Interactions of transition metal ions with His-containing peptide models of histone H2A

Journal of Molecular Liquids 118 (2005) 119 – 129 www.elsevier.com/locate/molliq

Interactions of transition metal ions with His-containing peptide models of histone H2A Marios Mylonasa, Artur Kre˛yelb, John C. Plakatourasa,*, Nick Hadjiliadisa,*, Wojciech Balc a

University of Ioannina, Department of Chemistry, Ioannina 45110, Greece b Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland c Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Available online 11 September 2004

Abstract The coordination properties of Ni(II), Cu(II) and Zn(II) ions towards the terminally blocked (CH3CONH– and –CONH2) hexapeptides – TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK–, which are all models of the C-terminal btailQ (–ESHH–) of histone H2A, were studied by potentiometric and several spectroscopic techniques (UV/Vis, CD, NMR, EPR). The peptides were chosen in such a way as to compare the effect of Glu, Ser and His residues on the stability, the coordination and hydrolytic abilities of the complexes formed. It was found that all peptides bind to the metal ions initially through one or two imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, a series of square-planar complexes are formed with Ni(II), which binds simultaneously through an imidazole and three amide nitrogens in an equatorial plane. This proposed conformation includes the participation of only one imidazole nitrogen, in the case of all peptides, in the coordination sphere of Ni(II) ions. Additionally, all peptides coordinate Cu(II) efficiently. At higher pH values, Cu(II) ions coordinate equatorially with the imidazole nitrogen of His-5 or His-4 and three amido nitrogens of the peptides –TESAHK– and – TESHAK–, while the second histidine residue of the peptides –TESHHK–, –TASHHK– and –TEAHHK– is additionally bound in the apical position. In contrast, the combination of potentiometric titrations and one- and two-dimensional 1H-NMR suggested no amide coordination in the coordination sphere of Zn(II) ions, over a wide range of pH. In basic solutions, the peptides –TASHHK– and –TESAHK– were hydrolyzed in a Ni(II)-assisted fashion, while no hydrolytic processes were noticed in peptides –TEAHHK– and –TESHAK– where the Ser or His-5 residues are replaced with the Ala residue. Moreover, CuH
1L complex with –TESHHK– reacts with H2O2 and the resulting reactive oxygen intermediate efficiently oxidizes 2V-deoxyguanosine. D 2004 Elsevier B.V. All rights reserved. Keywords: Histone H2A; Hexapeptides; Eukaryotic cells

1. Introduction It is well known that several metals have been found to be carcinogenic to humans and animals [1,2]. Nevertheless, the molecular mechanism by which metal carcinogenicity is exerted is not fully understood. Studies of several years support that neoplastic transformation of cells results from a heritable alteration in the genetic code, concluding that any molecule which can bind with constituents of the cell nuclei

* Corresponding authors. E-mail address: [email protected] (J.C. Plakatouras). 0167-7322/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2004.07.025

may affect the genetic code. Thus, it is believed that metal ions may cause changes to the genetic code by their binding to the proteins and DNA. Particularly, the abundance of histones inside the cell nuclei makes them the prime expectants for that role. In all eukaryotic cells, histone basic proteins provide a scaffold for DNA double helix and are organized in repeating units called nucleosomes. The nucleosome is composed of a histone octamer (containing two copies of each of histones H2A, H2B, H3 and H4) and a 170 base pairs DNA stretches [3]. The elucidation of metal’s toxicity and carcinogenicity can be approached by studying their interactions with model histone peptides. In this respect, the interaction of Ni(II)

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M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

ions with the blocked tetrapeptide –CAIH– representing the 110–113 residues of histone H3 was studied first [4–7]. In subsequent papers, the coordination properties of Ni(II) towards the hexapeptide –TESHHK– and bigger peptide models of the C-terminal btailQ –ESHH–, which corresponds to the 121–124 residues of a major variant of mammalian histone H2A, was also described [8,9]. In general, it was found that Ni(II) ions may form slightly distorted squareplanar complexes with –CAIH– motif above pH 6.5, bound through thiolate and imidazole donors, that could mediate oxidative damage to DNA [6]. In addition, Ni(II) ions are able to hydrolytically cleave the hexapeptide –TESHHK– at pH 7.4, producing new square-planar Ni(II) complexes with the SHHK– part of it [8]. The complex formed was stable enough to ensure a possible Ni(II) binding to nucleosome [10], and to promote oxidative degradations to DNA, via Fenton-like mechanisms [9]. In this paper, we review the results of a study of Ni(II), Cu(II) and Zn(II) ion interactions towards the blocked hexapeptides –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK–, models of the –ESHH– motif of histone H2A. It must be mentioned that these systematic changes in the peptide sequence of the studied hexapeptides may help in the better understanding of the mechanism of Ni(II)-assisted hydrolysis of –TESHHK– peptide [8,9] and to compare the influence of Glu, Ser and His residues against the stability, stoichiometry, oxidative ability, hydrolysis, etc. of the complexes formed. This study may provide an insight in the mechanism of histone hydrolysis and oxidation of DNA bases induced by metal ions.

