Copper(II) binding to Cap43 protein fragments

www.rsc.org/dalton | Dalton Transactions
PAPER
Copper(II) binding to Cap43 protein fragments
Maria Antonietta Zoroddu,*a Teresa Kowalik-Jankowska,b Serenella Medici,a Massimiliano Peanaa and
Henryk Kozlowskib
Received 21st May 2008, Accepted 7th August 2008
First published as an Advance Article on the web 24th September 2008
DOI: 10.1039/b808600a
The C-terminal 20 and 30 amino acid sequences of Cap43 protein were chosen as models to study their
interactions with Cu(II) ions. The behaviour of the 20 amino acid Ac–TRSRSH6 TSEG–
TRSRSH16 TSEG and 30 amino acid Ac–TRSRSH6 TSEG–TRSRSH16 TSEG–TRSRSH26 TSEG
peptides towards Cu(II) ions at different pH values and different ligand-to-metal molar ratios, was
examined. Spectroscopic (EPR, UV-Vis) and potentiometric techniques were performed to understand
the details of metal binding to the peptides. The study showed that, starting from pH 4.0, each
10 amino acid fragment T1 R2 S3 R4 S5 H6 T7 S8 E9 G10 was able to independently coordinate a single Cu(II)
ion. The coordination mode involved the imidazole nitrogen of histidine H6 residue, and three amidic
nitrogens from histidine H6 , serine S5 , and arginine R4 residues, respectively.
Introduction
Cap43 is a nickel- and calcium-inducible gene that has been
recognized to play a significant role in cancer metastasis and
invasion, as well as in the primary growth of malignant tumours,
possibly through its ability to induce differentiation.1–4 The Cap43
gene (also referred to as NDRG-1, N-Myc down-regulated gene
1) expresses a 3.0 kb mRNA that encodes a M r 43 000 cytoplasmic
protein. The expression of Cap43 protein is low in normal tissues;
however, Cap43 is overexpressed in cancer cells, including lung,
brain, melanoma, liver, prostate, breast, and renal cancers. The
low level of expression of Cap43 in some normal tissues compared
to their cancerous counterparts, combined with the high stability
of Cap43 protein and mRNA, makes the Cap43 gene an important
marker for diagnosing cancer diseases at the early stages.4–7
The most striking feature of this protein lies in its C-terminal
moiety, where a 10 amino acid monohistidinic sequence (TRSRSHTSEG) is repeated consecutively three times to give an interesting site for examination under metal-coordination conditions. The
C-terminal sequence, being external to the whole protein, is more
accessible for metal ion binding. For this reason, one of the aspects
we wished to study was the possibility of multiple coordination of
the C-terminus with different metal ions.
Our first studies included the assessment of the coordination
ability of Ni(II) and Cu(II) ions towards a 14 aminoacid, terminally
blocked fragment of Cap43 protein (Ac–TRSRSHTSEGTRSR–
Am). Results showed that the motif on the C-terminal portion
of our protein may serve as an efficient binding site for metal
ions.8 Particularly, Cu(II) starts coordination from the imidazole
nitrogen of the histidine residue in weakly acidic solution and, as
the pH was raised, the metal ion was able to deprotonate successive
peptide nitrogens giving the maximum of major species CuH-2 L
(3 N) at pH 7.0 and CuH-3 L (4 N) at pH 8.7.
Besides, one of our more recent studies concerned the investigation on the coordination of Ni(II) ions with the 20 amino
a
Department of Chemistry, University of Sassari, Italy. E-mail: zoroddu@
uniss.it
b
Faculty of Chemistry, University of Wroclaw, Poland
This journal is © The Royal Society of Chemistry 2008
acid Ac–TRSRSH6 TSEG–TRSRSH16 TSEG and 30 amino acid
Ac–TRSRSH6 TSEG–TRSRSH16 TSEG–TRSRSH26 TSEG C-terminal Cap43 sequences. This study pointed out that each 10 amino
acid fragment was able to effectively coordinate a Ni(II) ion,
thus suggesting a possible detoxification role of Cap43 within the
human organism.9
The present paper reports the results of combined spectroscopic and potentiometric studies on the copper(II) interaction
with the two and three repeats of the TRSRSHTSEG amino
acid fragment, namely the 20 amino acid (Ac–TRSRSHTSEG–
TRSRSHTSEG, peptide1), and the 30 amino acid (Ac–
TRSRSHTSEG–TRSRSHTSEG–TRSRSHTSEG, peptide2) sequences of the C-terminal part of Cap43 protein. The imidazole
nitrogen of the histidine residue is an essential binding site for the
copper(II) ion, and the 20 and 30 amino acid sequences contain
two and three histidine residues, respectively. The aim of this study
was to examine the binding abilities of these fragments to more
than one metal ion, in order to gather more experimental evidence
on the coordination behaviour of C-terminal portion of Cap43
protein.
