Interactions of Zn(II) Ions with Three His-Containing Peptide Models of Histone H2A

The interactions of Zn(ll) ions with the blocked hexapeptide models -TESHHK-, -TASHHK- and -TEAHHK- of the -ESHH- motif of the C-terminal of historic H2A were studied by using potentiometric and IH-NMR techniques. The first step of these studies was to compare the pKa values of the two His residues inside each hexapeptide calculated by potentiometric or H-NMR titrations. Hereafter, the potentiometric titrations in the pH range 5 11 suggest the formation of several monomeric Zn(ll) complexes. It was found that all hexapeptides bind to Zn(ll) ions initially through both imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, the combination of potentiometric titrations and one and two dimensional NMR suggested no amide coordination in the coordination sphere of Zn(II) ions. Obviously, these studies support that the -ESHH- sequence of histone H2A is a potential binding site for Zn(II) ions similarly with the Cu(II) and Ni(ll) ions, presented in previous papers.


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 that can bind with constituents of the cell nuclei 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. Especially, the abundance of histones makes them the prime expectants for that role. It must be noticed that the nucleosomes represent the first level of DNA compaction in eukaryotic nuclei and consist of 146 bp of core DNA wrapped around a cluster of eight histone proteins (containing two copies of each ofhistones H2A, H2B, H3 andH4), with 20-60 bp of linker DNA joining adjacent cores /3/. Obviously, the studies of metal interactions with the peptides or protein fragments, especially histones, can lead in explaining mechanisms related to cancer and toxicity caused by metals. As a consequence, the coordination properties of Ni(II) ions towards the tetrapeptide Ac-CysAlalleHis-am (-CAIH-) representing Marios Mylonas et al. Bioinot;ganic Chemistry and Applications
Changes of pH were monitored with a combined glass-silver chloride electrode calibrated daily in H + concentrations by HNO3 titrations/20/. The time to reach pH-equilibrium during titrations varied from 1-10 min, depending on the pH value. Sample volumes of 1.5 mL and concentrations of mM of the hexapeptides and 0.5 mM Zn(NO3)2"4H20 were used. The experimental data were analyzed using the SUPERQUAD program/21/. Standard deviations computed by SUPERQUAD refer to random errors.
NMR spectroscopy H-NMR experiments were performed on a Bruker AMX 400MHz spectrometer. The one dimensional experiments were carried out in 1:4 D20:HO mixtures at a peptide concentration of 5 mM of both free and Zn(ll)-bound hexapeptide -TASHHK-, in peptide Zn(ll) ratio 1.2:1 at pH* 10.5 and 25 C (The pH* reading of the electrode was not corrected for the isotope effect). TOCSY experiments was used to assign the spectra of both free and Zn(ll)-bound hexapeptide -TASHHK-, in peptide Zn(ll) ratio 1.2:1 (cL--15 mM) in the same pH* and temperature. Finally, H-NMR titrations were carried out for the hexapeptides -TESHHK-,-TASHHKand -TEAHHK-(c 5 mM) in 99.9 % D20 solutions covering the pH* range 2 11 at 25 C, used to calculate the pKa values of N3 nitrogen atoms of imidazole rings of the two His residues.

RESULTS AND DISCUSSION
Acid-base behavior of free hexapeptides Both the Nand C-terminal of the hexapeptides models (Scheme 1) were blocked by acetylation and amidation, respectively, to make the peptides more realistic models of the -ESHHmotif of histone H2A. As has already been written in the Experimental Section, ESI-MS and H-NMR techniques were used for the characterization of the hexapeptides but the data have been already reported in previous papers/12,13/.
