Zn(II) complexes of glutathione disulfide: Structural basis of elevated stabilities

Article abstract of DOI:10.1021/ic101212y

Glutathione disulfide (GSSG), a long disregarded redox partner of glutathione (GSH), is thought to participate in intracellular zinc homeostasis. We performed a concerted potentiometric and NMR spectroscopic study of protonation and Zn(II) binding properties of GSSG ((γECG)₂) and a series of its nine analogs with C-terminal modifications, tripeptide disulfides: (γECS)₂, (γECE)₂, (γECG-NH₂)₂, (γECG-OEt)₂, and (γEcG)₂; dipeptide disulfides, (γEC)₂ and (γEC-OEt)₂; and mixed disulfides, γECG-γEC and γECG-γEC-OEt. The acid-base and Zn(II) complexation properties in this group of compounds are strictly correlated to average C-terminal electrostatic charges. In particular, it was demonstrated that GSSG assumes a bent (head-to-tail) conformation in solution at neutral pH, which is controlled by electrostatic attraction between the protonated γ-amino groups of the Glu residue and the deprotonated C-terminal Gly carboxylates. This interaction modulates the ability of GSSG to coordinate Zn(II), both indirectly, by affecting the basicities of the amino groups, and directly, through the participation of the Gly carboxylates in the outer coordination sphere of the Zn(II) ion. A specific coiled structure of the major [Zn-GSSG]^2- complex is additionally stabilized by the formation of hydrogen bonds between glycinyl carboxylates and two Zn(II)-coordinated water molecules. The elevated stability of Zn(II)-GSSG complexes was demonstrated by competition with FluoZin-3, a fluorescent sensor with high Zn(II) affinity, commonly used in in vitro and in vivo studies. The potential biological functions and reactivity of GSSG complexes of Zn(II) ions are discussed.

Full text of DOI:10.1021/ic101212y

7
2 Inorg. Chem. 2011, 50, 72–85
DOI: 10.1021/ic101212y
Zn(II) Complexes of Glutathione Disulfide: Structural Basis of Elevated Stabilities
,†
‡
§
,‡,||
Artur Kr e- z_ el,* Jacek W ꢀo jcik, Maciej Maciejczyk, and Wojciech Bal*
†
Laboratory of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Tamka 2,
‡
5
0-137 Wroclaw, Poland, Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
§
Pawi nꢀ skiego 5a, 02-106 Warsaw, Poland, Department of Physics and Biophysics,
University of Warmia and Mazury, Oczapowskiego 4, 10-719, Olsztyn, Poland, and
||
Central Institute for Labour Protection;National Research Institute,
Czerniakowska 16, 00-701 Warsaw, Poland
Received June 17, 2010
Glutathione disulfide (GSSG), a long disregarded redox partner of glutathione (GSH), is thought to participate in
intracellular zinc homeostasis. We performed a concerted potentiometric and NMR spectroscopic study of protonation
and Zn(II) binding properties of GSSG ((γECG) ) and a series of its nine analogs with C-terminal modifications,
2
tripeptide disulfides: (γECS) , (γECE) , (γECG-NH ) , (γECG-OEt) , and (γEcG) ; dipeptide disulfides, (γEC)
2
2
2 2
2
2
2
and (γEC-OEt) ; and mixed disulfides, γECG-γEC and γECG-γEC-OEt. The acid-base and Zn(II) complexation
2
properties in this group of compounds are strictly correlated to average C-terminal electrostatic charges. In particular, it
was demonstrated that GSSG assumes a bent (head-to-tail) conformation in solution at neutral pH, which is controlled
by electrostatic attraction between the protonated γ-amino groups of the Glu residue and the deprotonated C-terminal
Gly carboxylates. This interaction modulates the ability of GSSG to coordinate Zn(II), both indirectly, by affecting the
basicities of the amino groups, and directly, through the participation of the Gly carboxylates in the outer coordination
2
-
sphere of the Zn(II) ion. A specific coiled structure of the major [Zn-GSSG] complex is additionally stabilized by the
formation of hydrogen bonds between glycinyl carboxylates and two Zn(II)-coordinated water molecules. The elevated
stability of Zn(II)-GSSG complexes was demonstrated by competition with FluoZin-3, a fluorescent sensor with high
Zn(II) affinity, commonly used in in vitro and in vivo studies. The potential biological functions and reactivity of GSSG
complexes of Zn(II) ions are discussed.
4
Introduction
cathepsin B activity. The latter function may be one example
of an emerging concept of redox signaling, as a major system
Glutathione disulfide (GSSG), also known as oxidized
glutathione, is the primary product of oxidation of GSH, the
predominant nonprotein cellular thiol, which is crucial for
antioxidant defense, xenobiotic metabolism, maintenance of
cellular membranes, and many other functions. The cyto-
solic concentration of GSSG, much lower than GSH, is
controlled by a specific export system. The earlier research
has therefore concentrated on GSH, with GSSG disregarded
as a mere byproduct of GSH reactivity. Novel findings stress
the importance of the actual GSH/GSSG balance. It is
estimated to account for over 90% of the control of the
5
of coordination of cell functions. The formation of mixed
disulfides in a reaction of GSSG with protein thiols is one of
several postulated pathways in this system. Another specific
function, discovered for GSSG, is the regulation of Zn(II)
content of metallothionein, which couples the redox state of
the cell with zinc metabolism.
trations of a eukaryotic cell range between approximately200
and 300 μM and do not vary significantly from one physio-
logical state to another. However, concentrations of thermo-
6
1,2
7-9
1,2
Typical total zinc concen-
9,10
dynamically and kinetically available zinc (“free” zinc) vary
3
intracellular redox potential, and as presented recently, it
controls the pacing of protein turnover, via the control of
(
(
4) Lockwood, T. D. Arch. Biochem. Biophys. 2003, 417, 183–193.
5) Kim, S. O.; Merchant, K.; Nudelman, R.; Beyer, W. F., Jr.; Keng, T.;
DeAngelo, J.; Hausladen, A.; Stamler, J. S. Cell 2002, 109, 383–396.
(6) Casagrande, S.; Bonetto, V.; Fratelli, M.; Gianazza, E.; Eberini, I.;
Massignan, T.; Salmona, M.; Chang, G.; Holmgren, A.; Ghezzi, P. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 9745–9749.
*
To whom correspondence should be addressed. Phone: þ48-71-375-2765
(
6
A.K.), þ48-22-592-2346 (W.B.). Fax: þ48-71-375-2608 (A.K.), þ48-22-
58-4636 (W.B.). E-mail: art@protein.pl (A.K.), wbal@ibb.waw.pl (W.B.).
(
(
1) Sies, H. Free Radic. Biol. Med. 1999, 27, 916–921.
(7) Jiang, L. J.; Maret, W.; Vallee, B. L. Proc. Natl. Acad. Sci. U.S.A.
2) Dickinson, D. A.; Forman, H. J. Biochem. Pharmacol. 2002, 64, 1019–
1
998, 95, 3483–3488.
1
026.
(8) Kr e- z_ el, A.; Maret, W. Biochem. J. 2006, 402, 551–558.
(9) Kr e- z_ el, A.; Maret, W. J. Biol. Inorg. Chem. 2006, 11, 1049–1062.
(10) Li, Y.; Maret, W. Exp. Cell Res. 2009, 315, 2463–2470.
(
3) Schafer, F. Q.; Buettner, G. R. Free Radic. Biol. Med. 2001, 30, 1191–
1
212.
pubs.acs.org/IC
Published on Web 12/08/2010
r 2010 American Chemical Society

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 73
by severalorders of magnitudeand are strongly dependent on
Scheme 1. Structures of Tri- and Dipeptide γ-Glutamyl Disulfides
Studied in This Work, Presented in Their Fully Protonated Forms
11-13
cellular compartmentalization.
Recent studies clearly
demonstrate “free” zinc concentration at a level of hundreds
of pM in various types of eukaryotic cells, indicating a strong
9,14-17
binding and tight buffering of this metal ion.
In
particular, studies made with HT-29 human colorectal ade-
nocarcinoma cells shed more light on correlations between
cellular redox states dependent on GSH/GSSG ratios
(
potential variation between -260 and -160 mV) and “free”
zinc, metallothionein (MT), and thionein (apometallothionein,
TR) levels.
8,9,18
The elevation of GSSG concentration in HT-29
cells induced by cell differentiation or apoptosis caused an
increase of “free” zinc concentration and a significant de-
crease of the T/MT ratio (due to the formation of MT and
8,9,19
oxidized thionin, Tox).
Studies in human cell lines in-
dicate that both GSH and GSSG have their separate roles in
20
zinc toxicity. It has also been hypothesized that zinc and
GSH, together with nitric oxide, collectively protect against a
21
host of diseases.
The overall GSSG concentration within the cell under
normal conditions is ca. 0.5% that of GSH. Taking into
account that the GSH level may reach 10 mM, the “normal”
GSSG concentration in the cell cytosol might be as high as
5
0 μM. Moreover, compartmentalization of GSH/GSSG
22,23
may lead to elevated GSSG concentrations locally.
GSH/GSSG ratios as low as 1:1 are thought to occur in
2
4
the endoplasmic reticulum. Also, under various stress
3
,4
conditions, the overall GSSG level may be elevated.
The low level of GSSG inside the cell is maintained by
active export of this molecule across the cellular membrane.
Therefore, high GSSG concentrations should be expected
1,2
locally near the outer cell surface. However, no quantita-
tive data are available in this respect.
A molecule of GSSG contains two amine functions and
four carboxylates. This donor set is qualitatively similar to
that of ethylendiaminetetraacetic acid (EDTA), although with
a much bigger separation of respective coordination sites
(Scheme 1). Therefore, GSSG can be expected to be a versatile
ligand for many metal ions. Despite that, not so many studies
of GSSG coordination abilities were published, all with the
(
11) Tomat, T. E.; Nolan, E. M.; Jaworski, J.; Lippard, S. J. J. Am. Chem.
Soc. 2008, 130, 15776–15777.
12) Colvin, R. A.; Laskowski, M.; Fontaine, C. P. Brain Res. 2006, 1085,
–10.
(
1
25,26
metal ions of the first transition row.
The amine/carbox-
(
13) Maret, W.; Li, Y. Chem. Rev. 2009, 109, 4682–4707.
14) Vinkenborg, J. L.; Nicolson, T. J.; Bollomo, E. A.; Koay, M. S.;
(
ylate donor set of the Glu moiety is the primary metal binding
site. This set, analogous to those present in simple R-amino
acids, is a unique feature of GSH and GSSG, due to the
presence of a γ-peptidic bond. The two such sites in GSSG
may bind metal ions in a concerted way, with an ML-type
stoichiometry (MH L, with n between 3 and -1), or inde-
pendently, resulting in M L-type complexes (M H L, with n
between 0 and -4). The equilibrium between ML-type and
M L-type complexes depends primarily on metal-to-ligand
Rutter, G. A.; Merkx, M. Nat. Methods 2009, 6, 737–740.
15) Atar, D.; Backx, P. H.; Appel, M. M.; Gao, W. D.; Marben, E. J.
Biol. Chem. 1995, 270, 2473–2477.
16) Ayaz, M.; Turan, B. Am. J. Physiol. Heart Circ. Physiol. 2006, 290,
H1071–H1080.
17) Haase, H.; Hebel, S.; Engelhardt, G.; Rink, L. Anal. Biochem. 2006,
52, 222–230.
18) Kirlin, W. G.; Cai, K.; Thompson, S. A.; Diaz, D.; Kavanagh, T. J.;
Jones, D. P. Free Radic. Biol. Med. 1999, 27, 1208–1218.
19) Kr e- z_ el, A.; Hao, Q.; Maret, W. Arch. Biochem. Biophys. 2007, 463,
88–200.
20) Walther, U. I.; Czermak, A.; Muckter, H.; Walther, S. C.; Fichtl, B.
(
(
(
n
3
(
2
2
n
(
2
1
ratios. The metal ions in an M L complex may, in principle,
(
2
Arch. Toxicol. 2003, 77, 131–137.
be the same, or different, but no heterometallic cases have
been reported to date.
(
21) Sprietsma, J. E. Med. Hypotheses 1999, 53, 6–16.
(
22) Soderdahl, T.; Enoksson, M.; Lundberg, M.; Holmgren, A.; Ottersen,
Only three preceding papers on Zn(II) coordination to GSSG
were published. Potentiometry was applied as the sole ex-
O. P.; Orrenius, S.; Bolcsfoldi, G.; Cotgreave, I. A. FASEB J. 2003, 17, 124–
1
2
1
26.
2
7,28
(
23) Monostori, P.; Wittmann, G.; Karg, E.; Turi, S. J. Chromatogr., B
perimental technique in two of them.
ZnHL, ZnL,
009, 877, 3331–3346.
27
and Zn L complexes were postulated to exist in solution.
2
(
24) Hwang, C.; Sinskey, A. J.; Lodish, H. F. Science 1992, 257, 1496–
502.
(
25) Kr e- z_ el, A.; Bal, W. Acta Biochim. Pol. 1999, 46, 567–580.
(26) Kr e- z_ el, A.; Bal., W. Bioinorg. Chem. Appl. 2004, 293–305.

