Oxidation of glutathione by [FeIV(O)(N4Py)]2+: Characterization of an [FeIII(SG)(N4Py)]2+ intermediate

Article abstract of DOI:10.1021/ic100439n

The mechanism of glutathione (GSH) oxidation by a nonheme ferryl species has been investigated. The reaction of [Fe^IV(O)(N4Py)]²⁺ (1) with GSH in an aqueous solution leads to the rapid formation of a green intermediate, characterized as the low-spin ferric complex [Fe ^III(SG)(N4Py)]²⁺ (2) by UV-vis and electron paramagnetic resonance spectroscopies and by high-resolution time-of-flight mass spectrometry. Intermediate 2 decays to form the final products [Fe ^II(OH₂)(N4Py)]²⁺ and the disulfide GSSG over time. The overall reaction was fit to a three-step process involving rapid quenching of the ferryl by GSH, followed by the formation and decay of 2, which are both second-order processes.

Full text of DOI:10.1021/ic100439n

Inorg. Chem. 2010, 49, 4759–4761 4759
DOI: 10.1021/ic100439n
Oxidation of Glutathione by [FeIV(O)(N4Py)]2þ: Characterization of an
[FeIII(SG)(N4Py)]2þ Intermediate
Ashley A. Campanali, Timothy D. Kwiecien, Lew Hryhorczuk, and Jeremy J. Kodanko*
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202
Received March 5, 2010
The mechanism of glutathione (GSH) oxidation by a nonheme
ferryl species has been investigated. The reaction of [FeIV(O)-
(N4Py)]2þ (1) with GSH in an aqueous solution leads to the rapid
formation of a green intermediate, characterized as the low-spin
ferric complex [FeIII(SG)(N4Py)]2þ (2) by UV-vis and electron
paramagnetic resonance spectroscopies and by high-resolution
time-of-flight mass spectrometry. Intermediate 2 decays to form the
final products [FeII(OH2)(N4Py)]2þ and the disulfide GSSG over
time. The overall reaction was fit to a three-step process involving
rapid quenching of the ferryl by GSH, followed by the formation and
decay of 2, which are both second-order processes.
process of GSH oxidation, and kinetic studies that have
elucidated the overall reaction mechanism. This study has
relevance to biology and the protection of cells by GSH against
damage by ferryl compounds.
Previous studies in our laboratory11 revealed that ferryl 1 is
rapidly quenched by the cysteine derivative Ac-Cys-NHtBu
in1:1 H2O/MeCN, whichleads to theformation ofa disulfide
product. During data collection, the generation of a new
green species with an absorbance maximum at 658 nm was
observed by UV-vis spectroscopy after the disappearance of
1 (λmax=680 nm), which was present for ca. 1 h. However,
only trace amounts of the species were observed, and the
formation suffered from low reproducibility. Because this
absorbance fell within the range of known S f FeIII ligand-
to-metal charge-transfer (LMCT) bands,12,13 we surmised
that the new species was likely a ferric thiolate. Therefore, we
investigated the reactivity of related thiols with ferryl 1 and
quickly settled upon GSH because the generation of an
intermediate green species was highly reproducible and the
reaction could be performed in an aqueous buffer, without
the need for MeCN. Interestingly, many other thiols failed to
form the green species, which may provide evidence for the
structure of the green intermediate (vide infra).
Glutathione (GSH) is a ubiquitous tripeptide involved in
many different aspects of cell growth and regulation (Figure 1).
Arguably, its most important role is as an antioxidant, where it
acts as a key player in defense and survival. GSH protects cells
against oxidative stress by rapidly quenching reactive oxygen
species, including hydroxyl radical and superoxide. This role
is well understood.1 In contrast, the reactivity of GSH with
metal-based oxidants such as ferryls [iron(IV) oxo species],
which are powerful oxidizing agents generated by heme2,3 and
nonheme enzymes,4 has received much less attention.5-7 This is
worth investigating because GSH is thought to be the primary
line of defense against detrimental ferryl species, such as ferryl
myoglobin, in cells.8 In this Communication, we report the
reaction of GSH with a synthetic ferryl [FeIV(O)(N4Py)]2þ
(1).9,10 Included are the characterization of a low-spin ferric
species [FeIII(SG)(N4Py)]2þ (2), which is an intermediate in the
The reaction of ferryl 1 with GSH was investigated in detail.