Experimental section
Peptide synthesis
Peptides were chemically synthesized using solid phase Fmoc
chemistry in an Applied Biosystems Synthesizer.10 The synthesis
was performed using a fivefold excess of Fmoc amino acid
derivatives, DCCI and HOBt as activating agents and a 60 min
coupling time. Side chain protecting groups included: triphenylmethyl group for His; pentamethyl-chroman-sulfonyl group
for Arg; tert-butyl group for Glu, Ser and Thr. Peptides were
deprotected and cleaved from the resin by treatment with 2.5%
H2 O, 5% triethylsilan in trifluoroacetic acid (TFA) for 2 h at
room temperature. After removal of the resin by filtration, peptides
were precipitated with tert-butylmethyl ether, centrifuged and the
pellets resuspended in 50% acetic acid and lyophilized. Crude
peptides were reconstituted in 1 ml 50% acetic acid in H2 O and
Dalton Trans., 2008, 6127–6134 | 6127
low molecular weight contaminants were removed by gel filtration
on Sephadex G-25. The materials eluted in the void volume were
lyophilized, reconstituted in 1 ml 50% acetic acid in H2 O and subjected to RP-HPLC on a Vydac column (250–22 mm, 10–15 mm).
The column was eluted at a flow rate of 9 ml min-1 by a linear
gradient of 0.1% TFA–acetonitrile on 0.1% TFA–H2 O, rising
within 60 min from 10% to 100%. The optical density of the eluate
was monitored at 220 or 280 nm. Fractions were collected and
analyzed by MALDI-TOF MS. Fractions containing the peptides
of the expected molecular weight were pooled and lyophilized.
Potentiometric measurements
Stability constants for protons and Cu(II) complexes were calculated from pH-metric titrations carried out in argon atmosphere
at 298 K using a total volume of 2 cm3 . Alkali (NaOH, 0.1 M) was
added from a 0.250 cm3 micrometer syringe which was calibrated
by both weight titration and the titration of standard materials.
The concentration of peptide1 was 2 ¥ 10-3 M and the ligandto-metal molar ratio was 1.1 : 1 and 1.1 : 2. For peptide2, the
concentration was 1.3 ¥ 10-3 M and the ligand-to-metal molar
ratio was 1.3 : 1, 1.3 : 2 and 1.3 : 3. The pH-metric titrations were
performed in ionic strength 0.10 M (KNO3 ) on a MOLSPIN pHmetric system using a Russel CMAW 711 semi-micro combined
electrode calibrated in hydrogen ion concentrations using HNO3 .11
The SUPERQUAD12 and HYPERQUAD13 computer programs
were used for stability constants calculations. Standard deviations
refer to random errors only. They are, however, a good indication
of the importance of a particular species in the equilibrium.
Samples were titrated in the pH region from 2.5–10.5. In whole pH
range, for the solutions containing Cu(II) ions and peptide1 and
peptide2 (1 : 1 and 1 : 2, ligand-to-metal molar ratios, respectively),
no precipitation was observed. However, for peptide2 at 1 : 3
ligand-to-metal molar ratio, occurrence of precipitation precluded
the analysis of the system.
Spectroscopic measurements
Solutions for EPR and UV-Vis measurements were of similar
concentrations to those used in the potentiometric studies. The
X-band EPR spectra were obtained at 77 K (liquid nitrogen)
on a Bruker ESP-300 spectrometer. Absorption spectra were
recorded on a Beckman DU-650 spectrophotometer (Beckman,
Palo Alto, CA) and Hitachi U-2010 (Hitachi instruments, Milan,
Italy), in the spectral range of 300–900 nm, using 1 cm cuvettes.
The temperature of the experiments was fixed at 25 ◦ C and the
spectra were recorded over pH range 3.0–11 at steps of 0.5 units
by addition of NaOH. The values of e were calculated at the
maximum concentration of the particular species obtained from
the potentiometric data.
Results and discussion
Protonation constants
The potentiometrically measured protonation constants for the
ligands studied and for comparable peptides are shown in Table 1
along with the calculated stepwise constants assigned to the
respective peptidic functions. The 20 amino acid sequence Ac–
TRSRSH6 TSEG–TRSRSH16 TSEG (peptide1) can be considered
as a H5 L ligand; deprotonation involves two histidine residues
(pK a = 6.70 and 5.89)8,9,14 and two g-carboxylic groups of the
Glu residues, together with the C-terminal COOH (pK a = 4.71,
4.20, 3.56).15 The 30 amino acid sequence Ac–TRSRSH6 TSEG–
TRSRSH16 TSEG–TRSRSH26 TSEG (peptide2) is a H7 L ligand;
deprotonation involves three histidine residues (pK a = 6.88, 6.26
and 5.71), three g-carboxylic groups of the Glu residues and a
C-terminal COOH (pK a = 4.76, 4.34, 3.91, 3.39).