7
4
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
1
13
29
The remaining study used H and C NMR. However, the
relatively high stability of the ZnL complex, apparent from
those studies, has neither been noted nor discussed in ther-
modynamic or structural terms. These issues have now
gained importance in the light of biochemical discoveries
regarding the potential physiological roles for Zn(II)-GSSG
interactions, mentioned above. The participation of the Gly
carboxylate in Zn(II) coordination was discussed on the basis
of 1D NMR spectra, with a conclusion that no interaction
25% piperidine in DMF. The coupling was monitored using the
Keiser (ninhydrin) test and TLC. The scale of synthesis was ca.
1
.3 mmol in terms of resin substitution; a 2.5-fold excess of
amino acids was used for additions. The cleavage was effected
using a mixture of trifluoroacetic acid, trifluoroethanol, dichloro-
methane, and dimercaptoglycol (v/v/v/v = 3:1:5:1) over a
period of 24 h, followed by precipitation with cold diethyl ether;
peptide cleavage from the resin; removal of the protecting
groups Mtt, Boc, and tBu; and washing with diethyl ether.
The yield of the crude unprotected peptides, complexed with
TFA, was 30-50%. The final purification was done using
HPLC (Hewlett-Packard) on an Alltech Econsil C18 10U pre-
parative column (22 ꢀ 250 mm, 5 μm grain), in a 0-100% 0.1%
TFA/water to 0.1% TFA/acetonitrile gradient, detected at
200 nm. The disulfides of all thiol peptides listed above were
synthesized either by oxidation with elemental iodine in water
solution or by oxidation with atmospheric oxygen in alkaline
water solution (controlled with NaOH) followed by purification
on HPLC, as described above. The pH value of 9.2 was chosen as
optimal for air oxidation of glutathione and its analogs, on the
2
9
occurred.
Our study on conformational preferences of GSH and its
analogs indicated a strong influence of the C-terminal residue
on acid-base properties of the N-terminal moiety in these
3
0
molecules. This property, overlooked in preceding works,
was demonstrated clearly in a series of C-terminally substi-
tuted peptides. Therefore, we decided to repeat this successful
strategy and undertook a systematic study of Zn(II) com-
plexation to GSSG and its analogs, using NMR and poten-
tiometry in a concerted fashion, to correlate thermodynamic
and structural properties of these complexes and thereby
obtain better insight into their possible interactions in vivo.
3
3
basis of our previous experiments. The mixed disulfides
γECG-γEC and γECG-γEC-OEt were obtained by oxygena-
tion of equimolar solutions of respective thiol peptides at
constant pH, 9.6 and 9.8, respectively. The reactions were
monitored and purified by HPLC as described above. The
purities and identities of all synthesized peptides for the purpose
of this study were finally confirmed by mass spectrometry, on a
Experimental Section
Materials. Peptides glutathione disulfide (GSSG), monoethyl
ester of glutathione (GSH-OEt), glutathione sulfonic acid
Finnigan MAT TSQ 700 instrument. The m/z values found/
calculated were for (γECS) : 673.1/673.2 (MþH) and 337.5/
þ
þ
(
GSO
ethylendiaminetetracetic acid disodium salt dihydrate (EDTA),
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),
NaOD (40% w/v in D O), 4-nitrophenyl acetate, and tris
4-nitrophenyl) phosphate were obtained from Sigma-Aldrich. The
peptides γ-EC and γ-EC-OEt were purchased from Bachem.
Zinc sulfate heptahydrate (ZnSO 7H O), potassium nitrate
), 0.1 M NaOH solution in water (titrant), methanol,
and ethanol were obtained from Merck KGaA. FluoZin-3
tetrapotassium salt was from Invitrogen. D O and DCl (30%
3
H) and S-methylglutathione (GSMe), L-glutamic acid,
2
2
þ
þ
338.1 (Mþ2H) ; for (γECE) : 756.8/757.2 (MþH) and 379.7/
2
2
þ
(
380.1 (Mþ2H) ; for (γECG-NH ) : 610.6/611.2 (MþH) and
2
2
2
þ
þ
þ
2
307.3/307.1 (Mþ2H) ; for (γECG-OEt) : 668.6/669.2 (MþH)
þ
2
2
2
(
and 336.3/336.1 (Mþ2H) ; for (γEC) : 499.3/499.1 (MþH)
2
þ
and 250.6/251.1 (Mþ2H) ; for γECG-γEC: 555.8/556.1
þ
2þ
4
3
2
(MþH) and 279.2/279.6 (Mþ2H) ; for γECG-γEC-OEt:
þ 2þ
(
KNO
3
640.9/641.2 (MþH) and 321.7/322.1 (Mþ2H) ; for (γEcG)2:
þ
2þ
.
613.5/613.2 (MþH) and 307.7/308.1 (Mþ2H)
2
Thermodynamic Studies. The protonation and Zn(II) stability
2
w/v in D O) were purchased from Cambridge Isotope Labora-
3
constants of GSSG in the presence of KNO (I = 0.1 M) were
tories, Inc. 3-(Trimethylsilyl)-1-propanesulfonic acid (DSS) was
from Dr. Glaser AG. Acetic anhydrate (Ac O), sodium hydro-
xide (solid), 35% aqueous hydrochloric acid, diethyl ether
2 2 2
(Et O), and hydrogen peroxide (H O , 30%, analytical grade)
were obtained from POCH (Gliwice, Poland).
determined at 19, 25, 30, and 37 °C using pH-metric titrations
over the pH range 2.5-11.0 (Molspin automatic titrator, Mol-
spin Ltd.) with 0.1 M NaOH as a titrant. Changes of pH were
monitored with a combined glass-Ag/AgCl electrode (InLab
2
4
22, Mettler Toledo) calibrated daily in hydrogen concentra-
3
4
Peptide Synthesis. The reduced thiol peptides γ-Glu-Cys-Ser
tions using 4 mM HNO3 titrations (I = 0.1 M). Sample
volumes of 1.5-2.0 mL and concentrations of 1-2 mM ligands
and 0-2 mM Zn(II) were used. The experimental data were
(
(
γECS), γ-Glu-Cys-Glu (γECE), and γ-Glu-Cys-Gly-NH2
γECG-NH ) were synthesized in the solid state according to the
2
3
0
35
Fmoc strategy, as described before. The γ-Glu-D-Cys-Glu
peptide (γEcG) was also synthesized using the Fmoc strategy,
The N-Fmoc-protected
analyzed using the SUPERQUAD software. Standard devia-
tions computed by SUPERQUAD refer to random errors. The
ΔH and TΔS values were obtained from linear fits of tempera-
ture dependences of ΔG values, calculated from values of ln
i
K (T) of individual protonation and complex formation reac-
tions. Standard deviations of these quantities derive from these
31,32
but on a 2-chlorotrityl chloride resin.
amino acids N-Fmoc-Gly-OH and N-Fmoc-D-Cys-(Mtt)-OH
were obtained from Nova Biochem (Merck KGaA), while the
N-terminal N-tBoc-Glu-(R-tBu) was obtained from Sigma-Al-
drich. The coupling agents 1-hydroxybenzotriazole (HOBt) and
dicyclohexylcarbodiimide (DCC) were purchased from Merck
KGaA; solvents trifluoroacetic acid, 2,2,2-trifluorotehanol, piper-
idine, acetonitrile, N,N-dimethylformide (DMF), and dichlor-
omethane (DCM) were obtained from Riedel-de Ha €e n GmbH
linear fits. The protonation and Zn(II) stability constants for
other compounds reported in this paper were obtained, at 25 °C
only, by the same methodology.
1
NMR Spectrometry. H NMR spectra of GSSG were re-
corded on a Bruker AMX-300 spectrometer at 300 MHz or a
UNITY500plus spectrometer (Varian Inc.), equipped with a
waveform generator unit Performa II, a high stability tempera-
(
Seeize, Germany). Fmoc protection groups were removed by
1
13 15
(
27) Varnagy, K.; Sovago, I.; Kozzowski, H. Inorg. Chim. Acta 1988, 151,
17–123.
28) Li, N. C.; Gawron, O.; Bascuas, G. J. Am. Chem. Soc. 1954, 76, 225–
29.
29) Postal, W. S.; Vogel, E. J.; Young, C. M.; Greenaway, F. T. J. Inorg.
ture unit, and a 5 mm H{ C/ N} PFG triple probe, at 500.606
MHz. D O was used as a solvent for all samples. The spectra are
1
2
2
(
expressed in δ (ppm) relative to DSS, which was used as an
(
Biochem. 1985, 25, 25–33.
(33) Kr e- z_ el, A.; Szczepanik, W.; Sokozowska, M.; Je z_ owska-Bojczuk, M.;
(
(
30) Kr e- z_ el, A.; Bal, W. Org. Biomol. Chem. 2003, 1, 3885–3890.
Bal, W. Chem. Res. Toxicol. 2003, 16, 855–864.
31) White, P. D.; Chan, W. C. In Fmoc Solid Phase Peptide Synthesis. A
(34) Irving, H.; Miles, M. G.; Pettit, L. D. Anal. Chim. Acta 1967, 38, 475–
Practical Approach; Chan, W. C.; White, P. D., Eds.; Oxford University Press:
New York, 2000; pp 9-39.
488.
(35) Gans, P.; Sabatini, A.; Vacca, A. J. Chem. Soc., Dalton Trans. 1985,
1195–1200.
(
32) Guy, C. A.; Fields, G. B. Methods Enzymol. 1997, 289, 67–83.

