Re-examination of the formation of dinitrosyl-iron complexes during reaction of S-nitrosothiols with Fe(II)

Article abstract of DOI:10.1016/S0020-1693(01)00402-9

The reaction of S-nitrosothiol compounds with ferrous ions in solution has been investigated and the generated dinitrosyl-iron complexes have been characterized. During the reaction of S-nitrosocysteamine with Fe(II) in water solution in the presence of a twofold excess (with respect to iron) of cysteamine hydrochloride (CSH), an EPR-silent dinuclear iron complex (complex A of formula [Fe₂(RS)₂(NO)₄]) was formed as the major species and was characterized by FAB MS⁺, UV-Vis, NMR and IR spectroscopies. In the presence of a large excess of CSH (CSH/Fe(II)=20:1), a green paramagnetic mononuclear complex (complex B of formula [Fe(RS)₂(NO)₂]^-) was formed. From EPR and UV-Vis data, and also on the basis of the few crystallographic structures known for similar complexes, complex B is proposed to display a distorted tetrahedral geometry (C_2v), approaching a trigonal bipyramid with a missing ligand, with the unpaired electron mainly localized on the d_z2 orbital of the iron characterized by a d⁹ electronic configuration.

Full text of DOI:10.1016/S0020-1693(01)00402-9

www.elsevier.nl/locate/ica
Inorganica Chimica Acta 318 (2001) 1–7
Re-examination of the formation of dinitrosyl–iron complexes
during reaction of S-nitrosothiols with Fe(II)
Simona Costanzo a, Stephane Me´nage b, Roberto Purrello a, Raffaele P. Bonomo a,*1,
Marc Fontecave b,*2
a Dipartimento di Scienze Chimiche, Uni6ersita` di Catania, Viale A. Doria 6, 95125 Catania, Italy
b Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, CEA, Grenoble, DBMS/CB, 17 A6enue des Martyrs,
F-38054 Grenoble Cedex 9, France
Received 23 September 2000; accepted 31 October 2000
Abstract
The reaction of S-nitrosothiol compounds with ferrous ions in solution has been investigated and the generated dinitrosyl–iron
complexes have been characterized. During the reaction of S-nitrosocysteamine with Fe(II) in water solution in the presence of
a twofold excess (with respect to iron) of cysteamine hydrochloride (CSH), an EPR-silent dinuclear iron complex (complex A of
formula [Fe2(RS)2(NO)4]) was formed as the major species and was characterized by FAB MS+, UV–Vis, NMR and IR
spectroscopies. In the presence of a large excess of CSH (CSH/Fe(II)=20:1), a green paramagnetic mononuclear complex
(complex B of formula [Fe(RS)2(NO)2]−) was formed. From EPR and UV–Vis data, and also on the basis of the few
crystallographic structures known for similar complexes, complex B is proposed to display a distorted tetrahedral geometry (C26),
approaching a trigonal bipyramid with a missing ligand, with the unpaired electron mainly localized on the dz orbital of the iron
2
characterized by a d9 electronic configuration. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Iron complexes; Nitrosyl complexes; Nitrosothiol complexes
1. Introduction
the function of transport and storage of NO [16].
RSNOs formed either by low molecular weight thiols or
thiol-containing proteins have been detected in vivo
[17,18] and were shown to elicit physiological and bio-
chemical responses similar to those elicited by NO
[19–21]. The DNICs are formed during the reaction of
NO with Fe(II) in the presence of low molecular weight
thiols, aminoacids, peptides or proteins (through their
cysteine and histidine residues). Such complexes were
first observed in biological systems about 30 years ago
by their characteristic EPR signal [22–24], but their
physiological significance is still a matter of investiga-
tion. They have been proposed as endothelium derived
relaxing factor (EDRF) candidates [25] and shown to
exhibit antiplatelet [26], vasorelaxant [27] and blood
pressure lowering activity [28]. Whereas the DNICs
bound to protein thiols occur only as paramagnetic
forms, DNICs with low molecular weight thiols are
known to exist in two forms, paramagnetic and dia-
magnetic, which are interconvertible [29].