For comparison, the potentiometric experimental data of the
14 a.a. sequence8 (the monohistidinic fragment of the Cap43
C-terminal), together with peptides containing two or more
Table 1 Protonation constants for Ac–TRSRSHTSEG–TRSRSHTSEG (peptide1), Ac–TRSRSHTSEG–TRSRSHTSEG–TRSRSHTSEG (peptide2)
and comparable peptides at 298 K, I = 0.10 M (KNO3 )
log b
Peptide
HL
H2 L
H3 L
H4 L
H5 L
H6 L
H7 L
peptide1
Ac–HGHGa
peptide2
Ac–HHGH–OHb
Ac–HAHVHc
Cap43 (14 a.a. sequence)d
6.70 ± 0.01
6.945
6.88 ± 0.01
7.73
6.83
6.35 ± 0.01
12.59 ± 0.01
13.264
13.14 ± 0.01
14.34
13.07
10.36 ± 0.01
17.30 ± 0.01
16.51
18.85 ± 0.01
20.29
18.74
21.50 ± 0.01
25.06 ± 0.01
23.61 ± 0.01
22.81
27.61 ± 0.01
31.86 ± 0.01
35.25 ± 0.01
NIm
COO-
COO-
COO-
COO-
4.71
3.24
4.76
2.52
4.20
3.56
4.34
3.91
Stepwise protonation constants
log K
Peptide
NIm
NIm
peptide1
Ac–HGHG
peptide2
Ac–HHGH–OH
Ac–HAHVH
Cap43 (14 a.a. sequence)
6.70
6.94
6.88
7.73
6.83
6.35
5.89
6.32
6.26
6.61
6.24
a
b
c
5.71
5.95
5.67
3.39
4.01
d
Ref. 16. Ref. 17. Ref. 18. Ref. 8.
6128 | Dalton Trans., 2008, 6127–6134
This journal is © The Royal Society of Chemistry 2008
histidine residues, Ac–HGHG,16 Ac–HHGH–OH17 and Ac–
HAHVH18 have been included in Table 1.
As we can see from the pK a values reported in Table 1, the
first imidazole nitrogen of histidine residues for both peptides
investigated is about 0.5 orders of magnitude more acidic than the
same nitrogen in the monohistidinic 14 amino acid fragment of
Cap43 C-terminal sequence.8
At the same time, the pK a value for the protonation of the
second and third imidazolic nitrogens of the histidine residues
for both peptides increases with the length of the peptide chain,
from 5.89 to 6.70 for peptide1, and from 5.71 and 6.26 to 6.88 for
peptide2; the average pK a for the imidazolic nitrogens (6.29 and
6.28 for peptide1 and peptide2, respectively) remains equivalent
to the value found for the monohistidinic sequence (6.35).
Cu(II) complexes containing one metal ion
Potentiometric experiments allowed us to detect a variety of Cu(II)
species in the pH range from 4 to 10.5 for 1 : 1 ligand-to-metal
complexes; their formation constants are listed in Table 2 together
with the log K* values (log K* = log b(CuHj L) - log b(Hn L)), the
protonation corrected stability constants, which are useful tools
to compare the coordination ability of different ligands when
coordination involves deprotonation.19
Complexation of Cu(II) ions starts around pH 4.0 for both of
the peptides and involves the histidines of the two fragments as the
anchoring sites for metal ions. By increasing the pH, three amidic
groups on the backbone are successively deprotonated so that
their nitrogen atoms can bind the metal to form a square planar
complex, in a typical fashion already evidenced for a number of
similar species.8,14,19
Both the ligands essentially behave in the same way towards
the copper ion, as it can be inferred by potentiometric and
spectroscopic results obtained. Sample solutions containing Cu(II)
ions and the ligands were, at acidic pH values, pale-blue, at neutral
pH, violet and, at basic pH, purple coloured. This behaviour is
reflected by the variation of both position and intensity of l max
UV-Vis signal, with the typical ‘‘blue-shift’’ as the pH increases.
Spectroscopic UV-Vis and EPR parameters for the major
species obtained are reported in Table 3. The UV-Vis spectra
are dominated by three main absorption bands whose intensity
depends on the pH. The first is located around 680 nm and prevails
at acidic pH conditions; the second has its maximum at 580–
600 nm and prevails around neutrality; the third, the most intense,
is located at 520–530 nm and dominates in the alkaline pH range.