(–TASHHK–), Ac-Thr-Glu-Ala-His-His-Lys-am (–TEAHHK–), Ac-Thr-Glu-Ser-Ala-His-Lys-am (–TESAHK–) and AcThr-Glu-Ser-His-Ala-Lys-am (–TESHAK–) were synthesized in the solid phase, as described previously [11–13]. The purity and identity of the peptides was finally confirmed using TLC chromatography, one- and two(TOCSY) dimensional 1H-NMR techniques (Bruker AMX400MHz spectrometer) and ESI-MS (Finnigan Mat TSQ 700 mass spectrometer) [12,13]. 2.3. Potentiometry The protonation and stability constants of Ni(II), Cu(II) and Zn(II) complexes of –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK–, in the presence of 0.1 M KNO3 were determined by using pH-metric titrations over the pH range 2.5–10.5, at 25 8C, with 0.1 M NaOH as titrant (Molspin automatic titrator, Molspin, Newcastle-upon-Tyne, UK). Changes of pH were monitored with a combined glass-silver chloride electrode calibrated daily in H+ concentrations by HNO3 titrations [14]. Sample volumes of 1.5 ml and concentrations of 1 mM of the peptides and 0.5–1 mM Ni(NO3)2d 6H2O or Cu(NO3)2d 6H2O or Zn(NO3)2d 4H2O were used. The experimental data were analyzed using the SUPERQUAD program [15]. Standard deviations computed by SUPERQUAD refer to random errors. 2.4. NMR spectroscopy 1

2. Experimental 2.1. Materials Ni(NO3)2d 6H2O, Cu(NO3)2d 6H2O, Zn(NO3)2d 4H2O, HNO3, KNO3, acetonitrile (HPLC grade), dicyclohexylcarbodiimide (DCC), HCl, and NaOH were obtained from E. Merck (Darmstadt, Germany). 1-Hydroxybenzotriazole (1HOBt), trifluoroacetic acid (TFA), trifluoroethanol (TFE), 3(trimethylsilyl)propionic acid sodium salt (TSP), anisole, D2O and DCl were purchased from Aldrich (Milwaukee, WI). Chelex 100 resin was purchased from Sigma (St. Louis, MO). Isopropanol, dimethylformamide, diethylether and dichloromethane were purchased (analytical grade) from Lab-Scan (Dublin, Ireland). The protected amino acids, Fmoc-His(Mtt)-OH, Fmoc-Lys(Boc)-OH, FmocSer(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH and Fmoc-Ala-OH and the resin H-Linker-2-chlorotrityl were purchased from CBL (Patras, Greece). 2.2. Peptide synthesis The blocked hexapeptides Ac-Thr-Glu-Ser-His-HisLys-am (–TESHHK–), Ac-Thr-Ala-Ser-His-His-Lys-am

H-NMR experiments were performed on a Bruker AMX-400MHz spectrometer. The one- and two- (TOCSY) dimensional experiments were carried out in free and metalbound peptides, as described previously [13,16]. 2.5. EPR spectroscopy The X-band EPR spectra were obtained at 77 K (liquid nitrogen) on a Bruker ESP-300 spectrometer, using Cu(II) concentrations of 3 mM and Cu(II)-to-peptide ratios of 1:1. Ethanediol aqueous solution (30% v/v) was used as solvent for these measurements to ensure homogeneity of the frozen samples. 2.6. UV/Vis spectroscopy UV/Vis spectra were recorded on a SHIMADZU UV2401PC spectrophotometer, at 25 8C, in the spectral range of 300–900 nm, in 1 cm cuvettes, using Ni(II) or Cu(II) concentrations of 1 mM and metal-to-peptide ratio 1:1. The samples were titrated with NaOH by adding very small amounts of concentrated solutions manually. Changes of pH were monitored with a combined glass-silver chloride electrode over the pH range 2–11.

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

Scheme 1.

2.7. CD spectroscopy CD spectra were recorded on a JASCO J-715 spectropolarimeter, in the spectral range of 250–800 nm, in 1 cm cuvettes, using Ni(II) or Cu(II) concentrations of 3 mM and metal-to-peptide ratios 1:1 at pH 2–11.

3. Results and discussion 3.1. Acid–base properties of free hexapeptides Both the N- and C-terminal of the hexapeptides models (Scheme 1) were blocked by acetylation and amidation, respectively, to make the peptides more realistic models of the –ESHH– motif of histone H2A, as has been reported previously [11–13,16]. The protonation constants and dissociation macroconstants of the peptides were measured by potentiometric [11– 13] and 1H-NMR [16] titrations are presented in Table 1. The highest pK a values between 10.48 and 10.28 can be easily assigned to the q-amino group of Lys residues and the lowest values at 3.85 and 4.10 of –TESHHK– and – TEAHHK–, respectively, can be assigned to the carboxyl group of Glu residues, according to the literature data

121

Fig. 1. An example of a species distribution diagram of Ni(II) complexes with –TEAHHK–, calculated for reagent concentrations used in UV/Vis spectroscopy, using stability constants obtained from potentiometry: c Cu=c L=1 mM. UV/Vis absorption (o) is overlaid. The dashed line presents the sum of NiH
2L+NiH
3L.