Nitric oxide (NO) is involved in a large number of
physiological processes including neurotransmission [1],
immune system regulation [2,3], smooth muscle relax-
ation [4–6] and platelet inhibition [7,8]. The high reac-
tivity of NO with regard to molecular oxygen [9],
superoxide anion [10], and heme [11] as well as non-
heme iron [12] and the ready availability of these
reactants in the plasma and cellular environment sug-
gest that NO is stabilized in vivo by incorporation into
a carrier molecule that prolongs its lifetime and pre-
serves its biological activity. In these regards, S-nitro-
sothiols (RSNOs) [14,13] and dinitrosyl–iron
complexes (DNICs) [15], have been claimed to subserve
1 *Corresponding author. Tel.: +39-095-738 5093; fax: +39-095-
58 0138; rbonomo@dipchi.unict.it
2 *Corresponding author. Tel.: +33-4-76-88 9103; fax: +33-4-76-
88 9124; mfontecave@cea.fr
0020-1693/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S0020-1693(01)00402-9

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S. Costanzo et al. / Inorganica Chimica Acta 318 (2001) 1–7
Metal ions have been shown to promote the decom-
cryostat for low temperature operations. High field
EPR experiments at 285 GHz were performed on aceto-
nitrile solutions of complex B at the temperature of 5 K
on a home-built spectrometer [37,38]. The concentra-
tion of complex B was measured by the double integra-
tion of the EPR signal using a solution of TEMPO of
known concentration as a standard. Simulations of the
spin hamiltonian parameters were carried out using the
Bruker Simphonia program for the room temperature
spectra and a program written by Weihe [39] for the
frozen solution spectra. In the case of high field EPR
spectra, g values were corrected by using the giso value
obtained by the X-band experiments. Instrumental set-
tings are reported in each figure caption associated with
the corresponding experimental EPR spectrum.
Absorption spectra were recorded both on a Varian
Cary 1-Bio double beam spectrophotometer and on a
Hewlett–Packard HP 8452 A diode array spectro-
photometer.
position of RSNOs [30–33]. Recently we showed that
the iron chelators greatly stabilized RSNOs in solution,
suggesting that iron ions, as well, were able to react
with RSNOs [31]. In this work, we report the results of
a study on the solution chemistry of reaction mixtures
of S-nitrosocysteamine (CSNO) and Fe(II) in water
and show that the DNICs, characterized by a variety of
spectroscopic methods, are formed spontaneously, in
agreement with the preliminary reports [34,35]. This
opens the possibility that NO can be transferred from
thiols to iron also in biological systems, thus defining
RSNOs and DNICs as the two major interconvertible
stable biological forms of NO.
2. Experimental
2.1. Materials and methods
1H NMR spectrum was obtained in D2O from a
Varian Inova operating at 500 MHz.
Cysteamine hydrochloride and HEPES were pur-
chased from Sigma; FeSO4·7H2O (99.999%) and
2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) were
purchased from Aldrich, tert-butyl nitrite (90%)
(tBuONO) was purchased from Fluka. 1-Amino-2-
methylpropane-2-thiol has been synthesized according
to Ref. [31]. Milli-Q ultrapure water has been used in
all experiments.
IR spectra were obtained from on a Bruker Vector 22
FTIR spectrophotometer using KBr pellets.
FAB MS+ spectral analyses were performed on
Kratos MS50 spectrometer equipped with a cesium ion
gun operating at 20 kV and interfaced with a MSS data
system. 3-Nitrobenzylalcohol has been used as a matrix.
Oxyhemoglobin (HbO2) was prepared from red
blood cells according to the procedure described in Ref.
[36]. Deoxyhemoglobin (deoxyHb) was obtained, after
removing most of the oxygen in solution to avoid side
reactions, by adding a slight molar excess of sodium
dithionite to a solution of HbO2 in Tris 20 mM pH 7.5.
CSNO was synthesized in a degassed water solution
by reacting 1 M CSH with 1.1 equiv. of tBuONO for
15 min at room temperature according to the method
described in Ref. [31]. Its concentration was evaluated
by measuring the intensity of the absorption band at
333 nm (m=793) [31]. Solutions are prepared, kept on
ice in the dark and used on the same day. The reaction
of CSNO with ferrous iron was investigated during
incubation of 2 mM CSNO with 1 mM FeSO4·7H2O
(prepared in deaerated water) in the presence of a
twofold and a twentyfold excess of CSH in 0.1 M
HEPES buffer pH 7.8 at room temperature. The same
results were obtained when the reaction was carried out
in water adjusting the pH at 7.8 with NaOH.