The first two hexapeptides -TESHHK-, -TEAHHKand the hexapeptide -TASHHKcontain four and three groups, respectively, which are capable of reversible proton binding. These groups are the carboxyl group of Glu residue, the N3 imidazole nitrogens of His residues and the e-amino group of Lys residue. The protonation constants and dissociation macroconstants of these groups were measured by potentiometric Vol. 2, Nos. 1-2, 2004 Interactions of Zn (11) Ions with Three His-Containing Peptide Models of Histone H2A titrations and are presented in Table I. The highest pKa values between 10.48 10.28 can be easily assigned to the e-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/9,22,23/.    The other values of macroconstants corresponding to the deprotonations of His residues were found to be separated by less than one log unit, suggesting a possibility of concurrent deprotonations at the two His residues. The comparison with the literature indicates that the lowest pKa value should mainly correspond to the protonation of the His residue closer to the N-termini of the peptide, while the closest His in the Cterminal has a higher basicity/24-26/. Table 2 Dissociation macroconstants (pK,) of the hexapeptides -TESHHK-, -TASHHKand -TEAHHKcalculated on the basis of potentiometric and spectroscopic ('H-NMR) data -TESHHK--TASHHK--TEAHHK-pKa pKa pK His-4 His-5 His-4 His-5 His-4 His-5 Potentiometry 5.90 6.78 5.74 6.62 6.00 6.68 Henderson-Hasselbalch method 6.35 (I) 6.41 (2) 6.29 (2) 6.30 (2) 6.42 (2) 6.48 (2) Rabenstein-Sayer method b 6.12 (4) 6.88 (5) 6.06 (3) 6.61 (4) 6.15 (3)  Obviously, the two His residues in these hexapeptides have multiple acid-base equilibria, it is known that potentiometric titrations cannot always resolve these overlapping proton dissociations. On the contrary, NMR spectroscopy investigates individual protons in peptides or proteins, facilitating the study of specific proton association-dissociation equilibrium in polyprotic systems. Thus, a series of one dimensional H-NMR Interactions of Zn (ll) Ions with Three His-Containing Peptide Models of Histone .H2A spectra of all hexapeptides at various pH* values were recorded for the further verification of the pKa values of the two His residues, calculated from the potentiometric data. Generally, it is known that the proton dissociation can be easily monitored from the chemical shifts of the neighboring protons at each pH* value.
In particular, the dissociation of the N3 imidazole nitrogen atoms can be calculated from the plot of chemical shifts of the adjacent protons of C2 and C5 carbon atoms of imidazole rings as a function of pH*.
It must be mentioned that the calculation of these pK, values was realized using two different methods, in the first one, the pK, values of the two His residues were calculated independently/27,28/. In contrast, in the second method the two pK, values calculated together/29/.
In Figure 2 an example plot produced from the chemical shifts of the imidazole protons of C2 of hexapeptide -TASHHKis presented. Similar plots were produced for all hexapeptides. Obviously, the pKa values of two His residues of all hexapeptides calculated using the first method are not equivalent with the comparable values calculated from the potentiometric titrations (Table 2). Thereby, the pK, values of two neighboring groups as it is occurred in the case of the hexapeptides -TESHHK-, -TASHHKand -TEAHHKcannot be extracted with rewarding accuracy using the first method. In contrast, the same pK, values calculated using the second method are similar with the comparable values calculated from the potentiometric titrations (Table 2). Additionally, the use of the second method led to more realistic and accurate values because for the calculations we took into account the presence of both His residues. Marios Mylonas et al.

Bioinorganic Chem.istry andApplications Zinc Complexation
Potentiometric titrations in aqueous solutions were carried out for the hexapeptides in the presence of zinc nitrate in peptide Zn(ll) ratios 1:1 and 2:1. The stability constants of Zn(ll) complexes, calculated from these titrations using the SUPERQUAD program are presented in Table 3. The species distribution diagrams presented in Figure 3 indicate the formation of four complexes (ZnHL, ZnL, ZnH_L and ZnH_2L) in the case of Zn(II) /-TESHHKand Zn(il) /-TEAHHKsystems and two complexes (ZnHL and ZnH.zL) in the case of Zn(II) /-TASHHKsystem. Binuclear complexes of type ZnzLx or complexes with two ligands were repeatedly rejected by SUPERQUAD program/21/and were eliminated from the model. As can be seen from Figure 3, the coordination of all hexapeptides starts in pH above 5 and the ZnHL complexes are formed. The higher values of their stability constants (Table 3) comparing with the analogues complexes with GlyHis/30/, AlaH is /31/, GlyHist/32L SarHist /32/ and with protected peptides -HisHis-/33/ and -HisProHis-/34/ support the coordination of all hexapeptides through both imidazole rings. It must be mentioned that for the ZnHL complexes with the above dipeptides and the protected peptides the imidazole or amino monodentate binding/30-34/have been suggested. Additionally, the carboxylate oxygen of Giu residue of-TESHHKand -TEAHHK-, which is deprotonated in the pH range of the formation of these t/ol. 2, Nos. [1][2]2004 hteractions of Zn (II) Ions with Three His-Containing Peptide Models of Historic H2A complexes, may also participate in the coordination sphere of Zn(II) ions forming a slightly distorted octahedral complexes similarly with the analogues Ni(II) and Cu(II) complexes with the same hexapeptides /11-13/. Increasing the pH, the complexes ZnL and ZnH_L with hexapeptides -TESHHKand -TEAHHKrelease additional protons with pKa 7.53 and 7.11 (ZnL), 7.97 and 8.66 (ZnH_L), respectively (Table 3). Although these pKa values are comparable with the pKa values belonging to His-containing unprotected peptides with the amide coordination and could be abstracted from amide nitrogens/30,31,36-38/, we do not support the amide coordination of both hexapeptides. Generally, it is known that amide deprotonations is more difficult to observe with Zn(ll) ions than other metal ions, including Cu(ll) and Ni(ll) ions/16/. At high pH values it is possible to observe this type of coordination but additionally competition from hydroxo complex formation is limitative. Thus, Zn(II) ions exhibit a stronger tendency to undergo hydrolysis than amide coordination.
