Uncatalyzed and copper(II) catalyzed oxidation of glutathione by Co(III)2 bound superoxide complex

Article abstract of DOI:10.1016/j.ica.2014.03.003

In acid media, glutathione (GSH) is oxidised by metal bound superoxo complex, [(NH₃)₅Co(III)(μ-O₂)Co(III)(NH ₃)₅]⁵⁺ (1) to GSSG. Complex 1 is reduced to its corresponding peroxo complex, [(NH₃)₅Co(III)(μ-O ₂)Co(III)(NH₃)₅]⁴⁺ (2) which readily decomposes in acid media to Co(III), NH₄⁺ and O ₂. The oxidation of GSH is profoundly catalyzed by the presence of Cu²⁺ ion. The observed rate constant k_o was found to be proportional to [GSH]² for both uncatalyzed and catalyzed reaction and for the latter k_o was also proportional to [Cu]_T ² ([Cu]_T is the analytical concentration of Cu ²⁺). The rate for uncatalyzed reaction decreases for with increasing ionic strength (I) of the reaction media whereas, k_o for catalyzed reaction is invariant of ionic strength (I). Both for the uncatalyzed and catalyzed reaction, k_o is proportional to [H⁺] ^-3. Under the reaction condition employed, it has been seen that GSH reduces Cu(II) to Cu(I) and forms either mononuclear Cu(I)-GSH or dinuclear (Cu(I)-GSH)₂ complex with Cu(I) with a 1:1 composition. Suitable mechanistic pathways for the uncatalyzed and catalyzed reactions through the intermediate formation of mononuclear or dinuclear Cu-GSH complex have been suggested. The activation energy for the uncatalyzed and catalyzed reaction has been calculated as 97.1 ± 5.7 and 29.9 ± 1.2 kJ M^-1 respectively.

Full text of DOI:10.1016/j.ica.2014.03.003

Inorganica Chimica Acta 418 (2014) 51–58
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Uncatalyzed and copper(II) catalyzed oxidation of glutathione by Co(III)2
bound superoxide complex
b
b
b,
Bula Singh a, , Ranendu Sekhar Das , Rupendranath Banerjee , Subrata Mukhopadhyay
⇑
⇑
a Department of Chemistry, Visva-Bharati, Santiniketan 731 235, India
b Department of Chemistry, Jadavpur University, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In acid media, glutathione (GSH) is oxidised by metal bound superoxo complex, [(NH3)5Co(III)(
Co(III)(NH3)5]5+ (1) to GSSG. Complex 1 is reduced to its corresponding peroxo complex, [(NH3)5
Co(III)(
-O2)Co(III)(NH3)5]4+ (2) which readily decomposes in acid media to Co(III), NH4+ and O2. The oxi-
l-O2)
Received 16 November 2013
Received in revised form 6 March 2014
Accepted 9 March 2014
l
dation of GSH is profoundly catalyzed by the presence of Cu2+ ion. The observed rate constant ko was
found to be proportional to [GSH]2 for both uncatalyzed and catalyzed reaction and for the latter ko
was also proportional to [Cu]2T ([Cu]T is the analytical concentration of Cu2+). The rate for uncatalyzed
reaction decreases for with increasing ionic strength (I) of the reaction media whereas, ko for catalyzed
reaction is invariant of ionic strength (I). Both for the uncatalyzed and catalyzed reaction, ko is propor-
tional to [H+]ꢀ3. Under the reaction condition employed, it has been seen that GSH reduces Cu(II) to
Cu(I) and forms either mononuclear Cu(I)–GSH or dinuclear (Cu(I)–GSH)2 complex with Cu(I) with a
1:1 composition. Suitable mechanistic pathways for the uncatalyzed and catalyzed reactions through
the intermediate formation of mononuclear or dinuclear Cu–GSH complex have been suggested. The
activation energy for the uncatalyzed and catalyzed reaction has been calculated as 97.1 5.7 and
29.9 1.2 kJ Mꢀ1 respectively.
Available online 3 April 2014
Keywords:
Cobalt(III)
Superoxide
GSH
Redox
Kinetics
Mechanism
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
lipid [9], proteins [9], carbohydrates [10] and DNA [11,12] resulting
in tissue injuries and diseases [5,9,13].