[8,17,18]. The rest values of macroconstants corresponding to the deprotonations of His residues, were found to be separated by less than one log unit suggesting the possibility of concurrent deprotonations at the two His residues. According to the literature data, the lowest pK a value may correspond to the protonation of the His residue closer to the N-terminal of the peptide, while the closest His in the Cterminal has higher basicity [19–21]. 3.2. Coordination properties of metal complexes 3.2.1. Ni(II) complexes Potentiometric titrations in aqueous solutions were carried out for the hexapeptides in the presence of nickel nitrate in peptide–Ni(II) ratios 1:1 and 2:1. Four metal complexes (NiHL, NiH
1L, NiH
2L and NiH
3L) can be fitted to the experimental titration curves obtained for the Ni(II)/–TESHHK– and Ni(II)/–TESAHK–, while in the case of the rest three binary systems five metal complexes (NiHL, NiL, NiH
1L, NiH
2L and NiH
3L) were found, in the pH range of 5.0–10.5 (Fig. 1). The stability constants of

Table 1 Protonation constants and dissociation macroconstants of –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK– (T=25 8C, I=0.1 M KNO3) HL

H2 L

H3L

log b a

pKa

log b a

–TESHHK–

10.28 (1)

10.28

–TASHHK–

10.48 (1)

10.48

–TEAHHK–

10.25 (1)

10.25

–TESAHK– –TESHAK–

10.37 (1) 10.41 (1)

10.37 10.41

17.06 (1) 6.78 pK a* 6.88 (5) 17.10 (1) 6.62 pK a* 6.64 (4) 16.93 (1) 6.68 pK a* 6.72 (2) 16.60 (1) 6.23 16.68 (1) 6.27

pKa

pK a, calculated from potentiometric titrations. K a*, calculated from 1H-NMR titrations. a b=[HjLk]/([H] j [L]k), standard deviations of the last digit are given in parentheses.

log b a

H4 L pKa

22.96 (1) 5.90 pK a* 6.12 (4) 22.85 (1) 5.74 pK a* 6.06 (3) 22.93 (1) 6.00 pK a* 6.15 (3) 20.64 (1) 4.04 20.71 (1) 4.04

log b a

pKa

26.81 (1)

3.85

–

–

27.03 (1)

4.10

– –

– –

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M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

Table 2 Stability constants of Ni(II) complexes with –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK– (T=25 8C, I=0.1 M KNO3) NiHL log b –TESHHK– –TASHHK– –TEAHHK –TESAHK– –TESHAK– a

NiL

a

14.04 14.20 14.10 13.02 13.61

log b (2) (1) (1) (1) (1)

NiH
1L a

– 5.86 (1) 6.00 (1) – 5.80 (1)

NiH
2L

a

pKa1

log b

– 8.34 8.10 – 7.81


2.16
1.52
2.62
3.00
3.12

(2) (1) (1) (1) (1)

pKa3

log b a


11.52 (1)
10.16 (1)
11.48 (1)
11.58 (1)
11.55 (1)

9.36 8.64 8.86 8.58 8.43


22.80
20.49
22.34
22.67
22.74

log b

pKa2 7.38 8.62 8.92

NiH
3L

a

pKa4 (5) (1) (1) (1) (1)

11.28 10.33 10.86 11.09 11.19

b=[NiiHjLk]/([Ni]i [H] j [L]k), standard deviations of the last digit are given in parentheses.

Ni(II) complexes with the studied hexapeptides, calculated from pH-metric titrations using the SUPERQUAD program are presented in Table 2. Ni(II) complexes were also characterized with UV/Vis, CD and 1H-NMR [13] spectroscopies in the pH range of 5.0–10.5 and peptide–Ni(II) ratio 1:1, the parameters of which have already been presented elsewhere. The UV/Vis and CD spectroscopic data of the complexes are presented in Table 3. Obviously, Ni(II) ions start binding with all peptides at about pH 5. The first complexes formed with all peptides is probably NiHL. The stoichiometry and the d–d transition at 636–685 nm of these complexes [13] clearly indicate the binding of one or two imidazole nitrogens to Ni(II) ions [22,23]. In previous studies, Bal et al. [8] proposed that the carboxyl group of Glu residue of –TESHHK– may also participate in the coordination sphere of Ni(II) ions forming a slightly distorted octahedral complex. At higher pH values, the complexes NiHL with the peptides –TASHHK–, –TEAHHK– and –TESHAK– release a proton with a pK a 7.81–8.34 (Table 2), abstracted from the amide nitrogen of one of the His residues, forming a stable six-membered chelate ring and resulting the complexes NiL. Their overlap with other complexes in the pH range of 6.5–

9.5 and also their low concentration did not manifest its presence in CD and UV/Vis spectra with satisfactory results, and so it was only characterized on the basis of its stoichiometry and the values of the stability constants. These values (Table 2) are comparable to other similar complexes with –AGGH– and its analogues [24], suggesting the coordination of Ni(II) ions through the imidazole nitrogen of one of the His residues and the amide nitrogen of the same histidine. The transition to NiH
1L and NiH
2L complexes are associated with a neighboring amide bond deprotonation with a pK a of 7.38–8.92 and 8.43–9.36 (Table 2), respectively, filling the equatorial positions in the coordination sphere of Ni(II) ions. It must be mentioned that the formation of the stable five-membered chelate rings by consecutive nitrogens is the driving force for this coordination process. As it has been discussed in case of NiL complexes, NiH
1L complexes cannot be detected spectroscopically with satisfactory results, although the values of their stability constants (Table 2) suggest the 3N {2N
am, 1Nim} coordination mode, due to their similarity with the values of other comparable complexes with –AGGH– and its analogues [24]. In contrast, the d–d transitions near 440–