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 75
internal standard in one-dimensional spectra at 300 MHz. The
1D and 2D spectra at 500 MHz were calibrated using the HDO
signal at 4.78 ppm. 10 mM GSSG and 0 or 10 mM Zn(II) were
with only harmonic restraints applied on distances between
Zn(II) and its four donor atoms. Then, 30 ps of high-temperature
(1000 K) molecular dynamics was performed, followed by a
process of cooling down to 100 K over 6 ps, and by a potential
energy minimization. The resulting structures were refined (20
ps dynamics at 300 K) and minimized. During the annealing and
refinement procedure, all restraints were applied. The full
Lennard-Jones potential and distance-scaled electrostatic inter-
actions were used in this refinement. The 30 lowest energy
structures were analyzed and subdivided into families. The
maximum backbone RMSD between any two members of one
36
1
1
used for H experiments at 300 MHz. In a separate set of H
experiments at 500 MHz, the 1:1 samples of GSSG and Zn(II)
were measured in a concentration range of 0.1-100 mM.
Simulations of 1D NMR proton spectra which yielded chemical
shifts and coupling constants of Gly protons were performed
with the program NMRSIM, kindly provided by professor W.
Danikiewicz, Institute of Organic Chemistry, Polish Academy
1
3
of Sciences, Warsaw, Poland. The 1D C spectra of GSSG and
its Zn(II) complex in D O, pH* (uncorrected readings in D O of
a pH-meter calibrated with standard buffers in H O) 8.2, at a 1:1
ratio, were measured on a UNITY500plus spectrometer at
25.889 MHz, in a concentration range of 25 mM to 100 mM.
The 2D NMR spectra of GSSG, GSSG-(OEt) , and their Zn(II)
˚
2
2
family was set to 0.5 A. The VMD 1.8.1 software was used for
structure visualization.
4
3
2
Fluorimetry. The Zn(II) competition between the Zn(II)-
sensitive fluorescent chelating sensor FluoZin-3 was studied
by fluorescence changes using Jasco FP 750 spectrofluorometrat
1
2
4
4
complexes were recorded for disulfide and Zn(II) concentra-
tions of 20 mM in D O, at pH* 8.4 for GSSG and 8.8 for
25 °C. Samples containing 0.1 μM FluoZin-3 tetrapotassium
2
and 0.05 μM ZnSO in a 50 mM HEPES buffer (pH = 7.4, I =
4
3
7,38
GSSG-(OEt) . The ROESY experiments
2
were measured in
56 increments and with 0.25 or 0.4 s mixing times. The
0.1 M from KNO ) were titrated either with a 30 mM or with a
300 mM solution of GSSG in fluorimeter quartz cuvettes (1 cm
2
3
1
13
H/ C}gHSQC experiments
39,40
{
decoupled mode, with a carbon spectral width of 30 k and 512
were performed in the proton
ꢀ1 cm) followed by 5 min of equilibration. Relative changes of
the Zn(II)-FluoZin-3 complex concentration were monitored
by fluorescence measurements (F). These data were used for
determination of the exact FluoZin-3 concentration and for
calculations of competition equilibria between FluoZin-3 and
GSSG. The fluorescent complex was excited at 492 nm and
measured in the range of 495-600 nm (maximum emission at
increments. The spectra were recorded at 25 °C.
Structure Calculations. Volumes of cross-peaks provided by
ROESY spectra were calibrated for interproton distances ac-
cording to the common formula V = A/R . The calculation of
6
parameter A was based on the fact that the highest cross-peak
˚
measured corresponded to a distance of 2.4 A (see Supporting
2
þ
517 nm). Concentration of free zinc ([Zn ]) was calculated as
described previously by independent calibration of fluorescence
Information Tables S1 and S2). In this way, the lowest cross-
˚
˚
peaks corresponded to distances of 5 A and 5.8 A for Zn-
II)-GSSG and Zn(II)-(γECG-OEt) , respectively. Thirteen
in the presence of ZnSO
4
excess (Fmax) and EDTA (Fmin
)
2
þ
(
according to the formula [Zn ] = K ꢀ (F - Fmin)/(Fmax
-
is a dissociation constant (8.9 nM) of the Zn-
2
d
structurally relevant restraints, including seven inter-residual
ones, were included in the simulated annealing procedure for the
Zn(II)-GSSG complex. The numbers of restraints for the Zn-
F
min). K
d
(II)-FluoZin-3 complex measured under the same conditions
used here (50 mM HEPES buffer, pH = 7.4, I = 0.1 M).
9
,44,45
(
II)-(γECG-OEt) complex were 14 and 6, respectively. Each
2
Results
restraint included restrictions for four distances (two intramo-
nomeric and two intermonomeric) in the simultaneous applica-
tion of average distances (type SUM using XPLOR software).
Acid-Base Properties of GSSG and Its Analogs. Pro-
tonation constants of GSSG (Scheme 1) were obtained by
potentiometry at four temperature values, 19, 25, 30, and
4
1
This fact stems from the symmetry of complexes studied. The
restraints enforcing the equivalence of both monomeric units of
complexes were additionally included for every NOE. Harmonic
distance restraints were applied for bonds connecting the Zn(II)
ion and its four donor atoms provided by GSSG or (γECG-
3
7 °C, to enable calculations of thermochemical para-
meters of protonation phenomena. These constants are
presented in Table 1 and are in good agreement with the
previous determinations at 25 °C
The constants determined at 25 °C were published pre-
viously in our study on Ni(II) complexes of GSNO and
27,46-48
49,50
and at 37 °C.
OEt)
water molecules and GSSG or (γECG-OEt)
the calculations. Two further dihedral angle restraints were
added for χ dihedrals of Cys residues. The χ dihedral governs
the values of the JAB coupling constants in the Cys residue,
2
. One should emphasize the fact that no restraints between
2
were included in
5
1
1
1
GSSG. A reliable determination of the sixth most acidic
constant by potentiometry was impossible under our
experimental conditions. Several potentiometric determi-
nations of this constant were published at 25 and
4
2
according to the formula given by Perez at al. A high value of
one of these coupling constants (11.4 ( 0.2 Hz for Zn-
(
2
II)-GSSG and 10.7 ( 0.2 for Zn(II)-(γECG-OEt) ) and a
2
7,47,52-54
37 °C.
The broad range of values obtained, from
small value of the second one (4.3 ( 0.2 Hz and 4.2 ( 0.2 Hz,
respectively), together limited the χ value to the regions around
1
(43) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14,
either -60 or þ180°. Six-hundred conformations were calcu-
3
3–38.
lated for each complex using a simulated annealing procedure
(
(
44) Kr e- z_ el, A.; Maret, W. J. Am. Chem. Soc. 2007, 129, 10911–10921.
1
with square-well potential constraints included, 300 with χ =
45) Pomorski, A.; Otlewski, J.; Kr e- z_ el, A. ChemBioChem 2010, 11, 1214–
1
80 and 300 with χ = -60. The initial structure was minimized
1218.
1
(46) Theriault, Y.; Cheesman, B. V.; Arnold, A. P.; Rabenstein, D. L.
Can. J. Chem. 1984, 62, 1312–1319.
(
36) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62,
(47) Noszal, B.; Szakacs, Z. J. Phys. Chem. B 2003, 107, 5074–5080.
(48) Piu, P.; Sanna, G.; Zoroddu, M. A.; Seeber, R.; Basosi, R.; Pogni, R.
J. Chem. Soc., Dalton Trans. 1995, 1267–1271.
(49) Micheloni, M.; May, P. M.; Williams, D. J. Inorg. Nucl. Chem. 1978,
7
512–7515.
37) Bothner-By, A. A.; Stephens, R. L.; Lee, J.-M.; Warren, C. D.;
(
Jeanloz, R. W. J. Am. Chem. Soc. 1984, 106, 811–813.
(
(
38) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 63, 207–213.
39) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114,
40, 1209–1219.
(50) Formicka-Kozzowska, G.; Kozzowski, H.; Je z_ owska-Trzebiatowska,
1
0663–10665.
40) Kontaxis, G.; Stonehouse, J.; Laue, E. D.; Keeler, J. J. Magn. Reson.,
Ser. A 1994, 111, 70–76.
41) Br u€ nger,A. T. X-PLOR, a System for X-ray Crystallography and
NMR; Yale University Press: New Haven, CT, 1992.
42) P ꢀe rez, C.; L o€ hr, F.; R u€ terjans, H.; Schmidt, J. M. J. Am. Chem. Soc.
001, 123, 7081–7093.
B. Acta Biochim. Pol. 1979, 26, 239–248.
(
(51) Kr e- z_ el, A.; Bal, W. Chem. Res. Toxicol. 2004, 17, 392–403.
(52) Powell, K. J.; Town, R. M. Austral. J. Chem. 1995, 48, 1039–1044.
(53) Shtyrlin, V. G.; Zyavkina, Y. I.; Ilakin, V. S.; Garipov, R. R.;
(
Zakharov, A. V. J. Inorg. Biochem. 2005, 99, 1335–1346.
(54) Pessoa, J. C.; Tomas, I.; Kiss, T.; Buglyo, P. J. Inorg. Biochem. 2001,
84, 259–270.
(
2