O2 molecules, in living aerobic organisms, are utilized as the
terminal oxidant in the mitochondrial respiratory chain where it
is finally reduced to water via the acceptance of one electron at a
time [1]. Often oxygen molecules are partially reduced by 1eꢀ to
To combat with this stress full condition, living organisms also
have developed elaborate defensive moves through the use of anti-
oxidant systems. One such effective antioxidant systems comprises
of thiol molecules which contain the sulfhydryl group (–SH)
attached to a carbon atom. Such molecules like cystine, cysteine
and reduced glutathione (GSH) play an essential role in the protec-
tion against ROS [14]. Among these, GSH which can be found in
both prokaryotes and eukaryotes cellular antioxidant defence sys-
tem have much more abundance in living cells than the other two
molecules [15]. Though GSH, the principal non-protein tripeptide
ꢀ
form the superoxide anion O2 as a result of the leakage of elec-
trons from different redox centres [2]. Superoxide anion itself is a
relatively weaker oxidant but it can act as the precursor to the for-
mation of other reactive oxygen and reactive nitrogen species (ROS
and RNS respectively) like peroxide (O22ꢀ), hydroxyl radical (ꢀOH)
and nitric oxide radical (ONꢀ) and its derivative peroxynitrite
(ONOOꢀ) anion [3–5]. ROS, produced in limited extent are an
integral part of different and essential cellular mechanisms like
the immune systems and signalling pathways [5–8]. But severe
problems arise when the ROS is formed beyond this limit of
cellular tolerance. The situation, called oxidative stress can imply
deleterious effects because ROS targets different bio-molecules like
(L-c-glutamyl-L-cisteinylglycine) thiol molecule, is mainly synthe-
sized in cell cytoplasm but can be found in other cellular compart-
ments also [16]. Apart from its antioxidant properties, GSH also
plays vital roles in various bio functions [15,17,18] and the ratio
of GSH to its oxidized form GSSG in cells is a predictor of cellular
toxicity [19] and fatal diseases like diabetes, cancer or HIV [20–22].
Thus the understanding and monitoring of the action of GSH on
ROS species have long been a subject of study and several reports
on the reaction between GSH with hydroperoxide, t-butyl
hydroperoxide (tBHP), radicals generated by xanthine oxidase
(in vivo and in vitro) and metal oxidant can be found [2,23–26].
⇑
Corresponding authors. Tel.: +91 94331 63075; fax: +91 33 2414 6223.
E-mail addresses: singhbula@rediffmail.com (B. Singh), smukhopadhyay@
chemistry.jdvu.ac.in, ju_subrata@yahoo.co.in (S. Mukhopadhyay).
http://dx.doi.org/10.1016/j.ica.2014.03.003
0020-1693/Ó 2014 Elsevier B.V. All rights reserved.

52
B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
Nevertheless, detailed investigation of the kinetic and mechanistic
aspects of the redox reaction of GSH with metal bound Oꢀ2 is scarce
and still worth an experimentation. Moreover, since transition
metal ions like Cu2+ can catalyze the oxidation of thiol molecules
[27–35], investigation of the mechanism of the title reaction cata-
lyzed by Cu2+ is also of potential importance.
study but addition of freshly prepared GSH to 1 gradually
decreased the absorbance of 1. Such a time resolved UV–Vis spec-
tral change of 1 is shown in Fig. 1. GSH slowly reduces the super-
oxo complex 1 to its corresponding peroxo complex, [(NH3)5Co(III)
(l
-O2)Co(III)(NH3)5]4+ (2) and GSH itself is oxidised to GSSG (vide
infra). The peroxo complex 2 is unstable in acid media and it read-
ily decomposes to Co(III), NH4+ and O2 [41]. This, rather slow oxida-
tion of GSH by 1 is readily catalyzed by the presence of Cu2+ ion
added externally. Thus both the uncatalyzed and catalyzed oxida-
tion of GSH by 1, needed meticulous study in absence and presence
of added Cu2+ ions in reaction media. Both uncatalyzed and cata-
lyzed reactions were studied in acid perchlorate media at a fixed
ionic strength (I), maintained by calculated addition of NaClO4
and over the temperature range (288–308 K). Moreover, in all the
kinetic runs for both uncatalyzed and catalyzed reaction, a large
excess of [GSH] over [1] was maintained. The final reaction mixture
for the uncatalyzed reaction were prepared by the addition of mea-
sured amount of 1, sodium perchlorate, and perchloric acid (to
maintain media acidity) followed by the addition of fresh aqueous
solution of GSH. Here it is noteworthy that all solutions were pre-
pared by the use of freshly fetched Milli-Q water which has a resis-
In this work we have chosen a metal bound superoxo complex
l
-superoxo-bis[pentaamminecobalt(III)]5+
,
[(NH3)5Co(III)(l-O2)
Co(III)(NH3)5]5+ (1) as a suitable example for ROS species and stud-
ied both the uncatalyzed and Cu2+-catalyzed kinetics of reduction
of 1 by GSH in acidic media under different reaction condition.
The suggested mechanistic details are expected to enlighten our
knowledge about the antioxidant behavior of GSH on ROS in
absence or presence of any metal ion.