Table 3 UV/Vis (c=1 mM) and CD (c=3 mM) spectroscopic data for the 1:1 nickel complexes with the hexapeptides Complexes

NiHL

–TESHHK– UV/Visa CDb

636 (19) 377 (29)

480 (
0.16) 411 (+0.07)

–TASHHK– UV/Visa CDb

636 (7) 378 (30)

481 (
0.09) 412 (+0.02) 312 (
0.03)

–TEAHHK– UV/Visa CDb

636 (6) 378 (30)

485 (
0.06) 417 (+0.03)

–TESAHK– UV/Visa CDb

682 (11) 409 (59)

477 (
0.11) 406 (+0.02) 297 (
0.03)




–TESHAK– UV/Visa CDb

685 (12) 380 (12)

482 (
0.06) 297 (
0.04)

433 (38)

a b c

NiL

NiH
1L




441 (45)

NiH
2L 561 490 415 318

(+0.17) (
0.81) (
0.18) (
0.02)

445 (157)

450 (150) c

630 (10)

435 (51)

488 (
0.46)

427 (61)

535 (+0.33) 423 (
0.74) 306 (
0.04)

432 (76)

489 (
0.23) 416 (+0.02) 300 (
0.03)

479 (
0.22)

442 (154)

442 (125)

477 (
0.22) 425 (
0.25) 308 (
0.03)

440 (154) c

NiH
3L 559 493 412 560 494 439 400 522 422 306 566 496 439 337 516 421 309

(+0.48) (
1.35) (
0.37) (+0.24) (
1.00) (+0.04) (
0.10) (+1.60) (
3.05) (
0.12) (+0.20) (
1.91) (+0.54) (
0.03) (+1.80) (
3.34) (
0.08)

k max nm (q/M
1 cm
1). k max nm (Dq/M
1 cm
1). Spectroscopic assignment was impossible due to low concentration of the species and overlap with other species.

445 (163)

450 (165)

441 (159)

442 (127)

440 (163)

560 493 409 560 495 441 400 522 422 307 567 497 439 337

(+0.44) (
1.48) (
0.38) (+0.45) (
1.48) (+0.20) (
0.17) (+1.71) (
3.36) (
0.10) (+0.18) (
2.06) (+0.68) (
0.02)

516 (+1.84) 421 (
3.45)

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

450 nm and the CD parameters of NiH
2L complexes [13] support the simultaneous coordination of an imidazole and three amide nitrogens equatorially {3N
am, 1Nim}, forming a stable square-planar complex [25–27]. The participation of only one imidazole nitrogen in the coordination sphere of Ni(II) ions, in the case of all hexapeptides, is supported from the comparable values of logK*=log b{NiH
2L}
log b{H2L} of the NiH
2L complexes (
28.58,
27.26,
28.41,
28.18 and
28.23 in the case of –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK–, respectively) [13]. These values are also very similar with the corresponding values of comparable 4N {3N
am, 1Nim} Ni(II) complexes with the peptides –AKRHRK– [27] and –TRSRSHTSEGTRSR– [26] having logK*
28.70 and
28.16, respectively. The same proposal of the 4N coordination mode of NiH
2L complexes resulted also from the correlation species distribution diagrams of Ni(II) ions between –TEAHHK–:–TESHAK– and –TEAHHK–: –TESAHK–, and is presented elsewhere [13]. These diagrams support that while in neutral and slightly basic solutions (pH range 6–9) Ni(II) ions prefer to bind to the peptides with two histidine residues, in more basic solutions Ni(II) ions may coordinate to both of the peptides with the same selectivity, indicating that in square-planar complexes NiH
2L with all hexapeptides, only one His residue is bound equatorially. Finally, the NiH
3L complexes are formed upon the deprotonation of a non-coordinated Lys amine, with little change in the spectra [13]. It must be noted that the coordination mode suggested above for the square-planar complexes in basic solutions supports the binding of imidazole of His at position 5, while the imidazole of His at position 4 remain and uncoordinated. The last proposal and generally the suggesting conformation of all complexes are in good agreement with the 1H-NMR data, presented and discussed previously [13]. It is noticed that Ni(II) complexation with the peptides –TESHHK–, –TESAHK– and –TASHHK– is accompanied