Concluding, the most probable hypothesis about the two deprotonations leading to the formation of ZnHL --ZnL --, ZnH_L complexes may be the successive deprotonation of already bound water molecules. This is in agreement with the preliminary analysis of the titration data which indicated titration of two more protons in the case of the hexapeptide Zn(II) systems than in the case of the free hexapeptides.
As can be seen in Table 3, the stability constants of ZnL complexes with the hexapeptides -TESHHKand -TEAHHKare higher than that of ZnL complexes with the protected dipeptide -HisHis-(log fl 4.19) /33/and with the cyclic dipeptide c-HisHis (log fl 2.55)/35/, in which the coordination of both imidazole rings to the metal ions were proposed. Obviously, ZnL complexes with the studied hexapeptides are considerably more stable than the corresponding complexes of the above reported and other His-containing peptides which are coordinated through one or two nitrogen atoms ( Table 3). The higher stability of the studied complexes may contribute from the carboxyl group of Glu residue which remained bound to the metal ions. It is worthy to note that the absence of Ser residue stabilize the ZnL complex with -TEAHHKcomparing with the corresponding complex with -TESHHK-( Table 3).
In contrast, the stability constants of ZnH.L complexes with the hexapeptides -TESHHKand -TEAHHKare similar to the related complexes with GlyHis/30/, AlaHis/3 l/, GlyHisLys/34/, GlyHisGly /35/and HmSHis /38/ which all correspond to the {NH2, N, Nm} donor set involved in the equatorial plane of Zn(II) ions (Table 3). Although amide coordination in ZnH.L complexes with the hexapeptides TESHHKand -TEAHHKis not suggested, the participation of the carboxyl group of Glu residue and the two imidazole rings in coordination sphere of Zn(II) ions may result in similar stabilization with the {NH,,, N N,,,} mode.
Finally, above pH 8 the deprotonation of the ZnH.L complexes with the hexapeptides -TESHHKand-TEAHHKtakes place with a pKa 10.30 and 10.34, respectively, forming the ZnHozL complexes (Table 3).
These pKa values are in good agreement to that for protonation of e-amino group of Lys residue, pK 10.28 and 10.25 (Table 1), in free ligands -TESHHKand -TEAHHK-, respectively. Obviously, the ZnH.2L complexes provide a similar coordination mode with ZnH.L complexes, differing to the deprotonated and uncoordinated e-amino group of Lys residue similarly with the analogues Cu(ll) and Ni(II) complexes with the studied hexapeptides/11-13/.
Fitting of the titration data of the Zn(II) -TASHHKsystem can be done only considering the ZnHL and ZnH.2L species. This may be due to simultaneous deprotonation of the coordinated water protons and the protonated e-amino group of Lys.
The proposed structures of some selected species are presented in Figure 4. In order to study further the proposed structures we decided to use NMR spectroscopy. Bearing in mind that the coordination sphere of all species with all hexapeptides is similar we tried to study ZnHL and ZnH.2L complexes. We chose Zn(ll) /-TASHHKsystem for these NMR studies because the above complexes exists without overlaps at their formation pH range only in the case of the hexapeptide -TASHHK- (Figure 3).