2. Experimental
2.1. Materials
Cobalt(II) chloride hexahydrate (SRL), ammonia (Merck), ammo-
nium peroxodisulfate (Merck), orthophosphoric acid (Merck),
perchloric acid (Merck), L-glutathione (Aldrich), copper(II) acetate
tance of 16.6 M
X and thus the solutions can be supposed to be
(SRL), sodium perchlorate (Aldrich), 5,50-dithio-bis-2-nitrobenzoic
acid (Aldrich), were used without further purification. All the
solutions for kinetic experimentations were prepared with ultra
pure Milli-Q water. Perchloric acid was standardized following
standard methods [36].
devoid of any significant amount of metal ions. The uncatalyzed
reaction was also carried out both in absence and in presence of
2.0 mM dipicolinic acid (dpa), an well known sequestering agent,
under otherwise strictly same reaction condition ([1] = 1.0 mM;
[GSH] = 20.0 mM; [H+] = 9.1 ꢁ 10ꢀ3 M; I = 0.5 M; T = 25.0 0.1 °C).
The corresponding ko values (12.3 ꢁ 10ꢀ4 and 12.6 ꢁ 10ꢀ4 sꢀ1
respectively) of the reduction reactions are comparable. The result
confirms that the aqueous reaction media is devoid of any signifi-
cant amount of interfering metal ions. During the study of cata-
lyzed reaction, thus, measured amount of aqueous solution of
copper(II) acetate ((CH3CO2)2CuꢂH2O) was additionally added to
the final reaction mixture to maintain [Cu]T in the range (3.0–
10.5) ꢁ 10ꢀ6 M before the addition of GSH. Since GSH molecules
rapidly reduce Cu2+ to Cu+ and/or can form complexes with both
Cu(I) and Cu(II) in different stoichiometric ratio (vide infra), it is
more convenient to express the analytical copper(II) concentration
as [Cu]T. Both the uncatalyzed and catalyzed reaction was followed
by the decrease in absorbance at 670 nm (Fig. S2) which is the
characteristic visible absorption peak of 1 and no other species in
the reaction mixture absorbs at this wavelength. The observed rate
constants (ko) for uncatalyzed and catalyzed reaction were deter-
mined using standard non-linear standard least-squares fit to the
first-order equation of such decay with time.
2.2. Preparation and characterization of complex 1
Superoxide complex (1) was synthesized following the literature
method [37] with little modification [38]. Initially, a
cobalt(III) complex is prepared from CoCl2, 6H2O, ammonia and oxy-
gen which is then further oxidized to -superoxo complex by
l-peroxo-
l
ammonium peroxodisulfate [37]. The chloride salt of 1 thus pre-
pared was converted to perchlorate salt by dissolving it in 40%
orthophosphoric acid followed by filtration; concentrated HClO4
was added drop wise to the filtrate to precipitate the perchlorate salt
of 1. Pure perchlorate salt of 1 was recrystallized from 10% HClO4.
Complex 1 was characterized by FTIR spectrum and the purity
was checked from UV–Vis absorption. FT-IR spectrum of complex
1 (Fig. S1) shows main absorption bands at 1604 (s), 1340 (s)
cmꢀ1 (s) for das (NH3) and 838 cmꢀ1 (s) for rr (NH3) which are in close
agreement with the literature values for similar di-bridged metallo
superoxide complex (das, asymmetric deformation vibration; rr
rocking vibration) [39]. The presence of the peak at 1087 cmꢀ1 (s)
confirms the presence of the bridging superoxo group [40].
Complex 1 shows characteristic UV–Vis absorption at 481 and
670 nm and purity of 1 was found to be more than 95% as mea-
sured from the molar extinction coefficient (
e) value at 670 nm (e
in Mꢀ1 cmꢀ1, found 860; reported 890 [37]).
0.8
2.3. Instrumentation
1
0.6
0.4
0.2
0.0
.
.
.
Change of UV–Vis absorbance of reaction mixture and kinetic
studies at different temperature were followed by the using a Shi-
madzu spectrophotometer (UV-1700) with 1.00 cm quartz cuvette
equipped with electrically controlled Shimadzu thermostat
( 0.1 °C). Fourier Transform Infrared (FTIR) data were collected
using a Shimadzu FT-IR 8400S. Mass spectrometry (ESI positive
mode) was done by Micro Mass Q-TOF Micro (LCMS) spectrometer.
20
400
500
600
wavelength (nm)
700
800
2.4. Kinetics
Both the
l-superoxo complex 1 and glutathione (GSH) are sta-
Fig. 1. Spectral change of 1 on reaction with GSH at an interval of 120 s. Condition:
[1] = 1.0 mM; [GSH] = 20.0 mM; [H+] = 9.1 ꢁ 10ꢀ3 M; I = 0.5 M; T = 25.0 0.1 °C.
ble in the acidic media ([H+] = 0.008–0.35 M) maintained in the

B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
53
2.5. Stoichiometry
at m/z = 878 Da and above in Fig. S4 probably results from different
aggregation of GSH molecules as (GSH)mn + (n, m P 1) [45].