On increasing the pH there is a lowering of g and an increasing
of the absolute value of A , suggesting the successively formation
of 4 N species and the deprotonation of side chain groups which
increases the whole negative charge on the copper complex.
Because of the low concentration or the overlaps of several
species, not all the potentiometrically observed species can be
spectroscopically well characterized; for that reason, only the wellseparated complexes for which it was possible to unambiguously
calculate spectroscopic parameters have been included in Table 3.
Potentiometry detects a range of complexes containing one
metal ion. Cu(II) forms seven monomeric species with peptide1
(Fig. 1a) and eight monomeric species with peptide2 (Fig. 1b).
From the potentiometric experiment of the Cu(II)–peptide1
system, CuHL, CuL, CuH-1 L, CuH-2 L, CuH-3 L, CuH-4 L and
CuH-5 L complexes fitted to the experimental titration curves
(Table 2).
Both potentiometric and spectroscopic results prove that complex formation between Cu(II) ions and peptide1 starts at around
pH 4.0 and the imidazol-N donor atom is the primary metal
binding site.
For peptide1, below pH 6.0, CuHL and CuL are visible.
CuHL, which corresponds to the monodentate coordination of
one hystidine residue, where a nitrogen atom from the imidazole
Table 2 Stability constants of Cu(II) complexes containing one metal ion with Ac–TRSRSHTSEG–TRSRSHTSEG (peptide1), Ac–TRSRSHTSEG–
TRSRSHTSEG–TRSRSHTSEG (peptide2) and comparable peptides at 298 K, I = 0.10 M (KNO3 )
log b
Peptide
CuH3 L
CuH2 L
CuHL
CuL
CuH-1 L
CuH-2 L
peptide1
Ac–HGHGa
peptide2
Ac–HHGH–OHb
Ac–HAHVHc
Cap43
(14 a.a. sequence)d
11.05 ± 0.01 5.93 ± 0.01 -0.28 ± 0.03 -6.22 ± 0.01
11.04
6.49
0.40
-6.13
22.04 ± 0.02 17.39 ± 0.02 12.72 ± 0.01 7.12 ± 0.01 0.86 ± 0.01 -6.04 ± 0.01
18.50
14.19
8.38
1.37
-6.73
13.08
8.08
1.27
-5.85
4.26 ± 0.01 -1.72 ± 0.02 -7.56 ± 0.01 -15.04 ± 0.01
CuH-3 L
CuH-4 L
CuH-5 L
-13.20 ± 0.01 -21.50 ± 0.01 -32.52 ± 0.02
-16.41
-13.92 ± 0.01 -23.65 ± 0.01
-16.66
-15.81
-25.06 ± 0.03
log K*e
Peptide
1N{NIm }
2N{NIm , N- },{2NIm }
3N{NIm , 2N- }, 3N{3NIm }
4N{NIm , 3N- }
peptide1
peptide2
Cap43 (14 a.a. sequence)a
Ac–HGHG
Ac–HHGH–OH
Ac–HAHVH
-1.54
-1.41
-2.09
-2.22
-1.79
-6.66
-6.13
-8.07
-6.54
-6.10
-5.66
-12.11
-11.73
-13.91
-13.07
-11.91
-19.09
-19.63
-21.39
-23.35
-22.61
-21.48
a
Ref. 16. b Ref. 17. c Ref. 18. d Ref. 8. e log K* = log b(CuHj L) - log b(Hn L).