123

by a hydrolytic cleavage of the peptides and formation of a square-planar complex with the resulting peptidic fragment. To prove this, a series of one-dimensional 1H-NMR spectra of freshly prepared samples containing each one of the hexapeptides with Ni(II) ions in a 1:1 ratio were recorded at pH* 10.8 at 37 8C during the time of incubation (about 8 h). The hydrolysis is also supported by synthesizing the tetrapeptides SAHK– and SHHK– which gave both identical square-planar complexes to the ones produced by hydrolysis (as indicated by their 1HNMR spectra at pH* 10.8). These NMR results indicate that while substitution of His in position 4 or Glu residue by an Ala residue has no influence on the hydrolysis reaction, the substitution of His in position 5 or Ser residue seems to be critical for this reaction (no hydrolytic ability). HPLC studies (Fig. 2) show the formation of the cleaved tetrapeptide. 3.2.2. Cu(II) complexes Similarly with the binary Ni(II)/peptide systems, potentiometric titrations in aqueous solutions were carried out for the hexapeptides in the presence of copper nitrate in peptide– Cu(II) ratios 1:1 and 2:1. The species distribution diagrams (Fig. 3) of the Cu(II)/hexapeptide systems indicate the formation of more than six species in the case Cu(II)/– TESHHK–, Cu(II)/–TASHHK– and Cu(II)/–TEAHHK– systems and five species in the case of Cu(II)/–TESAHK– and Cu(II)/–TESHAK– systems, in the pH range of 3.0–10.5. In general, it was found that the number of His residues inside the peptide sequence affects the Cu(II) binding towards the studied hexapeptides. Thus, it is similar in the case of the peptides containing two His residues (–TESHHK–, –TASHHK– and –TEAHHK–), while it is different and more simple in the case of the studied peptides containing only one His residue (–TESAHK– and –TESHAK–). The stability constants of Cu(II) complexes with the studied hexapeptides, calculated from pH-metric titrations using the SUPERQUAD

Fig. 2. HPLC graphs recorded during the incubation of a sample of the hexapeptide –TASHHK– in the presence of Ni(II) ions in 1:1 molar ratio (c=1 mM), at 37 8C, (a) pH=9.0, and (b) pH=7.4. (c) Correlation between the original incubated sample and the synthetic tetrapeptide in the presence of Ni(II) ions at the same molar ratio and buffer solution. Similar HPLC graphs were also recorded for the other hexapeptides.

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M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

Fig. 3. An example of a species distribution diagram of Cu(II) complexes with –TEAHHK–, calculated for reagent concentrations used in UV/Vis spectroscopy, using stability constants obtained from potentiometry: c Cu=c L=1 mM. UV/Vis absorption (o) is overlaid.

program are presented in Table 4. Cu(II) complexes were also characterized with UV/Vis, CD and EPR spectroscopies, in the pH range of 3.0–10.5 and peptide–Cu(II) ratio 1:1, the parameters of which have already been presented elsewhere [11,12]. The joint analysis of stability constants and spectroscopic parameters (UV/Vis and CD spectroscopic data of the complexes are presented in Table 5) allowed for an unambiguous assignment of number of nitrogen donors in the coordination sphere of the Cu(II) ion. The stoichiometry and pH range of existence of CuH2L complexes with the hexapeptides –TESHHK–, –TASHHK– and –TEAHHK–, indicates that it has one imidazole nitrogen bonded to Cu(II), comparable to other similar complexes with –AGGH– and its analogues [28]. The higher log b values of CuH2L complexes with the studied hexapeptides comparing with those of comparable complexes with –AGGH– and its analogues [28] suggest the participation of carboxyl group of Glu residue in the coordination sphere of Cu(II) ions. These complexes are superseded by CuHL complexes, in which two nitrogen donors are coordinated. CD spectra [11,12] indicate that there is no amide nitrogen coordination in these complexes, due to the low intensity of their CT band at 318 nm [28–30]. The d–d transitions as well as the CD and EPR parameters of CuHL complexes [11,12],

clearly indicate that the coordination is exerted by both imidazole nitrogens, similar to the analogous Ni(II) complexes [13]. In more basic solutions, CuL are formed, associated with a peptide bond deprotonation (CT band at 318–331 nm) [11,12]. Additionally, the lower and higher values of g|| and A|| constants [11,12], respectively, are characteristic of 3N copper(II) coordination in the equatorial plane [31]. It must be mentioned that the pH range of formation of these complexes is below the deprotonation range of His side chains in the free peptide. Thus, two complex formation patterns are possible: the first includes a deprotonation of one amide nitrogen and the second a deprotonation of two consecutive amide nitrogens and simultaneous release and reprotonation of one of the His residues. The second version involving the imidazole nitrogen of His-4 residue and the amide nitrogens of the Ser-3 or Ala-3 and His-4 residues seems more likely, because the symmetry and transition energies of the CD spectrum of the CuL complexes [11,12] very closely matches that of comparable complexes formed with AGGH [28]. Assuming the above interpretation, the formation of CuH
1L with –TESHHK– and –TEAHHK– is due to recoordination of the second histidine imidazole (His-5 residue). The values of spectroscopic parameters of both complexes are located between the 3N and 4N coordination mode, supporting an axial placement of the fourth nitrogen donor [11,12]. A transition to CuH
2L complexes is associated with a further peptide bond deprotonation. The EPR spectrum of these complexes exhibits a confirmatory nine line superhyperfine splitting pattern of four equivalent equatorial nitrogen donors [11,12], and the position of its d– d band indicates that the axial nitrogen remains bonded [30,32]. Finally, the CuH
3L complexes are formed upon the deprotonation of a non-coordinated Lys amine, with little change in the spectra [11,12], similar to the analogous Ni(II) complexes [13]. The scenario of consecutive deprotonations allows for an unambiguous assignment of His-5 and His-4 as equatorial and axial histidine donors, respectively. Obviously, the additional axial nitrogen coordination enhances the propensity of the peptide chain to wrap around Cu(II), evidenced by the lowering of the pK a values of CuH
2L formation by 2 log units, compared with AGGH [28].

