorbance
0.3
Wavelength (nm)
392 nm
430 nm
A
B
Absorbance
350 370 390 410 430 450 470 490
0.063
0.113
0.163
0.213
0.263
0.313
Fig. 2. Spectral transitions observed as a function of time upon the
fast addition of BuNOHG to iNOS
oxy
in the presence of ImH. ImH
concentration was 400 l
M. Final heme and BuNOHG concentra-
tions were 5 l
M and 2 mM, respectively. (A) Rapid-scanning
stopped-flow spectra recorded during the reaction. (B) Cross-sec-
tion of (A) variation in absorbance at 430 and 392 nm as a function
of time.
Substrate specificity of iNOS D. Lefe
`

vre-Groboillot et al.
3176 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS
as reference compounds and two pairs of non-a-amino
acid compounds, BuGua ⁄ BuNOHG and TFBGua ⁄
TFBNOHG were also studied. The determined values
of k
on
app
and k
off
app
are reported in Table 3. In the
studied range of concentrations, the k
obs
values were
higher for a guanidine than for the corresponding N-
hydroxyguanidine (Fig. 3). The k
on
app
values for the
guanidines were found to be 5–10 higher than those
for the corresponding N-hydroxyguanidines, and the
k
off
app
values for the guanidines were 25–60 times
higher than those for the corresponding N-hydroxy-
guanidines (Table 3). The k
on
app

values for the non-
a-amino acid guanidines BuGua and TFBGua were
found to be 7- and 25-fold lower than that for l-Arg,
and those for the non-a-amino acid N-hydroxyguani-
dines BuNOHG and TFBNOHG were 3- and 12-fold
lower than that for NOHA. The k
off
app
values for
BuGua and TFBGua were found to be 10 and 6 times
higher than that for l-Arg, and those for BuNOHG
and TFBNOHG were 20 and 5 times higher than that
for NOHA. Interestingly, k
on
app
values for the fluori-
nated compounds TFBGua and TFBNOHG are
3.8- and 3.6-fold lower than those for their nonfluori-
nated analogues BuGua and BuNOHG, respectively,
and the k
off
app
values for TFBGua and TFBNOHG
are 1.6 and 4 times lower than those for BuGua and
BuNOHG, respectively.
Discussion
Binding of guanidines and N-hydroxyguanidines
to iNOS
oxy
In the series of guanidines and N-hydroxyguanidines

studied here, the ratio between the K
app
or K
d
(calcula-
ted using Eqn 1¢) of a guanidine and that of its corres-
ponding N-hydroxyguanidine was always found to be
between 0.5 and 2 (Table 1). This was true for pairs
of compounds showing dissociation constants in the
micromolar range (l-Arg ⁄ NOHA) and pairs of
compounds showing K
d
in the millimolar range
(FPhGua ⁄ FPhNOHG). The difference between the
K
app
values for the guanidines and those for the cor-
responding N-hydroxyguanidines was, in most cases,
small and barely significant. However, we found that
the K
app
value for NOHA is slightly lower than that
for l-Arg (Table 1), and because such an observation
has also been previously reported by several authors
with nNOS [12,22,23] and iNOS [10], this difference is
probably significant. By contrast, we found that the
K
m
value for NOHA is higher than that for l-Arg
(Table 2), also in accordance with the literature data

on the three isoforms [29,31,32]. In the studied series,
Fig. 3. Plots of the rates of spectral transitions observed upon the
addition of guanidines or N-hydroxyguanidines to iNOS
oxy
in the
presence of ImH vs. the postmixing concentration of the studied
guanidine or N-hydroxyguanidine. Best linear fits are shown.
(A)
L-Arg and NOHA, (B) N-hydroxyguanidines BuNOHG and
TFBNOHG, (C) guanidines BuGua and TFBGua. See Table 1 for the
structure of compounds.
D. Lefe
`
vre-Groboillot et al. Substrate specificity of iNOS
FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS 3177
binding of an N-hydroxyguanidine moiety in the iNOS
heme pocket thus roughly involves the same binding
energy as binding of the guanidine moiety, and the
equilibrium constants are mainly determined by the
alkyl or aryl substituents of the compounds.
The crystal structures of mouse iNOS
oxy
–l-Arg and
bovine eNOS
oxy
–l-Arg complexes [33,34] have shown
that the guanidine moiety of l-Arg makes a salt bridge
with the side chain of a conserved glutamate residue
(E371 in mouse iNOS), and H-bonds with a backbone
carbonyl oxygen atom (W366 in mouse iNOS). The