3. Results
The reaction of CSNO with a ferrous salt in water at
pH 7.8 was studied and it was found that, under these
conditions, two different complexes were obtained, de-
pending on the excess of CSH present in the reaction
mixture. At low excess (twofold with respect to iron) a
yellow diamagnetic complex (A) was obtained, whereas
at large excess (at least twentyfold with respect to Fe),
a green paramagnetic complex (B) was the only product
of the reaction.
3.1. Spectroscopic properties of complex A
The optical spectrum (250–900 nm) of a reaction
mixture containing CSNO, ferrous iron and CSH in a
2:1:2 ratio displayed two bands at 305 and 362 nm, a
shoulder at 440 nm and a much less intense band at 755
nm (Fig. 1). Complex A could be precipitated, in the
form of a light brown powder, when a concentrated (40
mM Fe) solution of the complex in water was adjusted
to pH 7.8 by dropwise addition of NaOH. Dissolution
of that powder in water gave the same spectrum (Fig. 1)
showing that the great majority of the iron resided in
complex A. Unfortunately, it was not possible to fur-
ther purify the complex for getting crystals.
2.2. Spectroscopic measurements
Room temperature and frozen solution EPR spectra
were performed on a Bruker EMX spectrometer
equipped with an X-band bridge ER 041 XG and on a
Bruker ER 200 D X-band spectrometer, driven by the
ESP 3220 data system, both equipped with a Bruker

S. Costanzo et al. / Inorganica Chimica Acta 318 (2001) 1–7
3
The FAB MS+ spectrum showed a M+1 peak at
385 m/z (Fig. 2) which clearly indicated that complex A
was a dinuclear Fe complex, with two cysteamine moi-
eties and four NO groups, thus with the stoichiometry
Fe2(CS)2(NO)4. The presence of NO groups is also
supported by the appearance of two peaks at 355
(M+1−NO) and at 324 (M+1−H−2NO), corre-
sponding to two fragments of the molecule lacking one
and two NO groups, respectively. The peak at 284
(M+1−H−NO−SC) could be due to the loss of a
NO group and a cysteamine moiety from the molecular
ion, whereas the major peak at 153 m/z can be tenta-
tively assigned to the cysteamine disulfide.
The IR spectrum of complex A showed two bands at
1770 and 1730 cm−1, corresponding to the symmetric
and asymmetric stretching vibrations of the NO lig-
ands, respectively. These values, which are close to
those found by Butler et al. [40,41] for
Fe2(SBut)2(NO)4, do not allow to unambiguously con-
clude whether nitrosyl ligands should be regarded as
NO+ rather than neutral or negatively charged NO
ligands [42,43]. The presence of NO in the complex was
further confirmed by the observation that the reaction
of complex A with deoxyhemoglobin generated HbNO,
the ferrous hemoglobin–NO complex, characterized at
77 K by the typical rhombic EPR signal with a three
line hyperfine splitting in the parallel (gz) region (data
not shown).
1
The H NMR spectrum of complex A in D2O clearly
Fig. 1. UV–Vis spectrum of complex A in HEPES buffer (0.1 M, pH
7.8).
showed that the cysteamine moieties were bound to the
Fe center: two triplets at 3.24 and 3.34 ppm are as-
signed to the two methylene groups of cysteamine
which are shifted downfield with respect to the free
1
thiol (data not shown). The H NMR spectrum also
reveals the presence of a unique impurity identified as
the cysteamine disulfide.
3.2. Spectroscopic properties of complex B
Complex B is a mononuclear S=1/2 paramagnetic
species. Quantification of its EPR signal, using TEMPO
as a standard, demonstrated that all the iron present in
the reaction mixture was incorporated in the paramag-
netic complex upon reaction of CSNO with Fe(II) in
the presence of a large excess of CSH (twentyfold with
respect to iron). Complex B could also be generated
from complex A during reaction with 20 equiv. of CSH,
as shown from the appearance of the characteristic
EPR spectrum at room temperature. Under anaerobic
conditions complex B was stable but in the presence of
air or hydrogen peroxide it decomposed to generate
complex A, as shown by the changes in the light
absorption spectrum and from the decay of the EPR
signal.