Table 4 Stability constants of Cu(II) complexes with –TESHHK–, –TASHHK–, –TEAHHK–, –TESAHK– and –TESHAK– (T=25 8C, I=0.1 M KNO3)

–TESHHK– –TASHHK–b –TEAHHK– –TESAHK– –TESHAK– a b

CuH2L

CuHL

log b a

log b a

21.03 (1) 20.84 (1) 21.49 (1) – –

16.51 16.56 16.92 14.84 14.92

CuL

(1) (1) (1) (1) (1)

CuH
1L

CuH
2L

pK a1

log b a

pK a2

log b a

pK a3

log b a

4.52 4.28 4.57

11.11 (1) 11.53 (1) 11.20 (1) 10.19 (1) 9.54 (1)

5.40 5.03 5.72 4.65 5.38

4.19 – 4.37 4.83 3.64

(1)

6.92

(1) (1) (1)

6.83 5.36 5.90


3.73
2.25
3.40
3.31
5.58

b=[CuiHjLk]/([Cu]i [H]j [L]k), standard deviations of the last digit are given in parentheses. Moreover, formation of Cu2H
1L2 (log b=19.81 (1)) and Cu2H2L2 (log b=13.55 (1)) complexes.

(1) (1) (1) (1) (1)

CuH
3L pK a4

log b a

pK a5

7.92


14.09 (1)
11.81 (1)
12.99 (1)
13.26 (1)
16.56 (1)

10.36 9.56 9.59 9.95 10.98

7.77 8.14 9.22

Complexes

CuH2L

CuHL

–TESHHK– UV/Visa CDb

CuL

CuH
1L

550 (97)

589 (62) 731 (22) 614 (38) 638 (
0.38)

–TASHHK– UV/Visa CDb

612 (49) 720 (18) 647 (37)

650 (
0.30) 569 (78) 510 (+0.14) 320 (+0.44)

610 (
0.36) 632 (
0.14) 485 (+0.03) 331 (+0.42) 598 (86)

–TEAHHK– 620 (63) UV/Visa CDb 730 (18) 649 (44) 660 (
0.22)

–TESAHK– UV/Visa CDb –

720 (17) 677 (
0.06) 650 (44)

–TESHAK– UV/Visa CDb –

702 (24) 707 (
0.05)

a b

CuH
2L

633 (46)

k max nm (q/M
1 cm
1). k max nm (Dq/M
1 cm
1).

671 (
0.21) 548 (+0.17) 318 (+0.13)

665 323 683 516 328

(
0.23) 590 (87) (+0.16) (
0.12) 575 (97) (+0.02) (+0.08)

644 601 (
0.47) 548 485 (+0.14) 478 329 (+0.61) 312 563 (103) 558 (
0.36) 558 – 490 316 712 (
0.11) 556 (+0.30) 549 (92) 634 461 (
0.02) 492 353 (
0.10) 351 316 (+0.09) 316 558 (91) 564 603 (
0.68) 477 328 (+0.38) 308 644 (+0.02) 529 (127) 632 535 (+0.31) 496 357 (
0.03) 352 333 (+0.02) 317

CuH
3L

Cu2H
1L2 Cu2H
2L2

(+0.16) 550 (103) 655 (+0.08) (
0.46) 552 (
0.46) (+0.05) 484 (+0.15) (+0.62) 308 (+0.70) (138) 557 (
0.45) 590 (75) 578 (85) (+0.12) 490 (+0.16) (+0.44) 312 (+0.55) (+0.66) 527 (110) (
0.59) (
0.26) (+0.22) (
0.64) 558 (91) (+0.03) (+0.51) (+0.89) 526 (128) (
0.99) (
0.25) (+0.66)

636 500 347 312 557 482 308 630 494 350 317

(+1.24) (
1.42) (
0.36) (+0.57) (
0.59) (+0.09) (+0.56) (+1.25) (
1.33) (
0.37) (+0.66)

604 (
0.39) 491 (+0.06) 331 (+0.57)

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

Table 5 UV/Vis (c=1mM) and CD (c=3 mM) spectroscopic data for the 1:1 copper complexes with the hexapeptides