Thus, the stoichiometric equation representing the reaction of 1
and GSH in presence or absence of Cu2+ can be as follows:
The stoichiometry of the uncatalyzed and catalyzed reaction
has been determined by the estimation of unreacted 1 and unre-
acted GSH in separate experiments. In the first instance, stoichiom-
etric determinations were carried out following the initial (A0) and
final equilibrium absorbance (Ae) of the reaction mixtures at
670 nm where complex 1 was allowed to react with deficit
amounts of GSH in different ratio. The amount of unreacted 1
was determined from the difference between A0 and Ae.
5þ
2½ðNH3Þ5CoðIIIÞð
l
ꢀ O2ÞCoðIIIÞðNH3Þ ꢃ þ 2GSH þ 18Hþ
5
! 4Co2þ þ 20NH4þ þ 2O2 þ GSSG
ð1Þ
3.2. Observations for uncatalyzed reaction
3.2.1. Variation of ko with [GSH]
Similarly, complex 1 was allowed to react with excess GSH and
the unreacted amount of GSH was determined spectrophotometri-
cally by the action of Ellman’s reagent (5,50-dithio-bis-2-nitroben-
zoic acid, C14H8N2O8S2, DTNB) [42]. DTNB is a water-soluble
compound which specifically reacts with free sulfhydryl group
(–SH) containing molecule like GSH and quantitatively produces
a yellow-colored product (2-nitro-5-thiobenzoic acid, TNB) and
mixed disulfide at alkaline pH. In aqueous solution, TNB shows
an intense molar extinction coefficient of 13,600 Mꢀ1 cmꢀ1 at
412 nm which remains invariant in the pH range 7.6–8.6 [43].
Thus, by recording the absorbance values at 412 nm for the reac-
tions of Ellman’s Reagent with different known concentrations of
GSH at pH 8.0, a standard concentration versus absorbance curve
was prepared. Then, a measured aliquot of equilibrated reaction
mixture of 1 and excess GSH was allowed to react with Ellman’s
reagent at pH 8.0 and the amount of unreacted [GSH] was deter-
mined by the comparison of absorbance from the standard curve.
During this experimentation, it has also been ensured that except
GSH, no other species present in the reaction mixture reacts with
Ellman’s reagent.
The simple variation of [GSH] over [1] does not hold a linear
relationship with ko but results in a curved one where rate value
increases sharply with increase in [GSH] (Fig. 2, Table S1). A linear
relationship was only achieved when ko values are plotted against
[GSH]2 (Fig. 3) and the plot also passes through the origin. This is a
striking observation contrast to earlier reported ones [25,46,47]
where the uncatalyzed rate of oxidation of GSH depends on the
first power of [GSH]. This dissimilarity probably arises from the
selected range of [GSH] over which rates have been measured. In
Fig. 3, considering only initial few points ([GSH] 6 30 mM), ko
appears to not only vary linearly with [GSH] but also passes
through the origin. The square dependence on [GSH] becomes
apparent only when the concentration of GSH is high [26].
3.2.2. Variation of ko with media acidity
The observed rate constants (ko) for uncatalyzed reaction
decrease with increase in [H+] (Fig. 4, Table S2) suggesting reactiv-
ity of deprotonated species. Both complex 1 and GSH can be the
possible source of deprotonation. In complex 1 the ligating group
NH3 can release a proton forming its conjugate base [(NH3)5Co(O2)-
Co(NH3)4(NH2)]4+ (1-H) that could be the kinetically reactive spe-
cies. The reductant GSH molecule also can release the carboxyl
protons (pKa = 2.1 and 3.5 [46]) and the thiol proton (pKa = 8.73
[47]) under the present reaction condition. Since it has been found
that 1 is highly stable in acidic acetic acid buffer media [38], it is
evident that the carboxyl group cannot reduce 1. The only group
in GSH that has the potential to reduce 1 is thiol (–SH) group. Thus
we consider only the deprotonation of thiol group to form its con-
jugate base GSꢀ or its derivative (GSSG2ꢀ, vide infra) in subsequent
discussions. Under the present acidic condition, only a very small
amount of GSꢀ is expected to be formed [48] which accounts for
the much lower order of reaction rate (ꢄ10ꢀ4 sꢀ1) between GSH
Moreover, a gas is evolved during both the uncatalyzed and
catalyzed reaction of 1 with GSH and the volume of the gas was
collected by the downward displacement of water and corrected
to N.T.P (1.0 atm, 273.15 K) as usual. The oxidised products of
GSH of uncatalyzed and catalyzed reaction were characterized
from the LC–MS spectra (Figs. S3 and S4, respectively) of the
product mixture.