7
6
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
a
3
Table 1. Thermodynamic Description of GSSG Protonation at I = 0.1 M (KNO )
b
c
log β
i
pK
a
at 25 °C
thermochemical constants
d
e
f
e
species
reaction
19 °C
25 °C
9.695(4)
30 °C
9.552(3)
37 °C
potentiometry NMR
-ΔH
ΔS
-ΔG
3
-
, HL / L4- þ H
3-
þ
HL
K
1
K
2
K
3
K
4
K
5
K
6
9.831(6)
9.459(4)
9.695
8.888
3.941
3.165
2.409
9.71(4)
8.81(4)
3.96(2)
3.14(2)
2.40(2)
1.51(2)
37(4)
34(3)
63(12)
56(10)
119(4)
85(4)
243(4)
229
55.35(2)
50.74(2)
22.50(3)
18.07(4)
13.75(6)
2
-
2-
3-
þ
þ
H
2
H
3
H
4
H
5
H
6
L
L
L
L
L
, H
, H
2
L
L
/ HL þ H
18.806(5) 18.583(4) 18.340(3) 18.086(4)
22.685(9) 22.524(6) 22.320(5) 22.103(6)
-
-
2-
3
/ H
2
L
þ H
-13(1)
-7(1)
-
þ
,H
4
L / H
3
L
þ H
þ
25.81(1)
28.00(2)
29.39
25.690(7) 25.500(6) 25.303(8)
28.088(7) 28.10(1)
þ
þ
þ
, H
, H
5
L
L
/ H
4
L þ H
28.10(1)
29.71
-59(1)
2
þ
2þ
þ
g
g
g
g
g
g
g
g
9.2
6
/ H
5
L
þ H
29.88
30.10
1.61
-59
a
b
þ i
i i
β = [H L]/([L][H ] ). Calculated for individual protonation reactions at
d 47 e
c
Statistical errors of constant determinations are given in parentheses.
and their temperature dependencies. Measured in D
2
5 °C, using potentiometric values of pK
a
2
O and I ∼ 0.01, and recalculated. Units are kilojoules
-1
f
-1
-1
g
per mol (kJ mol ). Units are joules per Kelvin per mol (J K mol ). Beyond the measurement range, extrapolated on the basis of statistical
considerations (see text).
4
8,52,55
1
.60 to 2.34,
potentiometric determination of this constant, as pointed
corresponds to a low reliability of
5
3
out previously.
NMR provided an alternative method of establishing
all protonation constants of GSSG, including the most
5
6,57
acidic one.
However, NMR titrations provide data
on the acidities of individual groups, rather than overall
dissociation constants, produced by potentiometry. To
convert the former (Supporting Information Table S1) to
the latter, one can use a range normalization function
(eq 1) where p and u indexes indicate protonated and
unprotonated states, respectively.
F ¼ ðδ - δ Þ=ðδ - δ Þ
ð1Þ
p
u
p
This function was constructed for chemical shifts of R-
Glu (separately in low and high pH) and Gly protons
Figure 1. A comparison of the potentiometry derived species distribu-
tion of protonic species of GSSG at 25 °C and I = 0.1 (KNO ) with pH
dependencies of chemical shifts of nonexchangeable protons specific to
, K , K , and K (R-Glu, blue circles) and to K and K (Gly, red circles).
Protonation reactions assigned according to Table 1. The thick dark
3
(only low pH) and fitted to eq 2, valid for two overlapping
protonation processes.
K
1
2
5
6
3
4
5
8,59
2-
pH - pK1
2ꢀpH - ðpK1þpK2Þ
yellow line represents the main species H
2
L
presented at physiological
1
0
þ 2 ꢀ 10
pH, which contains four deprotonated carboxyl groups.
F ¼
ð2Þ
pH - pK1
þ 102ꢀpH - ðpK1þpK2Þ
Protonation constants established for GSSG by poten-
1
þ 10
tiometry and NMR are included in Table 1. The assign-
1
13
ment of H and C NMR spectra of GSSG, based on
homo- and heteronuclear correlation spectra, was in
2
9,60
agreement with the literature.
The match between
the NMR and potentiometry-derived values is excellent,
as presented in Figure 1, on which the NMR titration
curves are overlaid on the protonic species distribution
derived from potentiometric data.
A more detailed analysis of dependence of chemical
shifts on pH revealed a specific sensitivity of the Gly
protons to the amine deprotonation, shown in Figure 2.
The Cys protons did not show such dependence, although
they are located much closer to the amine residues in the
peptide sequence. The corresponding pK values are pro-
vided in Supporting Information Table S1.
The thermochemical parameters of GSSG protonation
reactions are presented in Table 1 as well. These reversible
reactions were defined as association processes to make
Figure 2. A comparison of pH dependencies of chemical shifts of
nonexchangeable protons of GSSG (10 mM, 25 °C) in the alkaline pH
range, underlining similarities between profiles for Glu (A) and Gly
(
B) protons, as opposed to Cys protons (C). Individual protons are
color-coded as follows: R-Glu (black), average of β-Glu (red), γ-Glu
(
yellow).
green), average of Gly (purple), R-Cys (blue), average of β-Cys (dark
(
55) Palmer, A. G., III; Cavanagh, J.; Wright, P. E.; Rance, M. J. Magn.
Reson. 1991, 93, 151–170.
(
(
(
56) Kr e- z_ el, A.; Bal, W. J. Inorg. Biochem. 2004, 98, 161–166.
the sign of ΔG negative. The ΔH and ΔS values were
57) Rabenstein, D. L.; Sayer, T. L. Anal. Chem. 1976, 48, 1141–1146.
ꢀ
58) Kr e- z_ el, A.; Szczepanik, W.; Swi a- tek, M.; Je z_ owska-Bojczuk, M.
calculated from linear relationships ln K (T).
i
Bioorg. Med. Chem. 2004, 12, 4075–4080.
The protonation equilibria of analogs and derivatives
of GSSG were studied by potentiometry at 25 °C. All
constants obtained are presented in Table 2.
(
59) Dor ꢁc ꢀa k, V.; Kr e- z_ el, A. Dalton Trans. 2003, 2253–2259.
(
60) Kr e- z_ el, A.; W oꢀ jcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun.
(
Camb.) 2003, 704–705.

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 77
a
Table 2. Cumulative Protonation Constants (log β
Analogs of GSSG at T = 25 °C, I = 0.1 M (KNO
i
Values) for Derivatives and
Table 4. Thermodynamic Description of Reactions of Zn(II) Complexation to
GSSG at T = 25 °C, I = 0.1 M (KNO )
3
b
a
3
)
peptide HA
H
2
A
H
3
A
H
4
A
H
5
A
H
6
A
reaction
thermochemical constants
b
c
b
association reactions
-ΔH
ΔS
-ΔG
log K
tripeptide disulfides
9.75(1) 18.43(1) 22.14(2) 24.73(2) 26.62(5) c
9.912(7) 18.878(7) 23.99(1) 28.25(1) 31.73(1) 34.40(2)
9.426(8) 18.117(7) 20.77(1) c
9.564(9) 18.305(8) 21.04(2) c
Zn þ HL / ZnHL-
2
þ
3-
-15(2)
21(3)
43.7(9)
23(3)
136(8)
84(11) 45.95(2)
58(3) 61.1(4)
25.7(4)
4.50
8.05
10.71
2.66
(
(
(
(
(
γECS)
2
2þ
4-
2-
c
Zn þ L / ZnL
γECE)
γECG-NH
γECG-OEt)
γEcG)
2
2þ
4-
2
Zn þL / Zn
2
L
L
2
)
2
2þ
Zn þ ZnL / Zn
2
-26(11) 15.2(4)
2
9.72(1) 18.51(1) 22.45(3) 25.78(3) 28.34(4)
protonation reactions
b
c
b
pKa
2
-
ΔH
ΔS
-ΔG
dipeptide disulfides
2þ
2-
/ ZnHL- þ H
þ
þ
Zn þ H
2
L
49(1)
-22(2)
-80(3) 25.1(4) 4.39
141(8) 19.9(5) 3.48
0.7(1) 115.4(3) 35.06(2) 6.14
36(2) 67(7) 55.63(2) 9.75
2
þ
-
Zn þ ZnHL / Zn
2
L þ H
(
(
γEC)
γEC-OEt)
2
9.799(3) 18.772(7) 22.05(1) 23.42(9) c
9.604(5) 18.358(5) 20.81(2) c
c
-
2-
þ
ZnHL / ZnL þ H
2
2
-
3-
þ
ZnL / ZnH-1
L
þ H
mixed disulfides
a
Statistical errors of constant determinations are given in parenth-
b
-1
c
γECG-γEC
9.76(1) 18.66(1) 22.53(2) 25.26(3) 27.68(6) c
γECG-γEC-OEt 9.65(1) 18.47(1) 21.82(2) 24.17(6) c
eses. Units are kilojoules per mole (kJ mol ). Units are joules per
1
Kelvin per mole (J K mol ).
-
-1
other
simplify the presentation. The primary role of the Glu
residue in Zn(II) binding at and above pH 6 is clear.
Figure 3 (right-hand panels) presents subtle effects in
chemical shifts exerted by the presence of Zn(II). The
complex formation removed the equivalence of β-Glu
protons and increased the signal separation of β-Cys
S-MeGSH
9.124(3) 12.682(5) 14.66(2)
9.284(2) 12.842(4) 14.76(3)
9.588(7) 13.790(7) 16.00(2)
3
GSO H
Glu
a
þ i
i i
β = [H L]/([H ] [L]). Statistical errors of constant determinations
c
b
are given in parentheses. log β beyond the measurement range.
protons. Also, one of the Gly protons (GlyH ) was
1
Table 3. Cumulative Stability Constants for Zn(II) Complexes with GSSG at
I = 0.1 M (KNO ) and Various Temperatures
3
specifically sensitive to Zn(II) binding. Figure 4 compares
selected chemical shift profiles with potentiometry-based
distributions of complexes at 25 °C, calculated for con-
a
b
log βijk
1
centrations used in H NMR titrations. This figure
species
19 °C
25 °C
30 °C
37 °C
demonstrates the match between the results of these two
experimental methods. One can clearly see that the R-Glu
profile reflects the formation of all four complex species,
-
ZnHL
14.24(4)
8.097(3)
10.86(5)
14.19(8)
8.049(3)
10.71(8)
14.08(2)
7.940(3)
10.57(6)
-1.674(5)
14.03(5)
7.894(3)
10.41(8)
-1.589(4)
2
-
ZnL
Zn
2
L
and that the GlyH proton is selectively sensitive to the
1
3
-
ZnH-1
L
-1.747(6)
-1.696(6)
2-
formation of the ZnL complex.
An absence of dimerization/oligomerization of the
complexes at a pH* of 8.2, which effectively corresponds
a
Statistical errors of constant determinations are given in parenthe-
βijk = [Zn H L ]/([Zn] [H ] [L] ).
i j k
b
i
þ j
k
ses.
5
6
2-
to a pH of 8.0 (the ZnL species predominates at ca.
90% of total Zn(II) under such conditions), was con-
firmed by the absence of changes in chemical shifts of
Thermodynamic Aspects of Zn(II) Complexation. The
analysis of potentiometric titrations, performed at var-
ious temperatures, uniformly indicated the formation of
four different complex species in the Zn(II)-GSSG system,
1
complexed GSSG in H NMR spectra recorded for
equimolar Zn(II) and GSSG in the range of concentra-
-
2-
3-
13
tions between 0.1 mM and 100 mM, and in the C NMR
monomeric ZnHL , ZnL , ZnH
L
1
, and a bimetallic
-
Zn L. The stability constants for these complexes are
spectra of the same samples, between 25 mM and 100 mM
(data not shown). The ROESY spectrum of the GSSG-
Zn(II) complex was recorded with the sample containing
2
presented in Table 3, while Table 4 presents thermody-
namic constants for formal and actual Zn(II) binding
reactions in the Zn(II)-GSSG system. The ΔH and ΔS
values for these reactions were calculated, as with pro-
20 mM GSSG and 20 mM Zn(II) in D O at a pH* of 8.4,
2
which effectively corresponds to a pH of 8.2 (pH chosen
in such way to match the highest complex occupancy).
5
6
tonation constants, from linear relationships ln K (T).
i
2
-
The coordination of Zn(II) to GSSG analogs was
studied only by potentiometry at 25 °C, except for the
complex with (γECG-OEt) , which was additionally stud-
ied by 2D NMR (see below). Stability constants of
complexes of disulfide analogs are presented in Table 5,
and those of the nondisulfide ones are given in Table 6.
These tables also contain competitivity index (CI) values,
which allow for a comparison of overall Zn(II) bind-
ing abilities of ligands forming complexes of various
stoichiometries.
Structural Aspects of Zn(II) Complexation. Figure 3
left-hand panel) presents the global comparison of pH
dependence of chemical shifts of individual GSSG pro-
tons without and with Zn(II). The signals from chemically
equivalent β-Glu and β-Cys protons were averaged to
This sample contained ca. 92% of the ZnL species. The
spectrum provided eight of nine expected intraresidual
crosspeaks, as well as nine interresidual crosspeaks: three
between the Glu and Cys residues, three between the Cys
and Gly residues, and three between the Glu and Gly
residues. The analogous ROESY spectrum was also re-
2
corded for 20 mM (γECG-OEt) with 20 mM Zn(II) at a
2
5
6
pH* of 8.8, corresponding to a pH of 8.55, at a max-
imum of ZnA complex formation (83%, major contam-
ination from the Zn A
6
0
2þ
species). This spectrum
2
contained 9 of 11 expected intraresidual crosspeaks, two
interresidual crosspeaks analogous to those seen for the
GSSG complex and four additional interresidual cross-
peaks, involving the methyl protons of the ethyl substi-
tuent on the Gly residue. The spectral assignments in the
(