Fig. 2. FAB MS+ spectrum of complex A in 3-nitrobenzylalcohol.
The UV–Vis spectrum (250–900 nm) of a water
solution of complex B (Fig. 3) displayed an absorption
band at 392 nm (m:3580 M−1 cm−1) and two dꢀd
bands at 603 (m:299 M−1 cm−1) and 772 nm (m:312
M
−1 cm−1), responsible for the green color of complex
B solutions. The room temperature X-band EPR spec-
trum (Fig. 4) showed a signal centered at giso=2.030
with a 13-line superhyperfine structure assigned to the
interaction of the unpaired electron, primarily localized
Fig. 3. UV–Vis spectrum of complex B 1 mM in HEPES buffer (0.1
M, pH 7.8).

4
S. Costanzo et al. / Inorganica Chimica Acta 318 (2001) 1–7
had only five lines (Fig. 5), reflecting the lack of the
hydrogen atoms on the methylene group a to the sulfur.
In fact, methyl groups are expected to exhibit very
small and probably irresolvable hyperfine interaction.
By lowering the temperature the isotropic signal lost its
shf structure but persisted down to 255 K, although the
solution at that temperature was already frozen (Fig.
6). Further decreasing the temperature, an anisotropic,
apparently axial, signal appeared at X-band (Fig. 6)
and remained unaltered down to 77 K with gÞ=2.042
and gꢀ=2.014, in agreement with the values reported
for similar complexes [45]. At high magnetic fields (285
GHz) the EPR signal (Fig. 7) revealed, in fact, that the
complex had a rhombic symmetry with gz=2.015, gx=
2.033, gy=2.041, thus explaining the broadness of the
X-band low field component at 160 K.
Fig. 4. EPR spectrum at room temperature of complex B. The
spectrum has been recorded on a Bruker EMX spectrometer and the
instrumental parameters are: microwave frequency, 9.66 GHz; mi-
crowave power, 10 mW; receiver gain, 6.32×104; modulation fre-
quency, 100 kHz; modulation amplitude, 0.2 G; and time constant,
4. Discussion
These iron complex species have been previously
reported by Vanin et al. [35] but the products of the
reaction were not fully characterized. Here, the use of a
variety of spectroscopic methods allowed us to identify
the complex species in solution coming out from the
reaction (UV–Vis as well as mass spectra and high filed
EPR spectra are shown for the first time).
5.120 ms. The calculated spin hamiltonian parameters are: giso
2.030, AN=2.35, and AH=1.21.
=
Fig. 5. EPR spectrum at room temperature of the dinitrosyl–iron
complex with 1-amino-2-methylpropane-2-thiol. The spectrum has
been recorded on a Bruker EMX spectrometer and the instrumental
parameters are: microwave frequency, 9.66 GHz; microwave power,
10 mW; receiver gain, 6.32×104; modulation frequency, 100 kHz;
modulation amplitude, 0.2 G; and time constant, 5.120 ms. The
calculated parameters are: giso=2.030 and AN=2.47.
on the Fe atom, with the nitrogen atoms of two NO
groups and with the four protons of the methylene
groups a to the sulfur atoms of the cysteamine moiety,
in agreement with the simulated spectrum using AN=
2.35 G and AH=1.21 G [44]. Accordingly, when CSH
was replaced by 1-amino-2-methyl propane-2-thiol dur-
ing the synthesis of complex B, the resulting EPR signal
Fig. 6. EPR spectrum at variable temperature of complex B. The
spectra have been recorded on a Bruker ER 200 D spectrometer and
the instrumental parameters are: microwave frequency, 9.41 GHz;
microwave power, 10 mW; receiver gain, 1.00×103; modulation
frequency, 100 kHz; modulation amplitude, 2 G; and time constant
327 ms.

S. Costanzo et al. / Inorganica Chimica Acta 318 (2001) 1–7
5
describe the electronic features of the [Fe(NO)2RS2]−
fragment, whose geometry, as suggested by Briar et al.