3. Results and discussions
3.1. Stoichiometry and reaction products
From the determination of unreacted [1] and [GSH], it can be
firmly said that in both uncatalyzed and catalyzed reaction, one
mole of complex 1 reacts with one mole of GSH and one mole of
oxygen gas is evolved. Since oxygen is evolved it is evident that
complex 1 has completely mineralized in acid perchlorate media
to Co2+, O2 and NH+4 [41].
and 1 compared to the order of reaction rate (ꢄ107 Mꢀ1 sꢀ1
)
between GSH and different radicals in alkaline media [49]. Thus
100
80
60
40
20
0
The LC–MS spectra of uncatalyzed and catalyzed product mix-
ture in positive ion mode (Figs. S3 and S4 respectively) carry the
evidence that the oxidation product of GSH is mainly GSSG. The
presence of GSSG (M = 613 Da, Da = 1 g Mꢀ1) is most prominent
in LC–MS spectrum of catalyzed product mixture (Fig. S4) as the
presence of peaks at m/z = 613 and 635 Da represent the species
[M+H]+ and [M+Na]+ respectively. Moreover, peaks in Fig. S4 at
m/z = 391 and 484 Da also bear the signature of GSSG as they
results from fragmentation of the same [44]. The LC–MS spectrum
of uncatalyzed product mixture (Fig. S3) does not show the molec-
ular peak of GSSG. Nevertheless, the presence of peak at m/z = 361,
460 and 478 Da indicates that GSSG (M) must have been formed
and these peaks must have originated because of the limited
fragmentation of the species [M+H]+ [44]. Otherwise, none of the
fragmentation of alone GSH molecule can be the origin of those
peaks. Here it can be mentioned that the presence of the peaks
0
20
40
60
103[GSH] (M)
Fig. 2. Variation of ko vs. [GSH]. Condition: [1] = 1.0 mM; [H+] = 9.1 ꢁ 10ꢀ3 M;
I = 0.5 M; T = 25.0 0.1 °C.

54
B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
100
80
60
40
20
0
45
40
35
30
25
0.2
0.4
0.6
0.8
I (M)
1.0
1.2
1.4
0
1000
2000
106[GSH]2 (M2)
3000
4000
Fig. 5. Variation of ko vs. I. Condition: Condition: [1] = 1.0 mM; [GSH] = 40.0 mM;
[H+] = 9.1 ꢁ 10ꢀ3 M; I = 0.5 M; T = 25.0 0.1 °C.
Fig. 3. Variation of ko vs. [GSH]2 passes through origin. Condition: [1] = 1.0 mM;
[H+] = 9.1 ꢁ 10ꢀ3 M; I = 0.5 M; T = 25.0 0.1 °C.
30
40
16
12
8
20
10
30
0
0
10
20
30
40
103[G S H ] (M )
20
10
0
8
9
10
11
12
0
400
800
1200
1600
103[H+] (M)
106[GSH]2 (M2)
Fig. 4. Variation of ko vs. [H+]. Condition: [1] = 1.0 mM; [GSH] = 20.0 mM; I = 0.5 M;
T = 25.0 0.1 °C.
Fig. 6. Variation of ko vs. [GSH] (inset) and ko vs. [GSH]2 for Cu2+catalyzed reaction.
Condition:
[1] = 1.0 mM;
[Cu]T = 6.0 ꢁ 10ꢀ6 M;
[H+] = 0.35 M;
I = 0.5 M;
T = 20.0 0.1 °C.
it can be concluded that deprotonation of both 1 and GSH can be
held responsible for the inverse proton dependence of ko.
(r = 0.995) which is close enough to the rate (1.9 ꢁ 10ꢀ4 sꢀ1) of
uncatalyzed oxidation of GSH by 1 under similar condition. The
square dependence of the observed rate on each of the concentra-
tions of reductant and catalyst is indeed rare [55].
3.2.3. Variation of ko with ionic strength (I)
The observed rate constants ko decrease with the increase in
ionic strength I (Fig. 5, Table S3), indicating reaction between
oppositely charged reactive species. Since the oxidant species,
the superoxo complex 1 bears positive charge, the active reductant
species derived from GSH molecule must have negative charge.
3.4. Proposed mechanism of the uncatalyzed reaction
The prime observations for the uncatalyzed reactions are the
linear dependence of observed rate constant ko on [GSH]2 and its
decreases with increase in [H+]. Moreover, two of the reactive spe-
cies that participate in the rate determining step are of opposite
charge as predicted from rate versus ionic strength data. Further-
more, no significant solvent isotope effect was found when the
aqueous media was enriched with D2O possibly indicating the
non-occurrence of any electroprotic pathway in the reaction
[56,57]. Thus based on these observations a mechanism for uncat-
alyzed reaction was proposed in Scheme 1. Under the prevailing
reaction condition, both 1 and GSH is deprotonated to generate
the conjugate bases 1-H and GSꢀ respectively (Eqs. (2) and (3)
respectively). In subsequent step two GSꢀ react to form the active
reducing agent GSSG2ꢀ (Eq. (4)). This is similar to the formation of
GSSGꢀꢀ (or GSS(H)Gꢀ) from the reaction between GSꢀ radical and
GSꢀ (or GSH) [26,48]. GSꢀ is generally produced when GSꢀ reduces
dissolved O2 molecule to Oꢀ2 . Under the present reaction condition
the formation of GSꢀ was supposed to be insignificant since the
observed rate (ko) was found to be same in presence or absence
3.3. Observations for Cu2+ catalyzed reaction
3.3.1. Variation of ko with [GSH] for catalyzed reaction
For the catalyzed reaction, the observed rate constant ko sharply
increases with [GSH] similar to uncatalyzed reaction and forms a
curve. ko increases linearly with [GSH]2 and as expected the linear
plot passes through the origin (Fig. 6, Table S4).