crystal structures of iNOS
oxy
–NOHA complexes
showed identical positioning of the N-hydroxyguani-
dine moiety of NOHA, with additional contacts
between the N-hydroxyguanidine hydroxy group and
the amide nitrogen of a conserved glycine (G365 in
mouse iNOS) [18,19,21]. The crystal structures of
eNOS
oxy
–ClPhNOHG and nNOS
oxy
–BuNOHG com-
plexes showed similar positionings of the N-hydroxy-
guanidine moiety of ClPhNOHG and BuNOHG
involving: (a) a salt bridge between a glutamate side
chain and the two nonhydroxylated nitrogens of the
N-hydroxyguanidine, and (b) a nonbonded contact
between the hydroxy group and a glycine nitrogen
[17,19]. It thus seems that in such a positioning, chan-
ging the N-hydroxyguanidine moiety into a guanidine
moiety does not strongly modify the energy of binding
to iNOS. Preliminary data show that this is also true
for nNOS (D. Lefe
`
vre-Groboillot, unpublished data).
The structure–affinity relationship for alkylguani-
dines and N-alkyl-N¢-hydroxyguanidines bearing non-
functionalized linear alkyl chains (ProGua, BuGua,
PentylGua, HexGua, ProNOHG, BuNOHG, Pentyl-

NOHG and HexNOHG) showed that the binding
affinity is maximal for the compounds bearing a butyl
chain, i.e. BuNOHG and BuGua, with K
d
values
around 20 lm (Table 1). Compounds bearing a
n-pentyl chain (PentylNOHG and PentylGua) still bind
well to the iNOS active site (K
d
around 100 lm) but
compounds bearing an n-hexyl chain (HexNOHG and
HexGua) interact with iNOS with low affinities
(K
d
> 150 lm). The crystal structure of the nNOS
oxy
–
BuNOHG complex showed that the butyl chain of
BuNOHG interacts with the side chain of a conserved
valine residue (V567), a conserved proline (P565) and
the amide moiety of a conserved glutamine (Q478)
[19]. Because BuNOHG has previously been reported
to be similarly efficiently oxidized into NO by both
iNOS and nNOS [4,6,9], the binding modes of this
compound for the two isoforms is expected to be sim-
ilar. We measured the K
d
for the binding of BuNOHG
to nNOS
oxy

and found a somewhat higher value of
100 lm (data not shown), suggesting that the binding
of BuNOHG to iNOS is favoured slightly over its
binding to nNOS. The K
m
value for the oxidation of
BuNOHG by iNOS and nNOS were also found to fol-
low the order iNOS < nNOS [4,6,9]. It appears that
the hydrophobic contacts such as those observed
between the butyl chain and the protein in the
nNOS
oxy
–BuNOHG crystal structure are sufficient to
allow compounds BuNOHG and BuGua to bind to
the active site of iNOS and nNOS with K
d
values in
the 20–100 lm range. Interestingly, the crystal struc-
ture of the nNOS
oxy
–BuNOHG complex revealed that
upon binding of BuNOHG the side chain of residue
Q257 has to shift from its position observed in other
complexes (including the nNOS
oxy
–NOHA complex),
in order to accommodate the terminal methyl group
of BuNOHG [19]. This is in agreement with the
fact that longer compounds such as PentylNOHG or
HexNOHG showed lower affinities for the iNOS active

site, because their binding may require an important
reorganization of the protein environment.
The introduction of both an amino function and a
carboxylate function on the terminal methyl group of
BuGua and BuNOHG in a configuration leading to
the natural substrates, l-Arg and NOHA, led to a
10-fold decrease in the observed equilibrium constants
(Table 1). The positioning of the a-amino acid moiety
of NOHA or l-Arg analogues appears to be critical
for binding to iNOS
oxy
. Indeed, the K
d
values for
l-Arg and NOHA were found to be in the 2–4 lm
range, in agreement with previously reported data
[10,11,13], whereas those for the longer analogues
homo-l-Arg and homo-NOHA were found to be in
the 10–20 lm range and that for the shorter analogue
Nor-l-Arg was found to be > 300 lm. This indicates
that the alkyl chains of l-Arg (or NOHA) optimally
position their guanidine (or N-hydroxyguanidine) and
a-amino acid moieties relative to each other in the
Table 3. Apparent association and dissociation rate constants (k
on
app
and k
off
app
) for the binding of guanidines and N-hydroxyguanidines