7
8
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
a
3
Table 5. Cumulative Stability Constants of Zinc Complexes of Disulfides at T = 25 °C, I = 0.1 M (KNO )
b
c
d
log βijk
-ΔG
CI0.01/7.4
peptide
ZnHA
ZnA
2
Zn A
ZnH-1A
ZnH-2A
ZnA
(GSSG 4.22)
tripeptide disulfides
(
(
(
(
(
γECS)
2
13.80(1)
15.08(9)
12.7(1)
13.13(9)
13.78(4)
7.75(3)
8.618(6)
7.01(3)
7.31(2)
7.69(1)
10.68(1)
10.84(7)
10.57(6)
10.64(6)
10.67(5)
-1.92(5)
-1.38(2)
-2.46(4)
-2.29
44.2(2)
49.20(3)
40.0(2)
41.7(1)
43.90(6)
4.18
4.58
3.87
4.00
4.07
c
γECE)
γECG-NH
γECG-OEt)
γEcG)
2
2
)
2
-11.60(3)
-11.92(4)
2
2
-1.86
dipeptide disulfides
(
(
γEC)
γEC-OEt)
2
14.46(7)
13.31(5)
8.132(4)
7.43(1)
10.79(9)
10.62(7)
-1.84(1)
-2.16(7)
46.42(2)
42.42(6)
4.22
4.02
mixed disulfides
γECG-γEC
γECG-γEC-OEt
14.31(3)
13.85(4)
8.075(8)
7.739(6)
10.77(3)
10.68(9)
-1.83
-1.93
46.10(5)
44.18(3)
4.26
4.15
a
b
i
þ j
k
c
-1
Statistical errors of constant determinations are given in parentheses.
Competitivity index calculated for pH 7.4 and disulfide L, Zn(II) and Z concentrations of 0.01 M. CI0.01/7.4 is log KMZ fulfilling the condition
Σ
βijk = [Zn H L ]/([Zn] [H ] [L] ). Units are kilojoules per mol (kJ mol ).
i j k
d
60
i j k
ijk([Zn H L ] = [ZnZ].
Table 6. Cumulative Stability Constants of Zinc Complexes of Non-Disulfide
a
Analogs at T = 25 °C, I = 0.1 M (KNO
3
)
a
b
log βijk
CI0.01/7.4
compound
ZnA
ZnA
2
ZnH-1A
2
ZnH-2
A
2
(GSSG 4.22)
S-Me-GSH 4.63(2) 8.43(3) -0.48(2) -10.12(2)
2.94
2.88
2.69
GSO
Glu
3
H
4.73(3) 8.75(3)
0.01(2)
-9.84(2)
4.83(2) 9.04(2) -0.43(3) -10.84(3)
a
Statistical errors of constant determinations are given in parenthe-
βijk = [Zn H L ]/([Zn] [H ] [L] ).
i j k
b
i
þ j
k
ses.
ROESY spectra are presented in Supporting Information
Table S2, while the lists of nondiagonal crosspeaks,
together with their integrals, are presented in Supporting
Information Table S3.
The molecular mechanics calculations of the structures
2
-
of ZnL and ZnA species from the above NMR data
were based on the following assumptions regarding their
structures: (i) The Zn(II) ion is coordinated to amino and
carboxylate donors of both Glu residues, set in four
adjacent corners of an octahedron. The Zn-N distances
were set at 0.2103 nm, the Zn-O distances were set at
0
.2036 nm, and the carboxylate groups were positioned as
monodentate donors. These distances and arrangements
were based on the X-ray structure of the Zn(II)-Glu
6
1
complex. (ii) Two additional water molecules were
coordinated in the remaining two octahedral positions
around Zn(II). The Zn-O (water) distances were set at
Figure 3. A global comparison of pH dependencies of chemical shifts in
free GSSG (red symbols) and Zn(II)-complexed GSSG (black symbols).
Concentrations of GSSG and Zn(II) were 10 mM; the spectra were
6
1
recorded at 25 °C. In panel A, the chemical shifts for individual CH
2
0
.207 nm. (iii) The C2 symmetry for both molecules was
groups are averaged, while in panel B they are shown separately for
nondegenerate cases of β-Glu (for Zn(II)-complexed GSSG only), β-Cys,
forced by distance symmetry restraints implemented in
the XPLOR program.
4
1
1 2
and Gly protons: 0, 9, H ; O, b, H (assignments arbitrary).
The 30 lowest energy conformers of Zn(II)-GSSG,
yielded by calculations, were divided into six families. The
of these families are presented in Supporting Information
Table S4. One can observe two types of coordination
patterns in calculated structures, which are labeled trans-
N (trans with respect to amine donors;family 1) and
trans-W (trans with respect to water molecules;families
application of the criterion of -60 or þ180° for the χ
1
3
angle of Cys, based on extreme values of measured J
HRHβ
couplings (11.4 and 4.3 Hz) for this residue, eliminated
three of these families. Structures of the lowest energy
members of each of three remaining families are shown in
Figure 5. The conformational properties and mean energies
2
and 3). They are shown at the bottom of Figure 5. Each
structure is stabilized by two pairs of hydrogen bonds.
The dihedral angle of the disulfide bridge assumes an
energetically optimal value of -90° for family 1. In the
(
61) Gramaccioli, C. M. Acta Crystallogr. 1966, 21, 600–605.

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 79
remaining two families, the deviations of values of this
dihedral angle from the optimum value of 90° (second
symmetric minimum) are at least 20°. In family 1, the
backbone dihedral of Cys is placed in the helical region
energies are presented in Supporting Information Table S5.
The lowest energy members of these superfamilies are
shown in Supporting Information Figure S1. This result
suggests that Zn(II)-(γECG-OEt) complexes in solu-
2
(
third quadrant) of the Ramachandran plot. In family 3,
tion exist in a conformational equilibrium between open
and closed structures.
this dihedral falls into the β structure region and, in family 2,
into the forbidden fourth quadrant (j>0). The structural
properties presented above, together with a large mean
energy difference between the first and the remaining two
families, strongly suggest that a “true” structure belongs
to family 1.
Discussion
Protonation equilibria. Values of protonation macro-
constants of GSSG, as mentioned above, agree well with
27,46,47,52,54
those determined previously.
determined by NMR, is in excellent agreement with the
most recent reports.
The value of pK6,
The 30 lowest energy conformers for Zn(II)-(γECG-
OEt) yielded by calculations had to be divided into as
many as 12 families. Applying a similar procedure to that
47,54
2
This value is lower than a
practical acidic limit for determinations using pH-metric
titrations of millimolar compounds, ca. 2.0. Errors in
previous pH-metric determinations of this constant
described above for Zn(II)-GSSG with a somewhat
3
broader criterion (the value of measured J
coupling
27,52
HRHβ
was here a little smaller, -10.7 Hz) allowed to discard five
of the families. Structural properties of the lowest energy
members of the remaining seven families and their mean
were most likely caused by ignoring this fact, as pointed
out previously.
53
We have confined our analysis of GSSG protonation to
macroconstants, because this level of analysis is sufficient
as a background for the study of Zn(II) complex forma-
tion, our main goal. Also, the macroconstants determined
in the present work agree well with the results of a recent
4
7
detailed study of GSSG microspeciation. However,
enthalpies and entropies related to GSSG protonation
were not reported previously and therefore require a brief
discussion. These thermodynamic parameters describing
protonation processes in peptides at moderate ionic
strengths were previously obtained mostly for dipeptides,
6
2,63
such as those composed of glycine and alanine.
The
63
tripeptide triglycine was also studied. These studies pro-
vide a suitable reference for the discussion of the C-terminal
carboxylate in GSSG. In turn, thermochemical studies of
protonation of simple amino acids, such as glycine, provide
6
4-66
a reference for the γ-Glu moiety.
In another thermo-
chemical study, protonation of GSH and triglycine was
Figure 4. The species distribution for Zn(II)-GSSG complexes, calcu-
lated for the conditions of NMR experiments (10 mM GSSG, 10 mM
67
investigated at a high ionic strength of 3 M NaClO4.
The deprotonation of a terminal carboxylic group in
simpledi-andtripeptidesisaccompaniedbysmallenthalpic
Zn(II), 25 °C). Chemical shifts of R-Glu (red circles) and GlyH
1
(olive
circles) are overlaid for comparison. The thick dark yellow line indicates
2-
2-
the main species ZnL ([Zn-GSSG] ) present at physiological pH.
Figure 5. Three types of coordination patterns. trans-W, waters in trans position; trans-N, amine nitrogens in trans position; trans-O, carboxyl oxygens in
trans positions (lower row) and the lowest energy members of three families of the Zn(II)-GSSG complex (upper row). Hydrogen bonds are shown in
orange.