3.3.2. Variation of ko with [Cu]T for catalyzed reaction
It has been seen that most of the transition metal ions of copper,
iron, zinc or mercury form complex with molecules having thiol
group [27–35,50–54]. The formation of metal–thiol complex,
particularly with copper ion has been found to accelerate the oxi-
dation of thiol molecules. In this present work the oxidation of GSH
by complex 1 is catalyzed by Cu2+ ion and the observed rate con-
stant ko was found to be proportional to [Cu]2T (Fig. 7, Table S5).
The linear plot shows an intercept value of (2.2 0.3) ꢁ 10ꢀ4 sꢀ1

B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
55
60
50
40
30
20
10
0
20
30
20
10
0
16
12
8
0
2
4
6
8
10 12
106[Cu]T (M)
4
0
0
4
8
12
16
20
0
20
40
60
80
100
120
10-5/[H+]3 (K-3)
1012[Cu]T2 (M2)
Fig. 8. Variation of ko vs. 1/[H+]3. Condition: [1] = 1.0 mM; [GSH] = 20.0 mM;
I = 0.5 M; T = 25.0 0.1 °C.
Fig. 7. Variation of ko vs. [Cu]T (inset) and ko vs. [Cu]2T for catalyzed reaction.
Condition: [1] = 1.0 mM; [GSH] = 20 mM; [H+] = 0.30 M; I = 0.5 M; T = 20.0 0.1 °C.
constant (kUK1K22K3) value of 2.3 ꢁ 10ꢀ6 M sꢀ1 under the reaction
condition: [1] = 1.0 mM; [GSH] = 20.0 mM; [H+] = 9.1 ꢁ 10ꢀ3 M;
I = 0.5 M; T = 25.0 0.1 °C.
of dissolved oxygen. In the rate determining step (Eq. (5)), GSSG2ꢀ
reduces a molecule of 1 by an electron to 2 and itself is oxidised to
GSSGꢀ. In the subsequent fast steps GSSGꢀ further reduces another
molecule of 1 to 2 and in acid solution 2 decomposes to Co2+, O2
and NH+4. Thus, GSSG2ꢀ is finally oxidised by two electrons to GSSG.
As in the process two molecules of 1 react with two molecules of
GSH, a overall stoichiometric ratio of [1]:[GSH] = 1:1 is followed.
Thus, the observed rate constant for the uncatalyzed reaction
appears to be as follows:
3.5. Proposed mechanism of the Cu2+ catalyzed reaction
For the mechanism of the catalyzed reaction, the important
clues are that ko not only varies linearly with [GSH]2 but also with
[Cu]2T. Another important thread of information is that ko is
independent of ionic strength of reaction media. Since the oxidant
species, complex 1 is cationic, the reductant species must be
devoid of charges, otherwise ionic strength could influence ko.
Similar to the uncatalyzed reaction, the catalyzed reaction rate
3
2
ko ¼ kUK1K2K3½GSHꢃ =½Hþꢃ
ð8Þ
2
Eq. (8) predicts a linear relationship between ko and [H+]ꢀ3 and
indeed the plot of ko versus [H+]ꢀ3 for uncatalyzed reaction yields a
straight line (Fig. 8). Eq. (8) also provides the approximate rate
III
III
III
III
5+
O
O
4+
Co(NH3)5]
H+
K1
Co(NH3)4(NH2)]
[(NH3)5Co
+
(2)
[(NH3)5Co
O
O
)
(1)
(1-H
K2
GS-
(3)
(4)
H+
GSH
+
K3
[GSSG]2-
GS-
GS-
+
III
Co(NH3)4(NH2)]
III
O
4+
III
[(NH3)5Co
III
O
[(NH3)5Co
3+
Co(NH3)4(NH2)]
O
O
(1-H
)
(2)
+
[GSSG]-
kU
+
(5)
slow
[GSSG]2-
III
Co(NH3)4(NH2)]
III
Co(NH3)4(NH2)]
III
[(NH3)5Co
III
4+
O
O
3+
O
[(NH3)5Co
O
(2)
+
(1-H
)
Fast
(6)
(7)
+
[GSSG]-
[GSSG]
III
Co(NH3)4(NH2)]
Fast
III
3+
2+ + 10 NH4+ + O2
O
11 H+
2 Co
[(NH3)5Co
+
O
(2)
Scheme 1.

56
B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
(ko) values are highly sensitive to [H+] in reaction media and ko
1.0
0.8
0.6
0.4
0.2
0.0
increases with the decrease in [H+] (data not shown).