to iNOS
oxy
in the presence of 400 lM ImH. See Table 1 for the
structure of compounds. The rates of spectral transitions (Fig. 2)
were fitted vs. the postmixing concentrations of the studied guani-
dine or N-hydroxyguanidine with a linear function, as shown on
Fig. 3. k
off
app
was defined as the y intercept and k
on
app
as the slope.
Compounds k
on
app
(s
)1
ÆM
)1
)k
off
app
(s
)1
)
L-Arg 120 000 ± 30 000 5 ± 2
NOHA 12 000 ± 1500 0.1 ± 0.06
BuGua 18 000 ± 3000 50 ± 10
BuNOHG 3600 ± 200 2.0 ± 0.4

TFBGua 4700 ± 800 30 ± 4
TFBNOHG 1000 ± 200 0.5 ± 0.4
Substrate specificity of iNOS D. Lefe
`
vre-Groboillot et al.
3178 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS
NOS active site. This also indicates that adding one
methylene in the l-Arg chain does not impede efficient
binding, whereas removal of one methylene group is
detrimental for the interaction between the protein and
the substrate. The crystal structure of the eNOS
oxy
–
homo-l-Arg complex (PDB entry 1DM7, C.S. Raman
et al. 1999) actually showed that homo-l-Arg interacts
with the active site of eNOS
oxy
in a manner similar to
l-Arg, involving roughly identical positionings of the
guanidine and a-amino acid moieties. However, the
longer alkyl chain of homo-l-Arg forms a small bulge
between the two heme propionates in contact with the
heme and the side chain of a conserved valine (V338).
The decrease of the affinity of homo-l-Arg and homo-
NOHA compared with l-Arg and NOHA (Table 1)
could be linked to this unfavourable bulging confor-
mation of the alkyl chain of homo-l-Arg.
The simultaneous presence of both the a-amino and
a-carboxylate moieties appears to be necessary because
the NOHA analogue bearing only an a-amino moiety

(NOHAgma) interacted with iNOS
oxy
with an affinity
(K
d
 250 lm) much lower than that found for
BuNOHG, and the NOHA analogue bearing only an
a-carboxylate moiety (NOHGPA) did not interact with
iNOS (K
d
> 500 lm). This suggests that the a-amino
and a-carboxylate groups cooperate to provide favour-
able binding enthalpy for the formation of the complex
between NOHA and the protein. The crystal structures
of l-Arg or NOHA in NOS active sites actually
showed that the a-amino acid moiety of these com-
pounds interacts with the protein via an H-bond net-
work involving one or two water molecules that links
the a-amino and a-carboxylate moieties one to each
other, and to protein residues [18,19,21].
Finally, six compounds bearing an aryl moiety,
namely FPhNOHG, TolNOHG, ClPhNOHG,
BzNOHG, FPhGua and ClPhGua (Table 1), exhibited
K
d
values > 250 lm. The crystal structure of the
eNOS
oxy
–ClPhNOHG complex showed that the phenyl
ring of ClPhNOHG is in close contact with the side

chain of the conserved valine, V338, and with a pro-
pionate of the heme [17]. The chlorine atom is also
involved in nonbonded contacts with the conserved
methionine M341. Because ClPhNOHG was previously
reported to be an iNOS-specific substrate [5,6], we also
measured the equilibrium constants for the binding of
these compounds to eNOS and nNOS (data not
shown). ClPhNOHG actually displayed significantly
higher affinities for the two constitutive isoforms than
for iNOS: the K
d
values for its binding to nNOS
oxy
and eNOS
oxy
were found to be around 50 and 95 lm,
respectively, whereas that for its binding to iNOS
oxy
was
found to be close to 350 lm. Similar higher affinities
for nNOS
oxy
compared with iNOS
oxy
were also
observed for TolNOHG, which is also an iNOS
specific substrate [5,6], and for FPhNOHG, which is a
substrate highly selective for iNOS [5]. These results
obtained with guanidines or hydroxyguanidines bear-
ing an N-aryl moiety recall the well-documented selec-