To generate 1,14,15 [FeII(N4Py)(MeCN)](ClO4)2 (2 mM)16
was dissolved in 100 mM acetate buffer (pH = 6.02) and
treated with 2 equiv of aqueous peracetic acid, which led to the
maximum generation of 1, as judged by UV-vis spectroscopy.
The reaction of 1 (1 mM) with GSH (5 mM) showed the rapid
formation of a green intermediate with λmax=650 nm (<45 s),
clearly distinct from 1 (see Figure S2 in the Supporting
Information), followed by a slower decomposition of the
*To whom correspondence should be addressed. E-mail: jkodanko@
chem.wayne.edu.
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B. J. J. Am. Chem. Soc. 1981, 103, 2884–2886.
(3) Denisov Ilia, G.; Makris Thomas, M.; Sligar Stephen, G.; Schlichting,
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(11) Abouelatta, A. I.; Campanali, A. A.; Ekkati, A. R.; Shamoun, M.;
Kalapugama, S.; Kodanko, J. J. Inorg. Chem. 2009, 48, 7729–7739.
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Commun. 2005, 1405–1407.
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r
2010 American Chemical Society
Published on Web 05/06/2010
pubs.acs.org/IC

4760 Inorganic Chemistry, Vol. 49, No. 11, 2010
Campanali et al.
Figure 1. Structures of 1 and GSH.
Figure 3. High-resolution TOF-MS of 2 with m/z 364.5944 (left) and a
calculated fit (right).
Figure 2. Reaction of 1 (1 mM) with GSH (5 mM); UV-vis data for the
decay of the intermediate with λmax = 650 nm and the regeneration
of [FeII(OH2)(N4Py)]2þ at λmax = 450 nm. Inset: Time course decay of
λmax = 650 nm and time course regeneration of λmax = 450 nm.
green species, which coincided with the regeneration of
[FeII(OH2)(N4Py)]2þ (λmax=450 nm; Figure 2). The yield of
[FeII(OH2)(N4Py)]2þ was 89% at the end of the reaction,
based on an absorbance measurement using its extinction
coefficient under these conditions (1620 M-1 cm-1). An
isosbestic point was observed at 510 nm, indicating that there
were no long-lived intermediates in the conversion of the green
species into the FeII product. Following the decay, the yield of
oxidized disulfide GSSG, based on GSH consumption, was
determined by Ellman’s reagent to be 90 ( 20% based on 1.
The formation of GSSG was verified by electrospray ioni-
zation mass spectrometry (MS). No other oxidized GSH
products, such as sulfenic, sulfinic, or sulfonic acids, were
detected.
Control experiments were performed to determine if the
green intermediate contained iron, GSH, and N4Py. The
absorption at λmax=650 nm, indicative of a S f FeIII LMCT
band rather than a d-d transition based on its extinction
coefficient (vide infra), was not observed in control experi-
ments with FeII(ClO4)2 or FeIII(ClO4)3 salts alone, which
suggests a role for the ligand N4Py in the complex. Further-
more, when the aliphatic or aromatic thiols EtSH or PhSH
were treated with 1, no species with an absorbance band in
the visible region were detected by UV-vis spectroscopy.
The reaction of GSH with [FeII(N4Py)(MeCN)](ClO4)2 did
not show the rapid formation of an absorption band at
650 nm, confirming a role for 1 in the formation of the green
species.17 However, if aqueous solutions of FeII(N4Py)-
(MeCN)](ClO4)2 were allowed to oxidize in air before GSH
was added, the green species was observed to generate
rapidly, which is consistent with the reaction of a ferric
intermediate with GSH leading to the formation of the green
species, as indicated in the proposed mechanism (vide infra).