The earlier study of Cu2+ catalyzed reduction of Ni(IV) complex
by 2-aminoethanethiol [55] reports the square dependence of the
observed rate on catalyst concentration and that was explained
by proposing the reaction between an adduct formed by Cu(I)
and Ni(IV) complex (Cu(I)Ni(IV)) with another Cu(I). In this present
study such an adduct formation between complex 1 and copper ion
is implausible [41] and thus the complexation between copper and
GSH was rather given much more importance.
Earlier studies have shown that GSH forms complex with Cu(I)
or Cu(II) [28–30,33,35,54] with a varying ratio of Cu(I/II):GSH from
1:1 to 1:2. Actually, GSH have been found to show a variation in
stoichiometry during the formation of complexes with metal ions
[52,58,59] depending upon the reaction condition. Thus it becomes
a necessity to take a quick glance to the nature of Cu–GSH complex
under the present reaction condition. The formation of a complex
0
1
2
3
4
5
[GSH]/[Cu]T
Fig. 9. Plot of absorption at 260 nm over different [GSH] to [Cu]T ratio. Condition:
[H+] = 0.30 M; I = 0.5 M; T = 25.0 0.1 °C.
K1
III
III
III
III
O
4+
5+
O
+
H+
Co(NH3)4(NH2)]
[(NH3)5Co
Co(NH3)5]
[(NH3)5Co
(2)
(3)
O
O
(1-H
)
(1)
K2
GS-
+
H+
GSH
Fast
Cu+
GS-
Cu2+
Cu+
+ GS
+
+
(9)
K4
GS-
[Cu(I)(GS)]
(10)
GS
Cu(I)
K5
2[Cu(I)(GS)]
Cu(I)
(11)
(12)
GS
III
Co(NH3)4(NH2)]
III
III
III
[(NH3)5Co
O
O
4+
3+
Co(NH3)4(NH2)]
[(NH3)5Co
O
O
)
kC,D
(2)
(1-H
+
GS
+
slow
+
GS
Cu(I)
Cu(II)
Cu(I)
Cu(I)
GS
GS
III
III
O
4+
III
III
O
3+
Co(NH3)4(NH2)]
[(NH3)5Co
Co(NH3)4(NH2)]
[(NH3)5Co
O
O
Fast
(1-H
+
GS
)
(2)
(13)
+
+
2+
GS
Cu(I)
Cu(II)
Cu(II)
GS
Cu(II)
GS
III
Fast
III
O
3+
2 Co2+
+ 10 NH4+ + O2
Co(NH3)4(NH2)]
11 H+
(7)
[(NH3)5Co
+
O
(2)
2+
GS
Fast
2 Cu+
Cu(II)
Cu(II)
(14)
+
GSSG
GS
Scheme 2.

B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
57
is certain from the increasing absorbance of the mixture of Cu2+
-6.0
-6.2
-6.4
-6.6
-6.8
-7.0
solution (1.5 mM) with GSH solution (1.5 mM) (Fig. S5) [30]. The
spectral pattern for the formation of Cu–GSH complex is alike in
presence or absence of dissolved air. The absorbance increases
and then it reaches an equilibrium after some time which depends
on the ratio of [Cu]T:[GSH].
Stoichiometry of the Cu–GSH complex was determined follow-
ing the Jobs continuous variation method [60]. Different amount of
Cu2+ ([Cu]T = 0.6–2.4 mM) and GSH ([GSH] = 2.4–0.6 mM) were
mixed keeping the total molarity constant at 3.0 mM and the final
and equilibrium absorbance of the mixture was noted at 260 nm
(Table S6) [30]. The absorbance first increases with the increasing
ratio [GSH]/[Cu]T and then it decreases with further increase in the
ratio (see Fig. 9).
3.20
3.25
3.30
3.35
103/T (K-1)
3.40
3.45
3.50
The lines befitting the increasing and decreasing arm intersects
at the point where [Cu]T:[GSH] = 1:1 indicating that per copper ion,
one molecule of GSH is required to form the complex. Thus the
resulting complex can be mononuclear (Cu–GSH) or dinuclear
(Cu–GSH)2 since both of which obey the ratio of 1:1. In these com-
plexes, determination of the oxidation state of copper is another
task. Since it has now been firmly established that Cu2+ ion is
promptly reduced by thiol molecules [30,34,61], the complexes
are expected to consists of Cu(I) system. Previous studies have
shown the participation of mononuclear (Cu(I)–GSH) complex in
catalytic cycle [28–30,34,62] but participation of dinuclear
(Cu(I)–GSH)2 is not known or ruled out [29]. Dinuclear (Cu(I)–
GSH)2 can be assumed to catalyze the reaction because similar
dinuclear complex (Fe(II)–GSH)2 has been found to catalyze the
reduction of oxygen molecule [50]. Here it is worth remembering
that whatever the nature of complex participating in rate
determining step, it is devoid of any charge since reaction rates
are independent of I of reaction media.
