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Inorganic
Biochemistry
Journal of Inorganic Biochemistry 98 (2004) 11511159
www.elsevier.com/locate/jinorgbio
Departamento de Qu_mica ICEx, Universidade Federal de Minas Gerais 31.270-901 Belo Horizonte MG, Brazil
University of Warwick, Coventry CV4 7AL, UK
a,*
Received 27 November 2003; received in revised form 19 March 2004; accepted 24 March 2004
Available online 21 April 2004
Abstract
Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of
infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be
ecient in the treatment of acute promye-locytic leukemia. The exact mechanism of action has not been
elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins.
Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism.
This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in
aqueous solution.
The behavior observed for the As(III)glutathione system is very similar to that of As(III)cysteine. In both cases, the formation of two complexes in
aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry.
The formation constants of the cysteine complexes [As(H _1Cys)3], log K 29:846, and [As(H_2Cys)(OH)2] , log K
3_
2_
12:019, and of the glutathione complexes [As(H _2GS)3] , log K 32:06, and [As(H_3GS)(OH)2] , log K 103
were calculated from potentiometric and spectroscopic data.
In both cases, the [As(HL)3] species, in which the amine groups are protonated, predominate from acidic to
neutral media, and the [As(L)(OH)2] species appear in basic medium (the charges were omitted for the sake of
simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom
of cysteine. In the [As(L)(OH)2] species, the coordination sphere is completed by two hydroxyl groups. In both
_
cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH) 2O]
excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present
as [As(HL)3].
1. Introduction
The discovery of an organoarsenic compound
by Ehrlich and co-workers in 1909, salvarsan
0162-0134/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2004.03.010
1152
2. Experimental
2.1. Reagents
Oxidized and reduced forms of
glutathione and DL-cysteine
hydrochloride were used as
obtained from Sigma. Stock
solutions of glutathione and
cysteine were prepared just before
use under nitrogen atmosphere to
prevent ligand oxidation. For NMR
experiments, the ligands were
dissolved in D2O. Stock solutions
of so-dium metaarsenite, also
from Sigma, contained 0.1 M of
perchloric acid.
2.2. Spectroscopic measurements
A Diode Array Hewlett Packard 8451
A spectrometer equipped with a
Masterline 2095 thermostat at 25 LC
was used for UV and visible
absorption measurements. Re-sults
are expressed in terms of the molar
absorption co-ecient e related to
the total concentration of the ligand.
The concentration of ligand used in
the spectroscopic measurements
_4
_2
8.78a
10.71a
8.33b
10.50b
1.88c
8.15c
10.29c
1.69(2)d
8.17(1)d
10.30(1)d
(b) As(III)Cys
Species
log K
pK1
pK2
pK3
2.44a
[As(H_1Cys)3]
29.84(6)d
[As(H_2Cys)(OH)2]_
12.01(9)d
pK1
pK2
pK3
pK4
2.60e
3.82e
9.16e
9.88e
3.59f
8.75f
9.65f
2.04g
3.54g
8.54g
9.42g
2.10(3)d
3.53(3)d
8.65(2)d
9.52(2)d
(d) As(III)GSH
[As(H_2GS)3]3_
32.0(6)d
[As(H_3GS)(OH)2]2_
10(3)d
Ref. [28].
Ref. [26].
Ref. [27].
1154
N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159
Table 2
d
Multiplicity
J (Hz)
Cysteine
Cys Ha
3.79
dd
HaHb(1) 5.60; HaHb(2) 4.20
Cys Hb(1)
2.90
dd
Hb(1)Hb(2) 14.8; Hb(1)Ha 5.60
Cys Hb(2)
2.84
dd
Hb(2)Hb(1) 14.8; Hb(2)Ha 4.20
Glutathione
Glu Ha
3.62
appt
Glu HaGlu Hb 6.30
Glu Hb
2.021.96
m
Glu Hc
2.452.31
m
Cys Ha
#
#
#
Cys Hb(1)
2.80
dd
Cys Hb(1)Cys Hb(2) 14.2
2_
Fig. 1. Titration curves of solutions containing: 1, [Cys] 10 mM and [As(III)] 2 mM; 2, [Cys] 10 mM and [As(III)] 5 mM; 3, [Cys] 5
mM and [As(III)] 5 mM; 4, [Cys] 5 mM and [As(III)] 10 mM at 25 LC (I 0:1 M) with a solution of NaOH (0.1 M).
[As(H_2Cys)(OH)2] , as [As(Cys)
_
of [As(Cys)(OH)2] from
[As(HCys)3] the number of protons
lost depends on the ligand excess.