[45], is better described as a trigonal bipyramid with a
missing ligand rather than a simple tetrahedron. In an
iron d9 configuration the HOMO of the complex is the
dz2 orbital, which does not point along the directions of
the metal–ligand bonds, and, therefore, low superhy-
perfine nitrogen constants were observed. The odd elec-
tron would have small capacity of transferring spin
density to the nitrogen nuclei of the donors in the
equatorial plane and only a dipolar mechanism should
operate. The 13 line EPR signal could be simulated
taking into account 4 equiv. hydrogen atoms of methyl-
ene groups and 2 equiv. nitrogen atoms of NO groups
with quite similar Aiso values (1.21–2.35 G). It is not to
Fig. 7. High field (285 GHz) EPR spectrum of complex B. The
spectrum has been recorded on a home-built spectrometer at the
temperature of 5 K and the instrumental parameters are: field modu-
lation frequency, 935 Hz; scan rate, 0.5 mT s−1; time constant, 300
ms; and modulation amplitude, 8.55 mA.
exclude that the dz ground state could receive contri-
2
bution from the closest levels, whose energies are func-
tions of variations in the NꢀFeꢀN or SꢀFeꢀS angles
[47].
Upon freezing the solution, the shf structure in the
EPR spectrum disappeared and a reversed pattern
gÞ(or gx,y)\gꢀ was observed confirming the structural
One of the complexes, complex B, is a S=1/2 para-
magnetic species, as shown by EPR spectroscopy. The
EPR signal at room temperature is consistent, also on
the basis of the literature [44], with that of a mononu-
clear iron complex in which Fe is coordinated to two
NO molecules and the sulfur atoms of two cysteamine
moieties. The complex [Fe(RS)2(NO)2]− has 17 elec-
trons, and its iron-nitrosyl unit can be described with
the formal electron assignment [Fe(NO)2]9 according to
the Enemark–Feltham formalism which stresses the
well-known covalency and delocalization in the elec-
tronically amorphous Fe(NO)2 unit without committing
to a formal oxidation state on Fe [46]. On the other
hand, EPR and UV–Vis data, taken altogether, are
rather consistent with a tetrahedral geometry with a d9
configuration for the iron atom. Accordingly, the two
bands in the UV–Vis spectrum at 603 and 772 nm have
m values higher than those found in octahedral com-
plexes and typical of complexes with a tetrahedral
geometry, due to the ‘d–p mixing’.
The accurate EHMO calculations made by Sum-
merville and Hoffmann [47] on the [Fe(NO)2X2]− frag-
ment (X=Cl−, I−), assuming a C26 symmetry, showed
that the angle between p acceptor nitrosyl groups was
larger than that between the Cl− or I− donors. They
estimated the NꢀFeꢀN angle to be about 124°, a value
which is in agreement with crystallographic data re-
ported in the literature [48–50]. Changing their refer-
ence axis system, as considered by Lever [51], simply
rotating by 90° and thus interchanging y with z
(C26(III) in Lever’s formalism) in the zy plane, the
peculiarities mentioned above, i.e. dz as the electronic
2
ground state and the geometry of the complex better
described by a trigonal bipyramid with a missing lig-
and. Although in the case of a trigonal bipyramidal
geometry the gꢀ value is expected to be equal to the free
electron value,4 in C26 symmetry group both dx
2 and
2
−y
dz2 belong to a1; thus a contribution from d
2 to the
2
dz ground state could explain the greatxer−gyꢀ value
2
(2.014) experimentally obtained. Moreover, by using
the rough perturbation formulas given by Goodman
and Raynor [52], considering a spin–orbit coupling
constant of −201 cm−1 (obtained by extrapolating
spin–orbit coupling data for iron, reported by Figgis
[53]) and an orbital reduction factor of 0.7, a gÞ value
of 2.048 was obtained, assuming Z=13 000 cm−1 5
.