Fig. 11. lnko vs. 1/T for Cu2+ catalyzed reaction. Condition: [1] = 1.0 mM;
[GSH] = 20.0 mM; [Cu]T = 6.0 ꢁ 10ꢀ6 M; [H+] = 0.30 M; I = 0.5 M.
[Cu(I)(GSꢀ)] forms a dinuclear [Cu(I)(GSꢀ)]2 similar to dinuclear
Fe(III)-GSH complex (Eq. (11)) [50]. The dinuclear complex
[Cu(I)(GSꢀ)]2 reduces complexes 1 to 2 in slow step (Eq. (12)).
The oxidised species [Cu(I)Cu(II)(GSꢀ)]2+ again reduces another
molecule of 1 through a fast step (Eq. (13)) and the resulting
[Cu(II)(GSꢀ)]22+ decomposes to GSSG and Cu+ (Eq. (14)) [50]. The
thiyl radical, GSꢀ generated in Eq. (9) also dimerizes to form GSSG
[26] but the concentration of GSSG formed in this step is limited
by the concentration of [Cu]T which has the order of 10ꢀ6 M. Thus
the main source of GSSG is Eq. (14).
Scheme 2 results in the following expression of rate constant:
3
2
T
2
ko ¼ kC;DK1K2K2K5½Cuꢃ ½GSHꢃ =½Hþꢃ
ð15Þ
2
4
Based on these observations two alternative mechanisms for
catalyzed reaction is proposed herein. Scheme 2 and Scheme S1
includes the formation of dinuclear copper complex and mononu-
clear copper complex respectively and in both the schemes the
presence of Cu(I)/Cu(II) cycle play crucial role. However, Scheme 2
where the dinuclear copper complex is the main active catalyst
form appears to be preferred over Scheme S1 where the mononu-
clear copper complex is the active form of catalyst (suggestion of a
reviewer). Scheme S1 thus seems less plausible and may not follow
the observed rate dependence on reactants and catalyst concentra-
tion (see detailed discussions in SI).
The reduction of Cu2+ by GSꢀ in Eq. (9) is assumed to be fast and
almost quantitative [30,34,61] and thus the analytical concentra-
tion of copper in Eq. (15) is represented by [Cu]T. The empirical rate
law Eq. (15) provides the rate constant (kC,DK1K22K42K5) value of
1.5 ꢁ 109 Mꢀ1 sꢀ1 under the following reaction condition:
[1] = 1.0 mM; [GSH] = 20.0 mM; [Cu]T = 6.0 ꢁ 10ꢀ6 M; [H+] =
0.035 M; I = 0.5 M; T = 20.0 0.1 °C.
Here it is important to note that both the active reductant in
Scheme 2 and S1 are neutral in charge. Moreover, both of the final
rate expression for catalyzed reaction predicts that ko is propor-
tional to [H+]ꢀ3 (Eq. (15) in Scheme 2 and Eq. (23) in Scheme S1
respectively) similar to the final rate expression of uncatalyzed
reaction (Eq. (8)). The exact dependence of ko on [H+]ꢀ3 was not
further experimentally verified.
In Scheme 2, GSꢀ quickly reduces Cu2+ to Cu+ (Eq. (9)) which
with another GSꢀ forms [Cu(I)(GSꢀ)] (Eq. (10)). Two of monomeric
-6.5
-7.0
-7.5
-8.0
-8.5
-9.0
-9.5
3.6. Effect of temperature on ko
The observed rate constant ko increases with temperature for
both catalyzed and uncatalyzed reaction (Tables S7 and S8). The
activation energy, Ea obtained from the Arrhenius plot of lnko ver-
sus Tꢀ1 (Figs. 10 and 11 respectively) of the uncatalyzed reaction
(97.1 1.7 kJ Mꢀ1) is almost three times more than of the catalyzed
reaction (29.9 1.2 kJ Mꢀ1).
4. Conclusion
The oxidation of glutathione (GSH) by superoxo complex 1
which is slow in acid media is catalyzed by Cu2+ ion. For both
the uncatalyzed and catalyzed reaction, the stoichiometric ratio
of [1]:[GSH] is 1:1. For uncatalyzed reaction, the observed rate con-
stant ko is proportional to [GSH]2 and [H+]ꢀ3 and for catalyzed reac-
tion ko is proportional to [GSH]2, [Cu]T2 and [H+]ꢀ3. The dependence
of ko on ionic strength of the media shows that the active reductant
3.20
3.25
3.30
3.35
103/T (K-1)
3.40
3.45
3.50
Fig. 10. lnko vs. 1/T for uncatalyzed reaction. Condition: [1] = 1.0 mM;
[GSH] = 20.0 mM; [H+] = 0.30 M; I = 0.5 M.

58
B. Singh et al. / Inorganica Chimica Acta 418 (2014) 51–58
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Acknowledgement
Authors thankfully acknowledge financial assistance from UPE
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2014.03.003.
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