After determination of what was responsible for the forma-
tion of the green intermediate, other characterization data
were collected.
Figure 4. X-band EPR spectra from the reaction of 1 (1 mM) with GSH
(5 mM). The sample was frozen in liquid N2 1 min after mixing. Inset:
Expansion of the spectrum showing a shoulder at g=2.19. Experimental
conditions: temperature, 120 K; microwaves, 1.0 mW at 9.34 GHz;
modulation, 2.0 G, receiver gain, 14000.
The reaction of 1 and GSH was followed by MS. At early
time points, the high-resolution time-of-flight (TOF)-MS
spectrum displayed a prominent ion cluster with a dominant
peak at m/z 364.5944. This cluster displayed an isotopic
distribution that fit with the molecular formula [FeIII(SG)-
(N4Py)]2þ (denoted as 2, Figure 3). In this dication, the ligand
derived from GSH was treated as a monoanion (i.e., GSH -
Hþ), as would be expected if the thiolate of GSH were
deprotonated and bound to FeIII and the rest of the GSH
ligand was formally neutral. In addition, a separate ion cluster
was observed with a dominant peak at m/z 728.1821 (see the
Supporting Information), which was fit to the formula
[FeIII(SG)(N4Py)]þ, where the GSH portion of the species
was treated as a dianion (i.e., GSH - 2Hþ), presumably
because of deprotonation of both carboxylate groups and
protonation of the amine on the terminus of GSH. After 1 h at
room temperature, these ion clusters gave way to a major ion
cluster with m/z 220.418, which is consistent with the mole-
cular formula [FeII(OH2)(N4Py)]2þ
.
The green intermediate 2 was characterized by electron
paramagnetic resonance (EPR) spectroscopy. Spectra were
acquired at multiple time points during the formation and
decay of 2 (see the Supporting Information). A new species
was detected with g values of 2.17 and 1.98 (Figure 4), which
formed and then decayed on the same time scale as the
absorption band with λmax=650 nm, consistent with its
assignment as 2. The EPR spectrum in Figure 4 shows an
apparent shoulder for the resonance at 2.17, suggesting that a
third resonance with a slightly higher g value may be present
but is not resolved, whichwould indicate rhombic rather than
axial symmetry. In either case, the EPR spectrum in Figure 4
is consistent with intermediate 2 being a low-spin FeIII species
(17) The oxidation of GSH by [FeII(N4Py)]2þ and the formation of 2 will
occur under aerobic conditions, but it is a much slower process; see Figure S3
in the Supporting Information.

Communication
Inorganic Chemistry, Vol. 49, No. 11, 2010 4761
because the data agree well with the related species
[FeIII(X)(N4Py)]2þ (X=OH, OOH).18
To probe the mechanism of GSH oxidation further, kinetic
data were collected for the reaction of 1 (1 mM) with variable
concentrations of GSH (5-25 mM), and the UV-vis data
were fit using the kinetic modeling software Dynafit.19
Because of the fast generation of 2 (<1 min to reach
completion), a stopped-flow apparatus was employed. Under
all conditions, greater than 50% decay of the ferryl 1 was
complete within the deadtime of mixing, which places a lower
limit for the rate constant of ferryl quenching (k1) by GSH
at 1 ꢀ 104 M-1 s-1. On the basis of previous studies with Ac-
Cys-NHtBu and our visual observation of a transient yellow
color within the first several seconds of the reaction, the
formation of the ferric intermediate [FeIII(OH)(N4Py)]2þ
(labeled I in eqs 1 and 2) is proposed for this first step.20
The next two stepsof the reaction, the formation and decay of
the green intermediate 2, were evaluated using several pro-
posed mechanisms (see the Supporting Information for
further details). First- and second-order processes were
considered for both steps, and the data fit best to the
following reaction scheme involving a second-order reaction
between GSH and I [k2=14.3(1) M-1 s-1, eq2], followed by a
second-order decomposition of 2 that had zero kinetic
Figure 5. Possible structures of 2 showing η1 coordination by the GS-
thiolate or an alternative chelate structure.
an aqueous solvent, as indicated by the faster rate of decay
that was observed for 2 in 1:1 H2O/MeCN versus an aqueous
buffer alone (see the Supporting Information). In the
mixed solvent where MeCN is in large excess, MeCN may
trap the [FeII(N4Py)]2þ intermediate and make homolysis
irreversible by forming the stable low-spin ferrous complex
[FeII(MeCN)(N4Py)]2þ. However, other effects, such as a
change in the dielectric constant of the solvent medium, as
well as other reaction mechanisms, cannot be ruled out at this
point.