tivity of N-arylamidines for inhibition of nNOS vs.
iNOS [35].
Relationship between the structure of N-hydroxy-
guanidines, their affinity for iNOS
oxy
and their
oxidation by iNOS with formation of NO
Previous data showed that a very large number of
monosubstituted N-hydroxyguanidines R-NH-C(¼NOH)-
NH
2
bearing an alkyl or aryl substituent R, neither
too small nor too bulky, led to the detectable production
of NO in the presence of iNOS [4–7,9,29]. Formation of
NO from the oxidation of an N-hydroxyguanidine by
iNOS is thus not specific to NOHA and can occur
with many N-hydroxyguanidines.
The rates of NO formation from the oxidation of a
great number of N-alkyl- and N-aryl-N¢-hydroxyguan-
idines by iNOS were found to be highly dependent
on their structure [4–7,9]. However, the k
cat
values
found for NO formation upon iNOS-catalysed oxida-
tion of the seven N-hydroxyguanidines mentioned in
Table 2 varied by less than a factor 2, whereas their
K
d
values varied by a factor 200 (Table 1). It thus
appears that the k

cat
of NO formation is not simply
related to the affinity of the substrate for iNOS. For
instance, the k
cat
of NO formation from FPhNOHG
oxidation is 83% of that found for NOHA, whereas
the K
d
of this substrate is 130 times higher than that
of NOHA.
As mentioned above and shown in Table 2, very dif-
ferent N-hydroxyguanidines leading to similar k
cat
val-
ues (58–100% of that found for NOHA) showed
widespread K
m
⁄ K
d
ratios (from  1to 20). This vari-
ation may be related to that in k
off
, as expected by
considering the relation K
m
⁄ K
d
¼ 1+k
cat

⁄ k
off
. From
a qualitative point of view, this is in agreement with
the variation in k
off
app
for NOHA (0.1 s
)1
),
TFBNOHG (0.5 s
)1
) and BuNOHG (2 s
)1
) (Table 3),
which is inversely related to that of K
m
⁄ K
d
for these
N-hydroxyguanidines (19.3, 8.1 and 3.0 for NOHA,
TFBNOHG and BuNOHG, respectively). Rigorous
and quantitative correlations could not be done imme-
diately, as K
m
and k
cat
, K
app
, k

on
app
and k
off
app
values
were measured under different conditions for experi-
mental reasons (different temperatures or the presence
of imidazole). However, our data provide a first gen-
eral basis to understand the structural factors that are
D. Lefe
`
vre-Groboillot et al. Substrate specificity of iNOS
FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS 3179
necessary for guanidines and N-hydroxyguanidines to
efficiently bind to iNOS.
Criteria for the formation of NO from the
oxidation of a guanidine by iNOS
Contrary to what is observed for the N-hydroxyguani-
dines, not all the guanidines that bind to iNOS lead to
the production of NO [5–9]. For example, all arylguani-
dines assayed to date, among them FPhGua and
TolGua, have failed to lead to any detectable amount
of NO, although their affinity for iNOS is not lower than
that for the corresponding N-aryl-N¢-hydroxyguani-
dines that lead to k
cat
values of formation of NO as high
as 83 and 69% that obtained for NOHA (Table 2).
As in the case of the N-hydroxyguanidines, the k

cat
values of NO formation from the oxidation of guani-
dines do not appear to be linked to the affinity of the
compounds for iNOS. For example, compound TFB-
Gua led to a k
cat
value of NO formation of 55% that
obtained with l-Arg (Table 2) even though it bound to
iNOS with a K
app
value 50 times higher (Table 1).
Interestingly, the k
cat
of production of NO by oxida-
tion of the studied guanidines followed the same order
l-Arg > homo-l-Arg  TFBGua > PentylGua > BuGua
as that found for the K
m
⁄ K
d
ratio of the correspond-
ing N-hydroxyguanidines: NOHA > homo-NOHA 
TFBNOHG > PentylNOHG > BuNOHG (Table 2).
This suggests that the variations in the k
cat
values
found for NO formation from the guanidines could
be related to those of the k
off
of the corresponding