Similar EPR results were recently obtained on solu-
tions of [Fe(NO)2(1-methylimidazole)2]+ [50], the crys-
tal structure of which showed
pseudo-tetrahedral geometry.
a
complex
The optical band at 772 nm could, thus, be assigned
to the electronic transitions, 2B (d )’2A (d ) and
2
2
yz
1
z
2A (d )’2A (d ), b and a being the orbitals which
2
2
xy
1
2
2
can ‘mix’ via spizn–orbit coupling when considering the
magnetic field perpendicular to the z-axis. Hence, the
observed slight anisotropy in plane could be due to the
energy difference between a2 and b2. The optical band,
following energy sequence for the
d orbitals is
found:
2a (d )\b (d )]b (d )\a (d )\1a -
1 2 yz 1 xy 2 xz 1
z
2
4 When the magnetic field is parallel to the z-axis there should be
no contribution from mixing with other d orbitals via spin–orbit
coupling.
(dx −y2).3 This reference system is more suitable to
2
3 The small discrepancy with Lever’s results depends on the fact
that his reference axis system was generated starting from the cube
axis system.
5 Z Represents the average energy separation between the orbitals
which contains the free electron (2a1,dz2) and the lower energy
orbitals (b2, dxz and a2, dyz).

6
S. Costanzo et al. / Inorganica Chimica Acta 318 (2001) 1–7
thiol present in solution, either a paramagnetic or a
diamagnetic complex is formed and the two species are
interconvertible (Fig. 8). The diamagnetic species is a
dinuclear iron-dinitrosyl species in which each iron
atom is coordinated to two NO groups and the two
iron atoms are bridged by the sulfur atoms of two
cysteamine molecules. The paramagnetic species is a 17
electron mononuclear DNIC with a tetrahedral geome-
try (C26 symmetry group) distorted towards a trigonal
bipyramid with a missing axial ligand and a d9 configu-
ration of the iron atom, with the dz2 orbital being the
HOMO of the complex. The mechanism of NO transfer
from the thiol to Fe remains to be investigated.
Whether NO is first liberated in solution and rapidly
trapped by the metal ions or whether a more concerted
and controlled mechanism (RSNO+Fe“RSꢀFeꢀNO)
occurs has to be established.
Fig. 8. Proposed structures for complexes A and B.
however, is particularly broad and did not reveal any
splitting. The band at 603 nm has to be assigned to
2A1(dx
2)’2A (d ), which is the other electric
1
2
2
dipole allowed tranzsition in C26 symmetry. Moreover,
since, in such a dynamic situation, the vibronic mecha-
nism could operate to allow the otherwise forbidden
transition and b1 and b2 levels have comparable ener-
gies, it would be better to assign the electronic transi-
tion of the optical band at 772 nm to 2B2(dyz),
2B (d )’2A (d ) (see Fig. 8).
−y
Acknowledgements
This work was supported by C.N.R., Progetto Final-
izzato Biotecnologie. We thank Dr. C. Toia for per-
forming high-field EPR spectra.
2
1
xz
1
z
The second complex, complex A, is an EPR-silent
dinuclear Fe complex, as shown from mass spectrome-
try. Its proposed structure, similar to that of the
Roussin red salt [54], is shown in Fig. 8. Each iron is
coordinated to two NO groups and the two iron atoms
are bridged by the sulfur atoms of the two cysteamine
molecules. The fact that the complex is EPR-silent
suggests that each iron-dinitrosyl unit either has the
diamagnetic formal assignment [Fe(NO)2]8 or the para-
magnetic one [Fe(NO)2]9, as in complex B, with the
resulting S=0 state deriving from an antiferromagnetic
coupling between the two units. It should be mentioned
that complex B generated complex A during reaction
with air or H2O2 and that A was converted back to
complex B in the presence of an excess of cysteamine.
One possible explanation, assuming that there is no
change in the spin state between A and B and that A
has [Fe(NO)2]9 units, is that A to B conversion is a gain
of thiolate, due to the excess of CSH, whereas B to A
conversion is a loss of thiolate, consumed by oxidation
with O2 or H2O2. Alternatively, A has [Fe(NO)2]8 units
and A to B conversion is due to reduction by excess of
CSH, whereas B to A conversion is due to oxidation by
O2 or H2O2.
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5. Conclusions
We have shown that during reaction of RSNOs with
ferrous ions, NO can be transferred quantitatively from
the sulfur atom to iron. Depending on the excess of free

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