This journal is © The Royal Society of Chemistry 2008
Dalton Trans., 2008, 6127–6134 | 6129
Table 3 Spectroscopic data for Cu(II) complexes of Ac–TRSRSHTSEG–TRSRSHTSEG (peptide1), Ac–TRSRSHTSEG–TRSRSHTSEG–
TRSRSHTSEG (peptide2) and 14 a.a. sequence
UV-Vis
EPR
3
-1
Type
l max /nm
e /dm mol cm
peptide 14 a.a.–Cu 1 : 1
pH 5.5
CuL
pH 7.0
CuH-2 L
pH 9.0
CuH-3 L
(1 N)
(3 N)
(4 N)
700
605
534
peptide1–Cu 1 : 1
pH 5.5
pH 6.5
pH 7.5
pH 9.5
CuL
CuH-2 L
CuH-3 L
CuH-4 L
(2 N)
(3 N)
(4 N)
(4 N)
peptide2–Cu 1 : 1
pH 7.5
pH 9.0
pH 10
CuH-2 L
CuH-3 L
CuH-4 L
peptide1–Cu 1 : 2
pH 7.0
pH 8.0
pH 9.5
peptide2–Cu 1 : 2
pH 6.0
pH 6.5
pH 7.5
pH 8.5
pH 10.5
pH
a
a
-1
A /Gc
g d
20
89
122
140
167
196
2.342
2.233
2.196
688
609
534
530
57
112
121
129
149
167
184
185
2.328
2.233
2.210
2.203
(3 N)
(4 N)
(4 N)
581
557
553
116
128
128
165
184
192
2.232
2.211
2.195
Cu2 H-4 L
Cu2 H-5 L
Cu2 H-6 L
(3 N ¥ 2)
(3 N + 4 N)
(4 N ¥ 2)
583
574
552
92
103
126
167
2.235
193
2.201
Cu2 H-2 L
Cu2 H-4 L
Cu2 H-5 L
Cu2 H-6 L
Cu2 H-7 L
(2 N ¥ 2)
(3 N ¥ 2)
(3 N + 4 N)
(4 N ¥ 2)
(4 N ¥ 2)
625
596
570
532
532
59
85
101
121
122
161
167
2.305
2.228
186
193
2.207
2.197
Species
b
± 5. b d–d transition, ± 3. c ± 3. d ± 0.002.
Fig. 1 Species distribution diagrams of Cu(II) complexes with (a) peptide1 and (b) peptide2, 1 : 1 ligand-to-metal molar ratio.
nitrogen of the histidine residue is involved in the coordination to
the metal ion,8,20–22 cannot be detected by spectroscopy because of
the low concentration and overlaps.
Maximum formation of CuL can be seen at pH 5.5. EPR
parameters (Table 3, Fig. 2a) A = 149 G and g = 2.329 and
d–d transition at 688 nm, e = 57 are similar to those found for
1 N species with 14 a.a. monohistidinic fragment,8 though the
spectroscopic parameters together with the calculated log K* may
suggest the formation of a 2 N complex in a slightly distorted
environment in agreement with the irregular geometry of a (2Nim )
6130 | Dalton Trans., 2008, 6127–6134
macrochelate, as recently reported in the literature for similar
species.18
When the pH is raised, additional protons are lost in a quick
sequence. The corresponding pK a values (6.21, 5.94 and 6.98,
respectively) suggest that Cu(II) ion promotes the deprotonation
of three amidic nitrogens, as already reported for analogous
systems.14,20
The next complex which can be spectroscopically characterized
is CuH-2 L species (Fig. 1a). The spectroscopic parameters are
similar to those found for the Cu(II)–14 a.a. fragment species for
This journal is © The Royal Society of Chemistry 2008
be attributed to the involvement of an arginine side chain close to
the metal coordination centre.
The deprotonation has no effect on the structure of the complex
formed, as seen from EPR and UV-Vis measurements.
At pH higher than 9.0, CuH-5 L species (pK a = 11.02) can
be seen. It likely comes from the deprotonation of the pyrrolic
nitrogen of the imidazole residue bound to copper ion,8,14 which
can lead to the formation of imidazolato-bridged polynuclear
species.23
The log K* values for 3 N species suggest a stabilization for the
20 amino acid peptide complexes of about two orders of magnitude
compared to those of the 14 amino acid sequence, and for 4 N
species about two orders of magnitude higher compared to those
of the 14 amino acid sequence and Ac–HAHVH, four and three
orders of magnitude compared to those of Ac–HGHG and Ac–
HHGH–OH peptides, respectively (Table 2).
The stabilization may result from a structural organization
which involves the bigger fragment of 20 amino acids after Cu(II)
coordination or, possibly, from an axial interaction of the second
histidine residue with copper(II) ions.
For the 30 amino acid fragment (Fig. 1b), eight complexes
can be fitted to the experimental titration curve: CuH3 L, CuH2 L,
CuHL, CuL, CuH-1 L, CuH-2 L, CuH-3 L and CuH-4 L. At pH
lower than 6.0 four species can be fitted from the curve: three
protonated species CuH3 L (15%), CuH2 L (17%), CuHL (45%)
and CuL (log K* = 1.41) which appears at pH 4.5. All these species
overlap each other and cannot be unambiguously characterized by
spectroscopy. Increasing the pH, spectroscopic parameters can be
calculated only for CuH-2 L, CuH-3 L and CuH-4 L. CuH-1 L (pK a =
6.26) is a 2 N {NIm , N- } species and appears at pH 5. The next
species which can be characterized is CuH-2 L complex. EPR parameters A = 165 G, g = 2.232 (Fig. 2b) may indicate a 3 N {NIm ,
2N- } binding mode. Above pH 7 a new deprotonation (CuH-2 L CuH-3 L + H+ , pK a 7.88) was observed, which can be attributed
to the metal-promoted deprotonation of a third amide nitrogen.