N-hydroxyguanidines. Accordingly, the order l-Arg >
TFBGua > BuGua found for the k
cat
of production of
NO from oxidation of these guanidines corresponds
well to the order NOHA > TFBNOHG > BuNOHG
found for 1 ⁄ k
off
app
of the corresponding N-hydroxygu-
anidines (Table 3). These results may suggest that a key
factor for a guanidine to lead to NO formation in the
presence of iNOS could be the ability of the corres-
ponding N-hydroxyguanidine to bind to the active site
without being released before being further oxidized.
They could explain why the compound TFBGua is so
far the best non a-amino acid NO precursor upon oxi-
dation by iNOS (Table 2), because the k
off
app
value of
the corresponding N -hydroxyguanidine TFBNOHG is
particularly low (Table 3). In a more general manner,
our data suggest that changes in the NOS–substrate
complex structure (changes of the substrate structure,
but also mutation or post-translational modification of
the protein) could likely lead to a shift of the activity of
NOS from NO synthesis to N-hydroxyguanidine syn-
thesis. Further investigations are currently underway to
test these hypotheses. Our results also suggest that the

rational design of guanidines as new NO donors upon
in situ oxidation by NOSs should take into account
both thermodynamic and kinetic characteristics of the
interaction of the protein not only with the guanidine,
but also with the corresponding N-hydroxyguanidine.
Experimental procedures
Chemicals and reagents
BH
4
was purchased from Alexis Biochemicals (COGER,
Paris, France) and l-Arg and homo-l-Arg were from Sigma
(Saint-Quentin Fallavies, France). Alkylguanidines were
obtained by reaction of the corresponding amine with
pyrazole-1-carboxamidine hydrochloride in the presence of
diisopropylethylamine following a previously described pro-
tocol [8]. Arylguanidines were obtained by reaction of the
amine with N,N¢-bis(tert -butyloxycarbonyl)pyrazole-1-carb-
oxamidine followed by acidic deprotection as previously des-
cribed [8]. N-Hydroxyguanidines were obtained, as well as
small amounts of the corresponding ureas, by the addition of
hydroxylamine hydrochloride to intermediate cyanamides in
anhydrous ethanol [4–7]. Cyanamides were obtained from
the amines by addition of BrCN in methanol containing
anhydrous sodium acetate [4,5]. The physicochemical charac-
teristics of N-(n-propyl)-N¢-hydroxyguanidine, N-(n-butyl)-
N¢-hydroxyguanidine, N-(n-pentyl)-N¢-hydroxyguanidine,
N-(n-hexyl)-N¢-hydroxyguanidine, N-(4-fluorophenyl)-N¢-
hydroxyguanidine, N-(4-chlorophenyl)-N¢-hydroxyguanidine,
N-(4-methylphenyl)-N¢-hydroxyguanidine, N-benzyl-N¢-hydro-
xyguanidine, n-butylguanidine, 4,4,4-trifluorobutylguanidine

and (4-methyl)phenylguanidine have been published previ-
ously [5,6,8]. NOHA and homo-NOHA were synthesized as
previously reported [29]. Other chemicals were from Aldrich
(Saint-Quentin Fallavies, France), Sigma or Across (Noisy le
Grand, France) unless otherwise indicated and were of the
highest purity commercially available.
Protein preparation
iNOS
oxy
(amino acids 1–498) containing a six-histidine tag
at its C-terminus was overexpressed in Escherichia coli and
purified in the absence of BH
4
as described previously [36]. It
was estimated to be more than 95% pure by SDS ⁄ PAGE.
The enzyme concentration was determined from the 444 nm
absorbance of its ferrous–CO complex by using an extinction
coefficient of 76 mm
)1
.cm
)1
[37]. iNOS
oxy
(heme concentra-
tion 1 lm) was incubated overnight with 25 lm BH
4
and
1mm dithiothreitol at 4 °C before use.
Assessment of NO formation
Initial rates of NO formation were determined at 37 °C