125

126

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

In contrast, hexapeptide –TASHHK– behaves differently, forming two possible dimeric complexes at pHN5. One of them, Cu2H
1L2 complex, could not be detected spectroscopically, because of its minor concentration and overlap with other species. The second dimer, Cu2H
2L2, should be a 4N-coordinated complex. The absence of EPR signal at pH 7.29, where it is the dominant species, strongly supports its formation [12]. In addition, the value of its stability constant (Table 4) and the d–d transition at 578 nm [12] are similar with the corresponding values of the dimer Cu(II) complex with HH [33], where Cu(II) ions coordinate with the terminal amino group, the amide and the imidazole nitrogen of the C-His of one molecule and with the imidazole nitrogen of the N-His of a second molecule. Thus, in the Cu2H
2L2 complex, it is excepted that Cu(II) ions coordinate equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues (identical to complex CuL), plus the imidazole nitrogen of the His-5 residue of a second peptide molecule after reprotonation. In more basic solutions, the complexes CuH
2L and CuH
3L are formed, which, based on spectroscopic evidences, have been assigned similar structures with the ones formed with –TESHHK– and –TEAHHK–. It must be mentioned that the assignment of the coordination modes of Cu(II) ions towards –TESAHK– and –TESHAK– was easier than in the case of –TESHHK–, –TASHHK– and –TEAHHK–, due to the presence of only one histidine residue inside their peptide sequence. The minor low-pH complex, CuHL, did not manifest its presence in CD spectra but they were detected by EPR spectroscopy [12]. The stoichiometry and the d–d transitions at 702–730 nm [12] suggest that only one imidazole nitrogen is bound with Cu(II) ions [30]. In addition, the values of the stability constants (Table 4), which are at least 2 log units higher than those of comparable complexes of Cu(II) with AGGH and its analogues suggest the participation of the carboxyl group of Glu residue in the coordination sphere of Cu(II) ions as well [28]. CuHL complexes are superseded by CuL complexes, whose d–d transition near 630–650 nm and the splitting and the presence of the CT band at 323–328 nm in CD spectra [12], clearly indicate the simultaneous coordination of the imidazole and the amide nitrogen of the His residue, forming a stable common six-membered chelate ring

[28,30,34,35]. The proposed 2N {1N
am, 1Nim} mode is also supported by similar EPR parameters [12] of comparable 2N Cu(II) complexes with AGGH and its analogues [28] and with GGGGH or GGGH [32]. In more basic solutions, CuH
1L complexes are associated with a further peptide bond deprotonation (Table 4). The d–d transitions, the CD and the EPR parameters of these complexes [12], suggest the equatorial coordination of both peptides through the imidazole and the amide nitrogen of the His residue and the neighboring amide nitrogen, forming two stable, six- and five-membered chelate rings. It must be noticed that the spectroscopic behavior of these complexes is similar to the comparable Cu(II) complexes with –AKRHRK– [27] and with AGGH and its analogues [28], which are typical of 3N {2N
am, 1Nim} coordination. Increasing the pH, CuH
2L and CuH
3L complexes with –TESAHK– and –TESHAK– are formed. The EPR spectra of the resulting CuH
2L complexes that exhibit a nine line superhyperfine splitting pattern of four equivalent equatorial nitrogen donors and their spectroscopic parameters [12], which are in a very good agreement with the corresponding 4N Cu(II) complexes with GGGGH and GGGH, respectively [32], indicate the 4N {3N
am, 1Nim} coordination. Accordingly, the formation of CuH
3L complexes release one more proton with a pK a of 9.95–10.98 (Table 4), which is abstracted from the amine of the Lys residue, which remains uncoordinated. The oxidation-mediating properties of Cu(II)-TESHHK were checked with H2O2 against 2-deoxyguanosine (dG), using several techniques. Only the combination of Cu(II)– TESHHK– and H2O2 resulted in the substantial dG oxidation. No reaction was detected in the absence of H2O2, and very little 8-oxo-dG was generated in the presence of Cu(II) alone or –TESHHK– alone. The oxidation reaction was remarkably fast, indicating that the initial complex with undamaged –TESHHK– was the source of oxidative power. The spectrophotometric experiments demonstrated that the formation of 8-oxo-dG was accompanied by the presence of a species characterized by an intense absorption band at 386 nm. The decrease of intensity of the d–d band of the starting Cu(II)– TESHHK– complex in the course of experiments with H2O2 indicated that the new complex species contains copper in a different oxidation state. The spectra indicate Cu(III). This is evidenced by the intense CT band at 386

Table 6 Stability constants of Zn(II) complexes with the hexapeptides –TESHHK–, –TASHHK– and –TEAHHK– (T=25 8C, I=0.1 M KNO3) ZnHL log b –TESHHK– –TASHHK– –TEAHHK– a

a

13.45 (2) 13.39 (2) 13.70 (2)

ZnL log b

ZnH
1L a

5.92 (2) – 6.59 (1)

a

pK a1

log b

7.53


2.05 (2)

2.07 (5)

7.11

b=[ZniHjLk]/([Zn]i [H]j [L]k), standard deviations of the last digit are given in parentheses.

ZnH
2L pK a2

log b a

pK a3

7.97


12.35 (8)
11.08 (2)
12.41 (3)

10.30

8.66

10.34

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

nm. The following oxidation mechanism has been proposed [11]: CuII H
1 L þ H2 O2 YCuIII H
1 L
S OH þ OH
þ CuIII H
1 L
S OH þ H2 O2 YCuII H
1 L þ O
2 þ H2 O þ H