The spectroscopic parameters, d–d transition at 557 nm (Fig. 3)
and A = 184 G, g = 2.211 indicate a 4 N {NIm , 3N- } species
(log K* = -19.63), which has its maximum formation at pH 8.6.
The relative low hyperfine coupling constants A and high g may
correspond to a distortion in the coordination sphere of Cu(II).
Fig. 2 Selected X-band EPR spectra for the peptide1–Cu(II) (a) and
peptide2–Cu(II) (b) complexes in a 1 : 1 ligand-to-metal molar ratio.
the 3 N {NIm , 2N- } binding mode. Above pH 6 (Fig. 1a), the
successive deprotonations of CuH-2 L and CuH-3 L species take
place, maximum formation at pH around 7.7. The deprotonation
of CuH-2 L and formation of CuH-3 L corresponds to the deprotonation and coordination to copper(II) ions of the third amide
nitrogen. The absorption band of the d–d transition at 534 nm
and EPR parameters A = 184 G, g = 2.210 (Fig. 2a, Table 3)
indicate the 4 N{NIm , 3N- } binding mode. Moreover, the EPR
parameters of CuH-3 L species correspond well to those of 4 N
complex in a slightly distorted coordination environment19 and
they are insignificantly different in comparison to those of the
CuH-3 L complex for Cu(II)–14 a.a. system (Table 3).
The calculated pK a = 8.3 relative to the CuH-3 L CuH-4 L +
H+ equilibrium, might involve the deprotonation of the guanidine
moiety of an arginine residue.8,14 The relatively low pK a value can
This journal is © The Royal Society of Chemistry 2008
Fig. 3 UV-Vis spectra of the system peptide2 : Cu(II) molar ratio 1 : 1 as
a function of the pH.
Dalton Trans., 2008, 6127–6134 | 6131
Also in this case we are able to detect a CuH-4 L species
starting from pH 7.5; we can attribute it to the deprotonation
(pK a = 9.73) of a guanidine group of an arginine residue or to
a further deprotonation of N amide. The changes in the EPR
parameters (A = 193 G and g = 2.195) may suggest changes
in the coordination environment, probably through the increase
of coordinated amide nitrogen donors so that a 4 N {4N- }
coordination mode is likely.24
The log K* values for peptide2 complexes from 1 N to 2 N
coordination mode are similar to those of Ac–HHGHOH, but for
4 N {NIm , 3N- } binding mode are about three orders of magnitude
higher in comparison to that of Ac–HHGHOH.
It is interesting to note that log K* values for the peptide2
species appear to be higher than those of the monohistidinic 14 a.a.
sequence. In general, peptide1 and peptide2 complexes seem to be
more stable with respect to those of the monohistidinic fragment,
up to about two orders of magnitude for 3 N and 4 N species.
The length of the peptide plays an important role in stabilizing its metal complexes, probably through the conformational
organization of the peptide itself which can better shield the
metal coordination site from the attack of water molecules. This
stabilization adds to that of the side-chain organization promoted
by the metal itself upon coordination.
Cu(II) complexes containing more than one metal ion
To evaluate the role in metal coordination of sets of histidines
within the same peptide, we studied the behaviour of Cu(II) ions
towards peptide1 and peptide2 also in a 1 : 2 ligand-to-metal molar
ratio. Unfortunately, it was not possible to collect any data for
a 1 : 3 peptide2–Cu(II) molar ratio since, under our conditions,
extended precipitation was observed.
For both 20 and 30 amino acid fragments, six metal complexes
with 1 : 2 ligand-to-metal molar ratio can be fitted to the experimental titration curves. In Fig. 4a and b speciation curves for these
complexes are reported.
Their formation constants are listed in Table 4 with the relative
values of log K*.
If we examine the speciation curve for peptide1–Cu(II) with a
1 : 2 molar ratio (Fig. 4a) we see that complexation of Cu(II) starts
Fig. 4 Species distribution diagrams of Cu(II) complexes with (a) peptide1 and (b) peptide2, 1 : 2 ligand-to-metal molar ratio.