8
0
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
-
1
effects, typically less than 10 kJ mol , together with
-
1
-1 62,63
sizable entropic contributions (40-70 J mol
Glycine carboxylate protonation reactions involve simi-
larly low ΔH values, with ΔS about 30 J mol
K ).
-
1
-1 56-60
K .
The R-amine protonation is controlled primarily by en-
-
1
thalpy (40-50 kJ mol ), with little entropy change
-
1
-1 62,63
(
usually less than 10 J mol
K
).
In turn, entropic
effects are significant for the amine of the Glu moiety in
-
1
GSH, and in glycine, with ΔS values of 40-60 J mol
-
1 64-67
K
.
This difference is due to a salt bridge formation
between the deprotonated carboxylate and the neighbor-
ing protonated amine in R-amino acid moieties. The
protonation of GSSG amines (top two lines in Table 1)
fits in this pattern, with significant ΔH and ΔS contribu-
tions. The thermodynamics of the Gly residue protona-
tions is similar to the dipeptide and tripeptide data,
although the enthalpic contribution to the free energy is
slightly negative and is accompanied by higher entropic
contributions (two middle lines in Table 1). The major
difference with simple models is demonstrated by the
Figure 6. A linear correlation between the average value of the amine
þ
group dissociation constant (av. pK
a
(RNH
3
C
)) and z , the stoichiometric
electronic charge on the C-terminal residue in H L species for all ligands.
2
Red, black, and blue circles represents di-, tri-, and mixed di-/tripeptide
disulfides, respectively.
with the same slope was calculated for all peptides with an
even higher significance (p = 0.0008, n = 10; Figure 6).
Notably, the difference of amine pK values, pK - pK
þ
þ
protonation of a Glu carboxylate (H L þ H / H L ).
4
5
a
1
2
It involves a very high negative enthalpy, compensated by
a very large entropy change, nearly 10-fold higher from
that determined for simple amino acids. The explanation
of this effect is provided by the crystal structure of the
did not correlate significantly with z (R = -0.55, p =
C
0
.09, n = 10).
The influence of z on amine pK is particularly strong
C
a
in the dipeptide disulfide (γEC) . The presence of nega-
6
8
2
H L species of GSSG, which indicates additional salt
4
tively charged C-terminal carboxylates next to the gluta-
mic acid residue results in the elevation of the average
amine pK by 0.09 log units, compared to (γECG) , while
-
þ
bridges between the COO and NH3 groups belonging
to different Glu moieties. A disorganization of this long-
range structuring is entropically favorable. Also, the
crystal of GSSG contains four ordered water molecules.
Two of them are located within the salt-bridged macro-
chelate loop, composed of the two Glu and two Cys
residues. The commercial polycrystalline GSSG samples
contain one water molecule per GSSG. It is therefore
reasonable to assume that one or two water molecules
remain bonded to GSSG after dissolution, and their
release contributes to both a decrease of enthalpy and
an increase of entropy accompanying the protonation of
a
2
the difference of these values between (γECG-OEt) and
2
(
γEC-OEt) is less than 0.02 log units. The effect in the
2
mixed disulfide γECG-γEC is intermediate, 0.04 log
units, vs (γECG) .
2
We interpret these results as evidence for a direct, through-
space electrostatic interaction between the protonated
amine and the deprotonated C-terminal carboxylate(s).
The lack of effect on pK - pK indicates that this interac-
1
2
tion is confined to monomeric units of disulfides, behav-
ing largely independently. The further confirmation of
this effect is provided by the specific sensitivity of Gly, but
not Cys protons, to the amine group deprotonation seen in
NMR spectra (Figure 2, Supporting Information Table S3).
the H L species of GSSG.
4
Solution Structures of Uncomplexed Disulfides. The
comparison of GSSG with its analogs demonstrated a
dependence of values of amine protonation constants on
Larger differences of pK values of the amine groups
a
electronic charges on their C-terminal residues, z . We
C
are observed for nondisulfide analogs with the modified
thiol, S-methylglutathione, and glutathione sulfonic acid.
This effect is consistent with our conformational model of
observed such an effect previously in a corresponding
series of GSH analogs. As shown in Figure 6, a good
3
0
linear correlation of the averaged amine pKa on zC
GSH, which includes the pK modifying effect, exerted on
a
includes all disulfides studied, but secondary effects re-
the amine by the deprotonated thiol group, or, in principle,
any charged group in its vicinity.
3
0
lated to peptide sizes are also present. A correlation
coefficient R = -0.95 (p = 0.003, n = 6) was found
in the series of tripeptides. The slope of this correla-
tion, -0.16 ( 0.03 pH units per unit charge, is very similar
to that observed in GSH analogs. The value of R = -0.88
Thermodynamics of Zn(II) Complexes with GSSG. The
Zn(II) complexation pattern in GSSG and its analogs
2
includes two major complexes, ZnL at neutral and
-
3
weakly alkaline pH and ZnH 1L under more alkaline
-
-
conditions, as well as two overlapping, minor species,
(
62) Gergely, A.; Nagypal, I. J. Chem. Soc., Dalton Trans. 1977, 1104–
108.
63) Brunetti, A. P.; Lim, M. C.; Nancollas, G. H. J. Am. Chem. Soc. 1968,
0, 5120–5126.
-
ZnHL and Zn L in weakly acidic solutions. The two
2
1
previous potentiometric studies of Zn(II) coordination to
GSSG were done under similar, although not identical,
(
9
(64) Bunting, J. W; Stefanidis, D. J. Am. Chem. Soc. 1990, 112, 779–786.
(65) Kiss, T.; Sovago, I.; Gergely, A. Pure Appl. Chem. 1991, 63, 597–638.
(66) Ferretti, L.; Elviri, L.; Pellinghelli, M. A.; Predieri, G.; Tegoni, M. J.
28
27
conditions (T = 25 °C, I of 0.15 or 0.20 ). The earlier of
these studies proposed only the ZnL complex. The
2
-
Inorg. Biochem. 2007, 101, 1442–1456.
67) Corrie, A. M.; Williams, D. R. J. Chem. Soc., Dalton Trans. 1976,
068–1072.
68) Jelsch, C.; Didierjean, C. Acta Crystallogr., Sect. C 1999, 55, 1538–
540.
model presented in the later one is similar to ours, except
for its omission of the ZnH 1L species, and slightly
lower formation constant values (ca. 0.4 log units per
Zn(II) ion). In contrast with the preceding studies, however,
(
3-
-
1
1
(

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 81
2
The high stability constant of the ZnL species is due
to a lowering of the amine pK by 3.56 log units, com-
a
-
Scheme 2. Schematic Representation of Structures of Zn(II)-GSSG
Complexes
pared to uncomplexed GSSG. NMR titrations support
the intuitive notion that this species contains both amine
nitrogen residues coordinated to the Zn(II) ion. The
2
þ
4-
2-
formal process Zn þ L / ZnL contains favorable
enthalpic and entropic contributions, but interestingly
-
2-
þ
the reaction ZnHL / ZnL þ H , which constitutes an
important pathway for the formation of this complex (see
Figure 4), is controlled purely by entropy (the enthalpic
contribution amounts to less than 1 kJ per mole).
3
-
The formation of the ZnH 1L complex under alka-
-
line conditions is confirmed by small, but evident changes
of chemical shifts of R-Glu, β-Cys, and Gly protons,
compared to those for the ZnL complex (Figures 3
2
-
3
-
and 4). The pK value for ZnH
a
L
-1
formation obtained
from glutamic and β-Cys protons was 9.37(3), only some-
what lower from the potentiometric value of 9.745(9). The
low magnitudes of these NMR effects indicate that the
2
-
binding mode of ZnL is retained in this complex, thus
excluding the possibility of an amide bond deprotona-
tion. Therefore, the hydrogen ion must be released from a
our potentiometric results were confirmed quantitatively
by H NMR spectroscopic titrations, as shown in Figure 4.
1
These results, in particular the magnitudes of chemical
shift effects, confirm the glutamic amines and carboxylates
as Zn(II) binding sites in all complexes (Figures 3 and 4).
water molecule associated with the complex. The pKa
value is rather low for a -2 charged complex (typical pK
values range between 8 and 10 positively charged com-
a
-
The stability constant of the ZnHL complex, corrected
for protonation of the uncomplexed amine (log KZnHL =
plexes and are usually above 10 for neutral or -1 charged
complexes).
7
2-75
log βZnHL - log βHL) at 25 °C, equal to 4.50, is quantita-
tively similar to log KZnA values with A representing
Correlations of Properties in GSSG Analogs. Similarly
to the case of protonation equilibria, a deeper under-
standing of Zn(II)-GSSG complexes can be gained from
the analysis of tendencies in the series of Zn(II) complexes
of disulfide analogs studied. As shown in Supporting
Information Figure S2, the stabilities of individual com-
plexes are very strongly dependent on basicities of amine
groups. Correlation coefficients R as high as -0.98 were
6
9,70
simple amino acids, ca. 4.2-4.7.
However, as seen
in Table 4, this value is a result of an unfavorable enthalpic
effect, as opposed to low, but favorable ΔH effects for
-
1
analogous amino acid complexes (-5 to -15 kJ mol ),
compensated by the entropic contribution, which is 2 to 3
-
1
-1
times higher (40-70 J mol
K ). These differences are
consistent with the breaking of the electrostatic bridges
between Glu moieties (enthalpic cost), compensated by
an increased disorder (likely including the release of water
molecules from the uncoordinated GSSG molecule). This
complex may be “unfolded”, with Zn(II) bonded solely to
one glutamic unit, or “folded”, with an additional co-
ordination of the carboxylate from the second glutamic
unit (see Scheme 2 for schematic structures of complexes
discussed here and below). The comparison of log a
KZnHL value of 4.50 with one of 4.17, the analogous value
determined for glutamine, the closest structural analog of
found for ZnHA, Zn A, and ZnA complexes. Only the
2
stabilities of ZnH A complexes were a little less corre-
-1
lated, with R = -0.91. Inspired by the existence of the
relationship between the amine basicities of disulfides and
their C-terminal charges, discussed above, we performed
analogous calculations, presented in Figure 7. Indeed,
stabilities of all complexes exhibited strong correlations
with z . The qualities of these linear fits for ZnHA, Zn A,
C
2
and ZnA complexes were somewhat lower than the pre-
vious ones, R = -0.92, -0.87, and -0.93, respectively.
Interestingly, the correlation with z was higher than that
C
7
1
the glutamic binding site in GSSG, is in favor of the
folded structure, containing an additional, albeit weak
Zn(II)-ligand bond. This issue is explored in more detail
in the next section.
with pK þ pK for the ZnH-1A complex, R = -0.93.
1
2
Figure 7 demonstrates a very strict relationship between
Zn(II) binding properties of ZnHA and ZnA complexes,
with R as high as 0.996. These results allow one to
conclude that the overall ability of GSSG analogs to bind
Zn(II) is controlled directly by amine basicities, and
indirectly by C-terminal charges. Nevertheless, significant
secondary effects are present among the studied disulfides.
The inspection of Supporting Information Figure S2
indicatesthattheimpactofaminebasicityonspeciesstability
2
þ
The addition of the second Zn ion to this complex to
2
þ
-
þ
form Zn L, Zn þ ZnHL / Zn L þ H , is entropy-
2
2
driven (Table 4). Two glutamic Zn(II) sites in Zn L carry
2
positive charges, causing them to repulse each other.
Hence, this complex cannot assume a folded structure.
A high increase of entropy, accompanying its formation,
provides further evidence for the folded conformation
-
of ZnHL .
(72) Delgado, R.; Sun, Y.; Motekaitis, R. J.; Martell, A. E. Inorg. Chem.
(
69) Arena, G.; Cali, R.; Cucinotta, V.; Musumeci, S.; Rizzarelli, E.;
Sammartono, S. J. Chem. Soc., Dalton Trans. 1983, 1271–1278.
70) Sovago, I.; Kiss, T.; Gergely, A. Pure Appl. Chem. 1993, 65, 1029–
1993, 32, 3320–3326.
(73) Gualtieri, R. J.; McBryde, W. A. E.; Powell, H. K. J. Can. J. Chem.
1979, 57, 113–118.
(74) Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc. 1994, 116,
8443–8449.
(75) Menif, R.; Martell, A. E. Inorg. Chem. 1989, 28, 116–122.
(
1
080.
(
441–2446.
71) Tewari, R. C.; Srivastava, M. N. J. Inorg. Nucl. Chem. 1973, 35,
2