Interactions of Zn (II) Ions with Three Hk-Containing Peptide
Firstly, H-NMR spectra of-TASHHKwere recorded, at pH* 7.3 and 25 C, in the absence and presence of Zn(II) ions (ratio 1:1, in D20:H20 1:4 mixture). Unfortunately, the spectrum in the presence of Zn(ll) was not helpful due to the extensive broadening of all signals which was derived from the high concentration of free metal ions ( Figure 3). Thus, H-NMR ( Figure 5) and TOCSY ( Figure 6) spectra of-TASHHKat pH* 10.30 were recorded, in the absence and presence of Zn(II) ions (peptide Zn(II) ratio 1.2:1, in D20:H20 1:4 mixture). The chemical shifts of IH (6, ppm) of free and bound -TASHHKat pH* 10.30 are presented in Table 4. Table 4 IH-NMR assignment of-TASHHK-, in absence or presence of Zn(ll) ions in peptide Zn(ll) ratio 1 It is well known that the complexation of several peptides to the metal ions produces significant chemical shift changes of the signal of the protons near the binding sites in NMR spectra due to the electron density shift to the metal ions. Obviously, the comparisons of H-NMR and TOCSY spectra between free and bound -TASHHK-(Table 4) indicated that the positions of the protons of Thr, Ala, Ser and Lys residues were almost not affected, suggesting that they were not involved in the coordination sphere of Zn(ll) ions. It must h.Tteractions of Zn (II) Ions with Three His-Containing Peptide Models of Histone H2A be noticed that remarkable chemical shift changes of the signals belonging to a proton were not observed, suggesting the absence of bound amide nitrogen from the coordination sphere of Zn(ll) ions. Bioinorganic Chemtst 0 and Applications contrast we must report that signals of the peptide hydrogens in the H-NMR spectrum of free -TASHHKwere not detected. Additionally, it was found that the two new peaks below 8.00 ppm, and two peaks which are overlapping with the already existing peaks of free hexapeptide, produced two new cross-peaks in the TOCSY experiment [lm C2-H Im Cs-H: 7.24 6.98 ppm and 7.19 6.95 ppm] (Figure 6) corresponding to the two His residues in bound hexapeptide. The differences and also the similarity of them in chemical shifts of the signals of the imidazole protons observed in both His residues (

CONCLUSIONS
The studies with the blocked hexapeptide models -TESHHK-, -TASHHKand -TEAHHKof the -ESHHmotif of the C-terminal of histone H2A, presented in this paper, support that this sequence is a potential binding site for Zn(ll) ions similarly with the Cu(ll) and Ni(ll) ions/I 1-13/.
Firstly, a combined use of potentiometric and IH-NMR titrations has allowed us to compare the macroconstants in protonation equilibria of the two His residues inside the sequence of the studied hexapeptides. Afterwards the potentiometric titrations in aqueous solutions of hexapeptide / Zn(ll) systems, indicated that several monomer Zn(ll) complexes are formed in the pH range 5 11. It was found that these complexes were apparently less stable than the corresponding Cu(ll) complexes with the same hexapeptides /11,12/. Besides it is well known that Zn(ll) complexes are kinetically labile, leading to several equilibria between complexes with different donor sets and distorted geometries or between coordinated and uncoordinated forms /39/. The potentiometric data suggested the initial coordination of all hexapeptides through both imidazole rings and additionally through the carboxylate oxygen of Glu residue in the case of-TESHHKand -TEAHHK-, forming a slightly distorted octahedral complexes similarly with the analogues Ni(ll) and Cu(ll) complexes with the same hexapeptide models/11-13/, in more basic solutions, the most probable interpretation of the predominated complexes is the deprotonation of bound water molecules and eamino group of Lys residue. The last proposed structures of complexes existing in basic pH values, have been studied also by one and two (TOCSY) dimensional NMR techniques leading to the same coordination features.
Obviously, binding of Zn(ll) ions to the Cterminal of histone H2A may inevitably change its conformation, disturbing the interactions of histone H2A inside the histone octamer with the other histones, DNA and other molecules. Additionally, it is well known that Zn(ll) ions are able to bind with several molecules inside the cells, including reduced glutathione (GSH) which is one of the most abundant molecules of life (c 10 mM intracellularly) /40/ and free histidine which also exists in high concentrations (c 0. mM) and it has been proposed as a carrier of Zn(ll) ions in some tissues/41/. These observations clearly indicate the great biological interest relative to the binding of Zn(ll) ions inside the cells. Thus, the next step in our studies needs to ascertain the ability of Zn(il) ions to catalyze the hydrolysis of the studied hexapeptides in physiological conditions, similarly with Ni(ll) and Cu(ll) ions/9,13,14/.