Table 4 Stability constants of Cu(II) complexes containing two metal ions with Ac–TRSRSHTSEG–TRSRSHTSEG (peptide1), Ac–TRSRSHTSEG–
TRSRSHTSEG–TRSRSHTSEG (peptide2) and comparable peptides at 298 K, I = 0.10 M (KNO3 )
log b
Peptide
Cu2 H2 L
peptide1
peptide2
Ac–HAHVHa
18.54 ± 0.03
Cu2 L
Cu2 H-2 L
Cu2 H-4 L
Cu2 H-5 L
Cu2 H-6 L
Cu2 H-7 L
10.50 ± 0.04
-3.28 ± 0.04
-0.80 ± 0.02
-1.08
-15.52 ± 0.01
-13.39 ± 0.02
-13.63
-23.04 ± 0.01
-20.41±.02
-23.82
-31.50 ± 0.01
-28.06 ± 0.02
-34.24
-42.20 ± 0.02
-36.78 ± 0.02
log K*
Peptide
2N¥2
3N¥2
3N+4N
4N¥2
peptide1
peptide2
Ac–HAHVH
Cap43 (14 a.a.)b
Ac–HAHVH
-15.87
-12.77
-28.11
-25.36
-25.54
-27.82 (-13.91 ¥ 2)
-21.32 (-10.66 ¥ 2)
-35.63
-32.38
-35.73
-35.30 (-13.91 + -21.39)
-28.12 (-10.66 + -17.47)
-44.09
-40.03
a
-16.14 (-8.07 ¥ 2)
-42.78 (-21.39 ¥ 2)
Ref. 18. b Ref. 8.
6132 | Dalton Trans., 2008, 6127–6134
This journal is © The Royal Society of Chemistry 2008
at pH about 3.5, with formation of the Cu2 H2 L species. With
increasing the pH three species dominate: Cu2 H-4 L, Cu2 H-5 L,
Cu2 H-6 L.
For the Cu2 H-4 L complex, with maximum formation at about
pH 7, the stoichiometry and the parameters of absorption UV-Vis
and EPR spectra (Fig. 5a, Table 3) clearly indicate the formation
of 3 N {NIm , 2N- } coordination of two metal ions to the peptide.
By raising the pH, consecutive deprotonations take place to give
Cu2 H-5 L and Cu2 H-6 L complexes (Fig. 4a). The stoichiometry
of Cu2 H-5 L complex suggests the 3 N + 4 N {NIm , 2N- } + {NIm ,
3N- } coordination mode. Above pH 8, Cu2 H-6 L species, maximum
formation at pH 9.5, can also be fitted to the experimental titration
curves. The stoichiometry of Cu2 H-6 L complex is in agreement
with a 4 N {NIm , 3N- } ¥ 2 binding mode of two metal ions
coordinated to the peptide. This binding mode of each metal ion
is supported by spectroscopic data (Table 3).
Fig. 5 Selected X-band EPR spectra for the peptide1–Cu(II) (a) and
peptide2–Cu(II) (b) complexes in a 1 : 2 ligand-to-metal molar ratio.
This journal is © The Royal Society of Chemistry 2008
Finally, the Cu2 H-7 L species forms at pH above 8.5. The
pK a = 10.7 relative to Cu2 H-6 L Cu2 H-7 L + H+ equilibrium
is compatible with the deprotonation of a guanidine residue.
UV-Vis parameters and EPR values recorded for the system of
ligand–Cu(II) at 1 : 2 molar ratio are consistent with those obtained
for the same system at 1 : 1 molar ratio, suggesting an identical
behaviour towards metal coordination in each of the two 10 amino
acid fragments of peptide1.
For peptide1–Cu(II) at 1 : 2 molar ratio, the log K* value
(log K* = -15.87) for Cu2 H-2 L complex 2 N{NIm , N- } ¥ 2 is
0.27 orders of magnitude higher compared to that evaluated when
each metal ion is coordinated independently to the monohistidinic
fragment (log K* = -16.14). On the other hand, the log K* value of
the successive complex Cu2 H-4 L {3N ¥ 2} (log K* = -28.11) and
Cu2 H-6 L {4N ¥ 2} (log K* = -44.09) are respectively 0.29 and 1.31
orders of magnitude lower in comparison to that obtained for the
monohistidinic fragment with the same binding mode (-27.82 and
-42.78, respectively) (Table 4). This means that the coordination
of two metal ions to the two repeated 10 amino acid sequence with
3 N {NIm , 2N- } and 4 N {NIm , 3N- } binding modes is not of the
cooperative type. The same result was obtained for the analogous
nickel complex, with the same 20 amino acid peptide1 in a 1 : 2
ligand–Ni(II) molar ratio.9
For the 30 amino acid fragment, peptide2, in 1 : 2 ligand–Cu(II)
molar ratio the situation is slightly different. In the pH range from
4.0 to 6.0 we are able to detect small amounts of highly protonated
species: CuH3 L, CuH2 L and CuHL complexes. By raising the pH,
CuL, CuH-1 L and Cu2 L species are also present (Fig. 4b). In
analogy with peptide1, for peptide2 we are able to detect the 2 : 1
metal : ligand molar ratio species from sequential deprotonation
of the amidic functions: Cu2 H-2 L, Cu2 H-4 L, Cu2 H-5 L, Cu2 H-6 L
species; the corresponding pK a values are 5.65, 6.29 and 7.34 and
the corresponding coordination modes are 2 N {NIm , N- } ¥ 2, 3 N
{NIm , 2N- } ¥ 2, 3 N + 4 N {NIm , 2N- }{NIm , 3N- } and 4 N {NIm ,
3N- } ¥ 2, respectively.