using the classical spectrophotometric oxyhemoglobin assay
Substrate specificity of iNOS D. Lefe
`
vre-Groboillot et al.
3180 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS
for NO [38] under conditions described previously [4,5,8].
In some assays, the level of NO formation was measured
by electron paramagnetic resonance spectroscopy following
the formation of the paramagnetic ferrous mononitrosyl
diethyldithiocarbamate complex under previously described
conditions [8,39].
Determination of the dissociation constants for
the complexes between BH
4
-containing iNOS
oxy
and guanidines or N-hydroxyguanidines
Studies were carried out at room temperature in an
UVIKON 942 spectrophotometer (Kontron Biotek), in a
1-cm path length cuvettes (150 lL total volume). Each
cuvette contained 1 lm iNOS
oxy
in 50 mm Hepes buffer,
pH 7.4, in the presence of 25 lm BH
4
and 1 mm dithio-
threitol. The amplitude of the observed difference spectra
DA(k
max
) k

min
) induced by the progressive addition of
ImH to an iNOS
oxy
solution was fitted (a) vs. [ImH] with a
hyperbolic function, and (b) 1 ⁄DA(k
max
) k
min
) vs. 1 ⁄ [ImH]
with a linear function. The two fits gave dissociation con-
stants (K
ImH
) for the NOS–ImH complex that were less dif-
ferent than the values obtained from two identical
experiments, indicating that a single binding site model
satisfactorily accounted for the binding of ImH to iNOS
oxy
.
A dissociation constant value of K
ImH
¼ 52±5lm was
found, very close to the values reported by others [11–13].
Maximum amplitude of the difference spectrum was
68±4mm
)1
cm
)1
.
The study of the binding of guanidines and N-hydroxy-

guanidines to 1 lm iNOS
oxy
was performed in the presence
of 400 lm ImH, a situation that allows the monitoring by
UV ⁄ Vis difference spectroscopy of the formation of a com-
plex between the protein and compounds which bind to the
substrate binding site [11,12,14,22,25,36,40–42]. The studied
guanidine or N-hydroxyguanidine (dissolved in buffer) was
added stepwise to the sample cuvette, and equivalent vol-
umes of buffer were added to the reference cuvette. All
experiments were carried out under conditions where the
concentration of bound ligand was much smaller than the
total concentration of ligand. In a first calcula-
tion, the amplitude of the observed difference spectra
DA(k
max
) k
min
) was fitted vs. the guanidine or N-hydroxy-
guanidine concentration [ligand] with a hyperbolic function.
In a second step, 1 ⁄DA(k
max
) k
min
) was fitted vs. 1 ⁄ [lig-
and] with a linear function. The two fits always gave appar-
ent equilibrium constants (K
app
) for the NOS–ligand
complexes that were less different than the values obtained

from two identical experiments. This indicated that a single
binding site model satisfactorily accounted for the observed
spectral changes. The maximum amplitude of the difference
spectra was 56 ± 6 mm
)1
cm
)1
and did not vary signifi-
cantly with the structure of the guanidine or N-hydroxy-
guanidine.
Rapid kinetic studies of the binding
of guanidines and N-hydroxyguanidines to iNOS
oxy
Experiments were performed at 15 °C using a stopped-flow
instrument equipped with a rapid-scanning diode array
detector (Hi-Tech MG 6000) and following a protocol pre-
viously described [23]. A solution of iNOS
oxy
containing
400 lm ImH was mixed with a solution of guanidine (or
N-hydroxyguanidine) also containing 400 lm ImH. Post-
mixing heme concentration was 5 lm. The reaction was
monitored by following the absorbance at 430 and 392 nm.
Variations of the absorbances at these two wavelengths
were fitted with monoexponential functions. Observed rate
constants k
obs
were obtained by averaging the values of the
rate constants measured at the two wavelengths over 5–10
shots.

Acknowledgements
The authors thank Sylvie Dijols (UMR 8601 CNRS,
Paris) for the synthesis of the guanidines and
N-hydroxyguanidines used in this study. DL-G thanks
Zhi-Qiang Wang, Chin-Chuan Wei and Koustubh
Panda (Cleveland Clinic Foundation) for their help
with the stopped -flow ex perimen ts, and J eroˆ me San tolini
(CEA Saclay, France) for his help in the preparation
of proteins and helpful discussions. This work was
supported by the French Ministry of Research (fellow-
ship grant to DL-G), and by National Institutes of
Health (grant CA53914 to DJS).
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