The Cu(III) species, which oxidized dG so rapidly in our experiments, can also be expected to oxidize guanine residues in DNA and produce other kinds of potentially mutagenic oxidative DNA damage. Therefore, the –TESHHK– sequence of histone H2A may form a complex with stray Cu(II) ions, which is capable of promoting genotoxic effects of endogenous oxidants in cells. 3.2.3. Zn(II) complexes For the study of the interactions of Zn(II) ions, we decided to choose only the hexapeptides containing two His residues from the five hexapeptides, because it is known that in most of the active centers of Zn(II)-containing enzymes, Zn(II) ions are bound in more than one His residue [36–38]. So, potentiometric titrations in aqueous solutions were carried out for the hexapeptides –TESHHK–, –TASHHK– and –TEAHHK–, in the presence of zinc nitrate in peptide– Zn(II) ratios of 1:1 and 2:1. The stability constants of Zn(II) complexes, calculated from these titrations using the SUPERQUAD program are presented in Table 6. The species distribution diagrams (Fig. 4) show the formation of four complexes (ZnHL, ZnL, ZnH
1L and ZnH
2L) in the case of Zn(II)/–TESHHK– and Zn(II)/–TEAHHK– systems and two complexes (ZnHL and ZnH
2L) in the case of the Zn(II)/–TASHHK– system [16]. The higher values of stability constants of the first ZnHL complexes which are formed (Table 6) comparing with the values of analogues complexes with other His-containing peptides [21,39–42], support the coordination of the three hexapeptides through both imidazole rings. Additionally, the carboxylate oxygen of Glu residue of –TESHHK– and

127

–TEAHHK–, which is deprotonated in the pH range of the formation of these complexes, may also participate in the coordination sphere of Zn(II) ions forming a slightly distorted octahedral complex similarly with the analogous Ni(II) and Cu(II) complexes with the same hexapeptides [11–13]. In higher pH values, the complexes ZnL and ZnH
1L with hexapeptides–TESHHK– and –TEAHHK– are formed. Although the pK a values of these complexes (Table 6) are comparable with the pK a values of His-containing unprotected peptides with the amide coordination and could be abstracted from amide nitrogens [39,40,43–45], we don’t suggest the amide coordination of both hexapeptides. Generally, it is known that Zn(II) ions exhibit a stronger tendency to undergo hydrolysis than amide coordination [16]. Thus, the two deprotonations leading to the formation of ZnHLYZnLYZnH
1L complexes may be the successive deprotonation of already bound water molecules. It must be noticed that the stability constants of ZnL complexes with hexapeptides –TESHHK– and –TEAHHK– are higher than those of ZnL complexes with other Hiscontaining peptides, which have been proposed in the coordination of both imidazole rings to the metal ions [21,46]. A good explanation for this higher stability may come from the carboxylate oxygen of Glu residue, which remained bound to the metal ions. On the contrary, the stability constants of ZnH
1 L complexes with the –TESHHK– and –TEAHHK– (Table 6) are similar with the related complexes with other His-containing peptides, which all correspond to the 3N {NH2, N
, NIm} donor set involved in the equatorial plane of Zn(II) ions. Although it is not suggested, amide coordination in ZnH
1L complexes with the hexapeptides –TESHHK– and –TEAHHK–, the participation of the carboxyl group of Glu residue and the two imidazole rings in the coordination sphere of Zn(II) ions may result in similar stabilization with the 3N {NH2, N
, NIm} mode. Finally, above pH 8 the deprotonation of the ZnH
1L complexes led to the formation of ZnH
2L complexes. As can be seen from Table 6, ZnH
2L complexes provide similar coordination mode with ZnH
1L complexes, differing from the deprotonated and uncoordinated q-amino group of Lys residue similarly with the analogous Cu(II) and Ni(II) complexes with the studied hexapeptides [11– 13]. It must be noticed that the proposed structures have been certified using one- and two-dimensional 1H-NMR spectra [16].

4. Conclusions

Fig. 4. An example of a species distribution diagram of Zn(II) complexes with –TEAHHK–.

All the peptides interact strongly with Cu(II), Ni(II) and Zn(II) metal ions. Cu(II) and Ni(II) promote the deprotonation and their subsequent coordination of peptide bonds next to histidine residues. The proposed structures of the species formed at relatively high pH are presented in Fig. 5.

128

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

Fig. 5. Proposed structures of the MH
2L (a–f), ZnL (g) and ZnHL (h) species as deduced from the spectroscopic data. (a) and (d): when R= –CH2OH and XAA=Glu the peptide is –TESHHK–, when R= –CH2OH and XAA=Ala the peptide is –TASHHK–, and when R= –CH3 and XAA=Glu the peptide is –TEAHHK–, (b) and (e): the peptide is –TESAHK–, (c) and (f): the peptide is –TESHAK–, (g) when XAA=Ser the peptide is –TESHHK–, when XAA=Ala the peptide is –TEAHHK–, (h) the peptide is –TASHHK–.

It was found that Ni(II) ions promote hydrolysis of the peptides –TASHHK– and –TESAHK– at the –X-Ser- sites and this has been assigned to the presence of the Ser residue

[47]. The hydrolysis is faster with Ni(II), but the oxidation of 8-dG to 8-oxo-dG is faster in the system Cu(II)– hexapeptide than Ni(II)–hexapeptide, as shown by prelimi-

M. Mylonas et al. / Journal of Molecular Liquids 118 (2005) 119–129

nary experiments. Both effects may cause mutations and cancer. Zn(II) on the other hand, does not seem to have similar effects on histone peptides.

Acknowledgements This work was carried out within the framework of COST D8 action dMetals in MedicineT. It was also supported by the MSc graduate program in dBioinorganic ChemistryT coordinated by Professor Nick Hadjiliadis, and financed by the Greek Ministry of Education (EPEAEK). This work was partially sponsored by the Polish State Committee for Scientific Research (KBN), Grant No. 3 T09A 06818 and by a NATO Grant.

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