8
2
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
Figure 7. A linear correlation between log β values of ZnHA (A), ZnA
B), Zn A (C), and ZnH-1A (D) complexes and z , stoichiometric
electronic charges of zinc-disulfide complexes.
(
2
C
is highest for ZnHA complexes, with a slope of the linear
correlation of 3.0 log βZnHA units per 1 (pK þ pK ) unit,
1
2
Figure 8. Correlations between stability constants of ZnHA and ZnA
2þ
2þ
followed by ZnA, a slope of 2.0; ZnH A, a slope of 1.3;
-
and Zn A, as little as 0.36 units. These differences cannot
species defined byreactions Zn þ H
2
L / ZnHL(A) and Zn þ H
2
L /
1
ZnL (B). Compounds are color-coded as follows: disulfides of tripeptides
with free C-terminal function (olive); disulfides of di- and tripeptides with
blocked C-terminal function, together with γEcG (black); mixed disul-
fides (red).
2
be explained by a simple electrostatic mechanism, present
in uncomplexed GSSG analogs, and require the presence
of an additional bonding mechanism.
Figure 8 presents the relationship between equilibrium
carboxyl groups. Structure B depicts a hypothetical spe-
cies with no C-terminal interaction. Such are clearly not
dominant in complexes studied. Panel C presents a species
with C-terminal groups interacting only within their
monomeric units, while panel D presents a species with
a “crossed” interaction, where each C-terminus may reach
toward the other monomeric unit. The gain of Zn(II)
binding stability of 1.5 log unit per 1 log unit of basicity of
each amine (slope of 3 vs pK þ pK ) clearly indicates the
2
þ
þ
constants for reactions Zn þ H A / ZnHA þ H (top)
2
2
þ
þ
and Zn þ H A / ZnA þ 2H (bottom) and amine
2
basicities. These reactions provide actual processes of
formation of these complexes, as H A is the predominant
2
protonation form of disulfides in the pH range of forma-
tion of these complexes (cf. Figure 1 vs. Figure 4). They
further demonstrate the presence of an additional factor,
which increases the stability of complexes of tripeptides
with unmodified C-terminal carboxylates. It accounts for
ca. 0.3 log units of the stability constant, corresponding to
a ΔΔG of 1.6 kJ/mol for both ZnHA and ZnA complexes.
The mixed disulfides in which one unit belongs to the
above family, and another is either shorter or modified,
exhibit average stabilities, compared to parent symmet-
rical disulfides. Therefore, the mechanism in question is
additive with respect to monomeric units of the disulfides.
The extremely tight correlation between ZnHA and ZnA
stabilities and the conservation of the unblocked tripeptide
effect further support the presence of similar conforma-
tions in these two species. The similarity of conformations
1
2
participation of structure D in ZnHA complexes, with
C-terminal groups providing multiple interactions with
the first coordination sphere around the Zn(II) ion. Structure
C might be assumed to correspond to ZnA complexes,
with a stability gain directly corresponding to amine
basicity. However, such a 1:1 relation would require a
phenomenally rigid species, incompatible with the non-
covalent character of C-terminal interactions. Therefore,
the reality of closed complexes is best depicted by mix-
tures of structures C and D, with a higher proportion of
structure C in ZnHL complexes.
A weak negative correlation was found between the
stability of ZnHA complexes and the stepwise constant of
formation of ZnA complexes, slope = -0.18 log units.
This effect is most likely due to a slight shortening of
Zn-N bonds, expected in stronger complexes, which has
to be compensated conformationally and induces a slight
strain in ZnA species.
between ZnA and ZnH 1A complexes was discussed
-
above for GSSG. Stabilities of these three species exhib-
ited strong, albeit varied correlations on pK þ pK ,
1
2
in contrast to that for Zn A. The weak dependence on
2
pK þ pK is therefore a consequence of the unfolded
1
2
structures in the Zn A complexes, due to the electrostatic
2
repulsion between þ1 charged Zn(II) sites in them.
The stronger dependence of the stability of ZnH 1A
-
complexes on z provides a further proof for the location
Notably, the highest relative abundance of Zn A com-
2
C
plexes occurs for the weakest ligands, which do not carry
negative charges on their C-termini, and is due to a
relative weakness of ZnHA and ZnA complexes with
these ligands.
of C-terminal groups in the vicinity of the Zn(II) binding
site. The correlations between the water pK and amine
a
basicity or ZnA stability are weaker, with an R of 0.86
(Supporting Information Figure S3), but the deviation
from linearity is mostly due to the anomalous behavior of
the D-Cys analog, where the Zn(II)-bonded water mole-
cule is much more acidic. In general, the value of the water
Scheme 2 presents the possible modes of interaction in
various complexes of GSSG and its analogs. The Zn A
2
complexes are depicted in panel A, with a possible weak
interaction between Zn(II) binding sites and C-terminal
pK is influenced by overall electrostatics, as well as
a

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 83
6,77
7
specific effects of the C-terminal carboxylates. The average
water pK value for the group of derivatives containing at
least one tripeptide carboxylate unit (three unblocked
tripeptides and two mixed disulfides) is 9.80, vs. 9.64 in
the remaining five analogues. This difference is statistically
significant only at a 0.19 level but becomes significant at a
in the solid state.
in several cases of small complexes in solution, based on
Such structures were also proposed
a
7
8-80
indirect arguments.
The overall gain of Zn(II) bind-
ing stability between the series with strong H-bonding to
the carboxylate and the series with weaker H-bonding to
carbonyl oxygens is low, 1.6 kJ/mol. As discussed exten-
sively above, the nondirectional electrostatic interaction
provides a major part of Zn(II) stabilization. Therefore,
an equilibrium between the H-bonded and non-H-bonded
species should be present in all complexes studied. Our
data do not allow for estimation of the corresponding
equilibrium constants. Neither experimental nor theore-
tical data on energetics of hydrogen bonds of Zn(II)-
bonded water molecules to noncoordinated carboxylates
are available. However, the above discussion of strong
correlations of charges, basicities, and complex stabilities
suggests a high proportion of structures C and D of
Scheme 2 vs structure B, especially in the complexes of
unblocked tripeptide disulfides.
0
.03 level upon removal of those compounds with the
strongest electrostatic effects (Zn(II)-GSSG from the
first group and Zn(II)-(γEC) from the second group
2
(
the average water pK values become then 9.75 vs 9.55)).
Table 5 presents also the competitivity index (CI)
a
values, calculated for 10 mM Zn(II) and disulfide ligands
at pH 7.4 (CI0.01/7.4). CI was developed by us to aid
comparisons of metal binding abilities of ligands and sets
60
of ligands possessing various stoichiometries. It is defined
as the logarithm of the conditional stability constant of
MZ, the binary metal complex of a hypothetical molecule
Z which takes up the same proportion of the metal ion M,
as all actual complexes under consideration. For sufficiently
strongly binding systems, where the amount of uncom-
plexed M is insignificant, this proportion is 50%. The
appropriate formula is provided in the footnote to Table 5.
Defined in this way, CI0.01/7.4 essentially combines
binding abilities of ZnHA and ZnA complexes. The span
of CI0.01/7.4 values is only 0.7 log units for all compounds
presented in Table 5, and most of them are confined
within 0.2 log units, confirming that Zn(II) binding is
mostly due to the pair of N-terminal chelates common to
all disulfides studied. The bonding in GSSG is particularly
effective, second only to that of the Glu analog, and equal,
within the experimental error, to that of dipeptide carbox-
ylates, indicating that specific interactions of Gly in GSSG
fully compensate the stronger electrostatics of dipeptides.
Structures of Zn(II) Complexes of GSSG and Its Gly-
The structure presented in Figure 5 also explains the
rigidity of the disulfide bridge region, which manifested
coupling con-
3
itself in the extreme value of the J
HRHβ
stant. The conformation with χ = -60° is strongly
1
stabilized sterically. The changes of these angles are
prevented on one side by a hindrance between β protons
of opposite Cys residues and on the other side by a
hindrance between β-Cys protons and peptide oxygens
of opposite Cys residues.
Biological Relevance of Zn(II)-GSSG Complexes.
Two factors should be taken into account when analyzing
the possible relevance of the Zn(II)-GSSG system in a
biological environment: one is its relative stability, which
governs the possibility of complex formation in vivo, and
another is the presence of special features, which might
make them important even at a low abundance. The first
issue can be approached with the use of the competivity
index (CI), because it provides a method of estimating the
relevance of particular complexes in a partially character-
cine Ester. A heavy overlap between the pH profiles of
formation of minor ZnHL and Zn L species and that of
the major ZnL precluded NMR-based characterization
of their structures. However, the above discussion proved
that Zn L has an unfolded structure, in which two Zn(II)
-
2
2
-
6
0
ized equilibrium. As mentioned above, the CI values are
concentration-dependent and therefore allow inclusion of
the effects of absolute and relative abundances of indivi-
dual competitors. The CI0.01/7.4 value calculated for
GSSG, 4.22 (Table 5), is lower than those calculated for
2
-
sites do not interact with each other, while ZnHL shares
.
2
the folded, coiled structure with ZnL and ZnH 1L
-
3-
-
2
-
The structure of the ZnL complex is fairly rigid, as
indicated by such NMR spectral features as differentia-
tion of Glu-β protons and a selective change of chemical
shift of one of the Gly protons, correlated with the
6
7,70
likely biological low molecular weight Zn(II) ligands,
ATP (5.05), GSH (4.73), and His (4.83), but the difference
is not overwhelming. A direct calculation involving all
four ligands in an equimolar mixture indicates the following
distribution of Zn(II): GSSG, 3.7%; ATP, 22.4%; GSH,
33.9%; His, 40.0%. Furthermore, taking into account
that all three ligands exist intracellularly at concentra-
tions much higher than GSSG and available Zn(II) levels
2
-
formation of the ZnL complex (Figure 3). Both of these
features result from the spatial fixing of these geminal
protons in nonequivalent distances relative to their ad-
jacent carboxylate oxygens. The mechanism of this fixing
is provided by the structure presented in Figure 5, where
Zn(II) is coordinated directly by Glu carboxylates and
indirectly by Gly carboxylates, through hydrogen bonds
between their oxygen atoms and Zn(II)-bonded water
molecules. The latter interaction adds the stability of
Zn(II) binding specifically in those tripeptide disulfides
which contain unblocked C-terminal carboxylates, in
contrast with weaker H-bonding available in those disulfides
which have carboxyl functions modified by esterification
or amidation. Hydrogen bonding of Zn(II)-coordinated
water molecules to noncoordinating carboxylates is common
9
,14-17,19,44,45
are in the nano- to picomolar range,
these
calculations indicate that the formation of Zn(II)-GSSG
complexes intracellularly is unlikely.
(
77) Fu, X. C.; Li, M. T.; Wang, C. G.; Wang, X. Y. Acta Crystallogr.,
Sect. C 2006, 62, m13–m15.
78) Nagypal, I.; Farkas, E.; Gergely, A. J. Inorg. Nucl. Chem. 1974, 37,
(
2145–2149.
(
79) Pettit, L. D.; Swash, J. L. M. J. Chem. Soc., Dalton Trans. 1976,
2
416–2419.
(
80) Kiss, T. In Biocoordination Chemistry, coordination equilibria in
(
76) Hambley, T. W.; Christopherson, R. I.; Zvargulis, E. S. Inorg. Chem.
biologically active systems; Burger, K., Ed.; Ellis Horwood: Chichester, U. K.,
1990; pp 56-134.
1
995, 34, 6550–6552.