A green intermediate was observed in the reaction of 1 with
GSH and not with other thiols. Several models for the
structure of 2 are possible (Figure 5), including the simplest
model that wouldinvolve η1 binding of the GS- thiolate to an
octahedral FeIII center. Thiolate binding to the iron center is
supported by the absorption band at 650 nm. However, it is
possible that additional groups of GSH, such as the carbonyl
of the Cys residue, are coordinating as well. The existence of a
chelate structure is supported by the fact that coordination
numbers higher than 6 have been observed for N4Py com-
plexes18 and because GSH and not simple thiols form the
green intermediate. Future studies will be needed to under-
stand the coordination mode of 2.
order in GSH [k3=1.68(1) M-1 s-1, eq 3]. A value for ε650
=
1870 M-1 cm-1 was calculated for 2 by extrapolating the
linear portion of 1/A vs t to t=0, which agreed well with the
response factor that was optimized by the Dynafit software.
1
2þ
½FeIVðOÞðN4PyÞꢁ ð1Þ þ GSH f I þ GSSG ðk1Þ ð1Þ
2
2þ
I þ GSH f ½FeIIIðSGÞðN4PyÞꢁ ð2Þ þ H2O ðk2Þ ð2Þ
2þ
2þ
2½FeIIIðSGÞðN4PyÞꢁ ð2Þ þ 2H2O f 2½FeIIðOH2ÞðN4PyÞꢁ
þ GSSG ðk3Þ
ð3Þ
In conclusion, the kinetics of GSH oxidation by the ferryl 1
has been investigated, and the intermediate ferric thiolate 2
has been identified by EPR, high-resolution TOF-MS, and
UV-vis spectroscopy. Compound 2 is the first example of a
sulfur donor bound to the iron center of an N4Py complex.
The rapid quenching of ferryl 1 and subsequent oxidation of
GSH are relevant to biology and the protection of cells
against damage by metal-based oxidants. Further studies
concerning the oxidation of GSH and other biologically
relevant thiols are underway in our laboratory.
Given the observed second-order decay of 2, homolysis of
the FeIII-SG bond and dimerization of two GS• radicals to
form GSSG are proposed as the mechanism for eq 3.21 The
N-rich ligand set of N4Py may provide an extra driving force
for this homolysis step by favoring the FeII state over FeIII.
However, Fe-S bond homolysis is likely to be reversible in
(18) Roelfes, G.; Vrajmasu, V.; Chen, K.; Ho, R. Y. N.; Rohde, J.-U.;
Zondervan, C.; la Crois, R. M.; Schudde, E. P.; Lutz, M.; Spek, A. L.; Hage,
R.; Feringa, B. L.; Muenck, E.; Que, L., Jr. Inorg. Chem. 2003, 42, 2639–
2653.
(19) Kuzmic, P. Anal. Biochem. 1996, 237, 260–273.
(20) Collins, M. J.; Ray, K.; Que, L., Jr. Inorg. Chem. 2006, 45, 8009–
8011.
(21) Second-order decays have been noted for copper(II) thiolates of
cysteine. See: Baek, H. K.; Holwerda, R. A. Inorg. Chem. 1983, 22, 3452–
3456.
Acknowledgment. We thank Wayne State University
for its generous financial support of this research.
Supporting Information Available: Experimental procedures,
UV-vis, MS, and EPR spectra, and kinetic fits. This material is
available free of charge via the Internet at http://pubs.acs.org.