The spectroscopic data are consistent with the results obtained
from the potentiometric data calculations.
The log K* values of these complexes (-12.77, -25.36, -32,38
and -40.03) lie between 2.46 and 3.37 orders of magnitude higher
than that evaluated for the system where each metal ion is
coordinated to the monohistidinic fragment (14 a.a. sequence)
in an independent way (Table 4).
The evidence collected for all the species relative to the interaction of peptide2 with two metal ions suggests that the coordination
mode of our peptide to the Cu(II) ion is of the cooperative type.
The side chain organization promoted by the second metal ion
coordinated to the three-repeated peptide may play an important
role in stabilizing the Cu2 –peptide2 complexes. The stabilization
may also result from a possible additional interaction of the third
imidazole nitrogen with Cu(II) ions.
Finally, Cu2 H-7 L species forms at pH above 7.0, whose maximum (100% of Cu(II) in solution) lies at pH ~ 10.5. It likely may
come from the deprotonation and coordination of fourth amide
nitrogen to metal ions (pK a = 8.72). This is in agreement with the
change in the EPR parameters going from Cu2 H-6 L to Cu2 H-7 L,
with the increasing of A (A = 193 G) and the decreasing of g
(g = 2.197).24
It should also be mentioned that the log K* values for the
Cu2 H-4 L (3 N ¥ 2) complex of peptide2 and Cu2 H-5 L (3 N + 4 N)
Dalton Trans., 2008, 6127–6134 | 6133
complex of peptide1 are comparable to that of Ac–HAHVH
peptide.
Conclusions
The results obtained clearly indicate that the acetylated 20 amino
acid sequence Ac–TRSRSH6 TSEG–TRSRSH16 TSEG and
30 amino acid sequence Ac–TRSRSH6 TSEG–TRSRSH16 TSEG–
TRSRSH26 TSEG, fragments of Cap43 protein, bind Cu(II) ions
effectively. Potentiometric studies showed that Cu(II) can form
stable complexes with the peptides over the pH range from 3.5 to
11.0, while spectroscopic techniques assisted the identification of
each species.
Coordination of the metal ion starts at low pH values from
the imidazole nitrogen atom of the histidine residue and, with
increasing the pH, Cu(II) ions are able to deprotonate, in both
peptides investigated, successive peptide nitrogen atoms forming
Cu(II)–N- bonds and giving, at physiological pH, the 3 N {NIm ,
2N- }, CuH-2 L (1 : 1 ligand–Cu(II) molar ratio) and Cu2 H-4 L (1 : 2
ligand–Cu(II) molar ratio) as the major species, while, at pH higher
than 7, the 4 N {NIm , 3N- } CuH-3 L (1 : 1 ligand–Cu(II) molar
ratio) and Cu2 H-6 L (1 : 2 ligand–Cu(II) molar ratio) are dominant
species. The driving force leading the coordination process is the
formation of two five-membered chelate rings around the metal
ion, in the way depicted in Scheme 1.
Scheme 1 4 N {NIm , 3N- } coordination mode for the single monohistidinic fragment TRSRSHTSEG.
The Cu(II) complexes for 1 : 1 ligand-to-metal molar ratio with 1
N {NIm } and 4 N {NIm , 3N- } coordination mode of our fragments
are more stable by about 0.55 and 2.3 orders of magnitude in
respect to those of monohistidinic fragment, respectively. This
stabilization may result from a structural organization of the
peptide in copper(II) complex due both to the self-organization of
the longer peptidic chain and to the reorganization of the peptide
side-chain induced by metal coordination. The stabilization may
6134 | Dalton Trans., 2008, 6127–6134
also be increased from the additional interaction with copper(II)
ions with imidazole nitrogen of His residue in axial position.
It is also interesting to note that, at physiological pH, all the
Cu(II) ions are involved in the formation of complex species with
both the peptides investigated.
Acknowledgements
This work was supported by the Fondazione Banco di Sardegna,
Sassari, Sardegna, Italy.
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