The formation constants of the
arsenic(III) hydroxo complexes
_
2_
Fig. 2. Species distribution curves for the systems: (a) AsCys (1) As(OH)3; (2) As(OH)2O ; (3)
2 _
As(OH)O 2 ;
2 _
As(OH)O 2 ;
(4) [As(HCys)3]; (5) As(Cys)(OH)2] ; and (b) AsGSH (1) As(OH)3; (2) As(OH)2O ; (3)
3_
2_
(6) [As(HGS)3] ; (7) [As(GS)(OH)2] . In both cases the total ligand concentration is 15 mM
and the total As(III) concentration is 5 mM.
1156
concentration indicates a
stoichiometry equal to 1:3
metalloid-to-ligand (inset Fig. 3).
The curve reaches a plateau at
arsenic-to-cysteine molar ratio
equal to 2.
2 _
containing 1.5 _ 10 M of
cysteine and various
concentrations of As(III) at pH 7.0
have been recorded (Fig. 3). By
increasing the concentration of
As(III) the b-methylene doublets of
doublets overlap, become broad
and deshielded, indi-cating
coordination to the sulfur atom.
The a-methine proton is also
deshielded but to a lesser extent.
The broadening of the peaks
probably results from the
75
2_
_2
2_
and 46% as
Fig. 5. UVVis spectra of solutions containing 15 mM GSH and As(III) concentrations varying
from 3 to 120 mM at 25 LC l 0:1 cm (I 1:5 M).
3_
(a) pH 7.0. Inset: Molar absorptivities of the complex species [As(HGS) 3] . (b) pH 9.5. Inset:
2_
1158
(OH)2]
form H2GS .
The proposed molecular structures
of the arsenic(III) complexes of
glutathione are represented in Fig.
6. In both cases the geometry
around arsenic is pyramidal. The
main dierences between these
complexes and the corresponding
ones formed by cysteine are the
net charge and the bulk. The
larger volume of glutathione does
not hinder the formation of these
complexes, be-cause of its
flexibility.
The equilibrium model proposed
explains the system arsenicglutathione consistently with some
previous lit-erature results and
clarifies some remaining
controver-sial points. It is already
known that arsenic(III) coordinates
to glutathione at low pH values
[16,31]. Delnomdedieu et al.
As(glutathione)3 complex is
formed. Although the au-thors did
not calculate the stability
constants for the species formed
in solution, they observed that the
complex was stable from pH 1.5 to
7.0. When the pH was raised, they
observed the appearance of free
gluta-thione in the solution
indicating the dissociation of the
complex, which led to the
proposition that at higher pH
values no complexation would
occur [16]. Here we identify for
the first time the presence of a
complex species in weakly basic
medium. According to our re-sults,
in the pH range 37 the main
species is the [As(HL)3] but above
pH 7.5, hydroxo ions displace two
ligand molecules from the arsenic
coordination sphere leading to the
formation of an [AsL] species.
Therefore, our results can explain
those of Delnomdedieu et al.
because when the pH of a solution
containing the As(glutathione)3
complex is raised above 7.5, the
pres-ence of free GSH is detected
due to the liberation of two ligand
molecules per complex to form the
1:1 complex. Our results indicate
that, under physiological conditions, arsenic(III) interactions with
glutathione should
also occur. Indeed, Kazuo et al.
[32] have identified by HPLCICPMS (high performance liquid
chromatog-raphyinductively
coupled argon plasma mass spectrometry) the presence of free and
GSH-conjugated arsenite
metabolites in the bile and urine
of rats.
Despite the importance of the
interactions with cys-teine side
chains in proteins for the
biochemistry of ar-senic, we did
not find equilibrium reports for this
system in the literature. However,
very early studies of the synthesis
of compounds for the treatment of
trypano-somiasis describe an
As(cysteine)3 complex that was
characterized by elemental
analysis [33]. Our results
demonstrate that at physiological
pH arsenic(III) forms a quite stable
complex with cysteine and
glutathione suggesting that
arsenic interactions with cysteine
resi-dues in polypeptides or
proteins in vivo may not be
neglected.
Despite the increasing interest in
arsenic chemistry in biological
systems, this work is the first to
present values for equilibrium
constants of the species formed
between the metalloid and the
amino acid cysteine or the intracellular tripeptide glutathione. The
knowledge of these complexes
stability is very important to the
evaluation of the role of thiolcontaining molecules in arsenic
biochemistry.
4. Abbreviations
GSH glutathione
Cys
cysteine
Acknowledgements
This work was supported by grants
of CNPq (Con-selho Nacional de
Desenvolvimento Cient_fico e Tecnologico,_ Brazil) and FAPEMIG
(Fundac~ao de Amparo _a
Pesquisa de Minas Gerais, Brazil).
Nicol_as A. Rey is grateful to CNPq
for the fellowship. The authors
thank Prof. Jos_e D. Souza Filho
and Ivana S. Lula for the
assistance during the NMR
experiments and Prof. H_elio A.
Duarte for helpful discussions.
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