8
4
Inorganic Chemistry, Vol. 50, No. 1, 2011
Kr e- z_ el et al.
To further test whether or not GSSG can compete with
zinc components of biological fluids, we applied FluoZin-3,
a fluorescent chelating sensor commonly used in in vivo
and in vitro studies of Zn(II) distribution, as a thermo-
9
,19,44,45
dynamic model of a zinc pool.
highly fluorescent upon Zn(II) binding and has moderate-
The sensor becomes
to-high affinity for Zn(II) (K = 8.9 nM). Figure 9
d
presents the fluorescence decrease of partially saturated
FluoZin-3 with Zn(II) (0.1 μM FluoZin-3 and 0.05 μM
Zn(II)) in the presence of a wide range of GSSG concen-
trations. Initial strong emission became significantly
quenched (33%) after the addition of 1 mM of GSSG,
2þ
while pZn (-log[Zn ]) increased from 8.29 to 8.46. The
addition of GSSG in the range of its intracellular con-
centration hardly affected the FluoZin-3 fluorescence.
FluoZin-3 was demonstrated to interfere with the intra-
cellular zinc pool, and therefore such a competition
experiment may be considered as a benchmark for sig-
nificant participation of a competing ligand in Zn(II)
9
,19,44,45
distribution.
The absence of such an effect clearly
confirms that GSSG cannot be a significant Zn(II) che-
lator in intracellular fluids. However, the situation may
be quite different in the extracellular space, where avail-
8
1-83
able Zn(II) pools may exceed micromolar levels,
and
there are much fewer competing ligands (e.g., ATP and
GSH are absent). The lack of quantitative data in this
respect for this quickly changing and highly variable
milieu prevented us from performing appropriate calcu-
lations, but the potential presence of GSSG-Zn(II)
complexes will be possible to test in cell culture studies.
The above results allowed us to limit the discussion of
Figure 9. The competition for Zn(II) between FluoZin-3 and GSSG.
Initial concentrations of FluoZin-3 and Zn(II) were 0.1 and 0.05 μM,
respectively. Fluorescence spectra were recorded at 25 °C after the
addition and equilibration of 0-9.6 mM GSSG (A). pZn values at each
particular step were calculated according to the following equation: pZn
=
-log(K
d
ꢀ (F - Fmin)/(Fmax - Fmin)), where Fmax and Fmin were
2-
measured after the addition of 100 μM ZnSO and 100 μM EDTA(dotted
4
special features of Zn(II)-GSSG complexes to the ZnL
line), to the initial sample (B).
complex. Two features of this complex appear to be
important and may be functionally related to each other.
One is the presence of Zn(II)-bound water molecules
susceptible to deprotonation, and another is the folding
of Gly residues back on them. Such water molecules,
readily convertible into hydroxyl ions, are crucial for the
action of a great number of Zn(II) enzymes, such as
peptidases, esterases, phosphatases, etc., as well as their
Therefore, the presence of interactions between zinc-
bound water molecules and Gly carboxylates in GSSG
may be treated as a measure to quench the catalytic
activity of Zn(II). The blocking of this interaction low-
ered the pK of water by ca. 0.2 log units, increasing the
a
abundance of the potential catalyst by 60%. On the other
hand, one may conceive of the existence of specific targets
for Zn(II)-GSSG, complexes, e.g., specific hydrolysis
substrates which may activate Zn(II)-bound water by
pulling the carboxylates away. The very specific struc-
tures of Zn(II) complexes of GSSG certainly warrant
further studies of these issues.
8
4
synthetic models. We tested whether the Zn(II)-GSSG
complex might act as an esterase or phosphatase at pH
7
and tris(4-nitrophenyl) phosphate, respectively, accord-
.4, on commonly used substrates 4-nitrophenyl acetate
8
5,86
ing to standard procedures and conditions.
The gain
in hydrolysis rate was within the experimental error in
each case (data not shown), most likely due to a very low
abundance of the ZnH L complex, which possesses the
Conclusions
3-
-
1
The correlated thermodynamic and NMR structural de-
scription of protonation equilibria and complex formation
between Zn(II) ions and GSSG and its analogs revealed the
role of the Gly residue in complex stabilization and structur-
ing. Thespecific, coiledstructuresof major GSSGcomplexes,
including specifically bound water molecules, result from the
concerted action of electrostatic interactions and hydrogen
bonding. A similarly intricate network of interactions is pre-
catalytic hydroxyl ion at this pH (ca. 0.4% of total zinc).
(
81) Frederickson, C. J.; Giblin, L. J.; Balaji, R. V.; Masalha, R.;
Frederickson, C. J.; Zeng, Y.; Lopez, E. V.; Koh, J. Y.; Chorin, U.; Besser,
L.; Hershfinkel, M.; Li, Y.; Thompson, R. B.; Kr e- z_ el, A. J. Neurosci.
Methods 2006, 154, 19–29.
(
82) Frederickson, C. J.; Giblin, L. J.; Kr e- z_ el, A.; McAdoo, D. J.;
Mueller, R. N.; Zeng, Y.; Balaji, R. V.; Masalha, R.; Thompson, R. B.;
Fierke, C. A.; Sarvey, J. M.; de Valdenebro, M.; Prough, D. S.; Zornow,
M. H. Exp. Neurol. 2006, 198, 285–293.
sent in the structure of the ternary Zn(II) complex with GSH
and L-histidine presented by us previously. Such an inter-
56
(
83) Opoka, W.; Sowa-Ku ꢀc ma, M.; Kowalska, M.; Ba ꢀs , B.;
Gozembiowska, K.; Nowak, G. J. Physiol. Pharmacol. 2008, 59, 477–487.
play of individually weak interactions is more and more
frequently found to govern strong thermodynamic effects.
The Zn(II)-GSSG complexes were not found to be active as
87
(
84) Parkin, G. Chem. Rev. 2004, 104, 699–768.
(
85) Verpoorte, J. A.; Mehta, S.; Edsall, J. T. J. Biol. Chem. 1967, 242,
4
221–4229.
86) Goldberg, D. P.; diTargiani, R. C.; Namuswe, F.; Minnihan, E. C.;
Chang, S.; Zakharov, L. N.; Rheingold, A. L. Inorg. Chem. 2005, 44, 7559–
569.
(
(87) Schlund, S.; Schmuck, C.; Engels, B. J. Am. Chem. Soc. 2005, 127,
11115–11124.
7

Article
Inorganic Chemistry, Vol. 50, No. 1, 2011 85
hydrolases against nitrophenyl ester and phosphate sub-
strates, but as argued above, studies into their potential
biological functions and reactivities should be continued.
Supporting Information Available: The pK values of chemical
shifts of nonexchangeable protons of GSSG; H chemical shift
1
2
of Zn(II)-GSSG and Zn(II)-(γECG-OEt) complexes; inte-
grals of nondiagonal crosspeaks in ROESY NMR spectra;
families of lowest energy conformers of Zn(II)-GSSG and
Acknowledgment. This work was sponsored in part by
the Polish Ministry of Science and Higher Education,
Grant No. N N302 137735, and The Foundation for
Polish Science, Grant No. HOM/2008/8B. The authors
thank Ms. Katarzyna Pi a- tek, Dr. Edyta Kopera, and Mr.
Adam Pomorski for their assistance in experiments.
2
Zn(II)-(γECG-OEt) ; the lowest energy conformers of the
Zn(II)-(γECG-OEt) complex; correlations between log β va-
2
lues of ZnHL, ZnA, Zn A, and ZnH L complexes and log β
2
-1
2
values of H L species; correlation between log β values of the
ZnL complex and the pKOH values. This material is available
free of charge via the Internet at http://pubs.acs.org.