Journal of Inorganic Biochemistry 103 (2009) 1214–1220

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Journal of Inorganic Biochemistry

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Copper and nickel binding in multi-histidinic peptide fragments

Maria Antonietta Zoroddu *, Serenella Medici, Massimiliano PeanaDepartment of Chemistry, University of Sassari, 07100 Sassari, Italy

a r t i c l e i n f o

Article history:Received 6 April 2009Received in revised form 22 June 2009Accepted 22 June 2009Available online 3 July 2009

Keywords:Multi-histidinic peptidesCu(II)Ni(II)NMREPRCD

0162-0134/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jinorgbio.2009.06.008

* Corresponding author.E-mail address: zoroddu@uniss.it (M.A. Zoroddu).

a b s t r a c t

Multi-histidinic peptides have been investigated for Cu(II) and Ni(II) binding. We present spectroscopicevidence that, at low pH and from sub-stoichiometric to stoichiometric amounts of metals, macrochelateand multi-histidinic Cu(II) and Ni(II) complexes form; but, from neutral pH and above, both copper andnickel bind to individual histidine residues. NMR, EPR, UV–Visible (UV–Vis) and UV–Visible CD spectros-copy were used to understand about the variety of complexes obtained at low pHs, where amide depro-tonation and coordination is unfavoured. A structural transition between two coordination geometries, asthe pH is raised, was observed. Metal binds to Nd of histidine imidazole when main-chain coordination isinvolved and coordinates via Ne under mildly acidic conditions and sub-stoichiometric amounts of met-als. From EPR results a distortion from planarity has been evidenced for the Cu(II) multi-histidinic macr-ochelate systems, which may be relevant to biological activity. The behaviour of our peptides wascomparable to the pH dependent effect on Cu(II) coordination observed in octapeptide repeat domainin prion proteins and in amyloid precursor peptides involved in Alzheimer’s disease. Changes in pHand levels of metal affect coordination mode and can have implications for the affinity, folding and redoxproperties of proteins and peptide fragments.

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1. Introduction

Recently, histidine containing peptides have drawn consider-able attention as models for the study of molecular phenomenaassociated with metal ion binding in proteins involved in fatalneurodegenerative disorders such as Alzheimer’s and prion dis-ease or involved in metal detoxification mechanisms, thus pro-tecting against deleterious redox activity by sequestering excessmetal ions [1–6]. Despite an increasing body of evidence linkingcopper with neurodegenerative diseases, the precise coordinationgeometry, the affinity and stoichiometry of binding, as well aswhat triggers multi-histidinic amyloid-b peptide to convert fromits soluble form to an amyloidogenic form, are yet to be estab-lished [7].

We have used two peptides, the two and the three repeats Ac-(T1R2S3R4S5H6T7S8E9G10)n–NH2 sequence (n = 2, 20aa; n = 3, 30aa),as models to investigate further the role of multi-histidine sites incoordinating metals. In fact, the presence of multi-histidine sites inpeptides determines, in addition to the usual coordination mode, ahigh structural variety of complexes, as formation of macroche-lates or bent structures which can strongly affect activity. It hasbeen suggested that the three histidines coordination mode ofamyloid-b peptide controls the redox activity of copper ions [8].

ll rights reserved.

Presence of His-rich domains, similar to those in prion protein,can contribute to the understanding of crucial role of poly-imidaz-ole centres in the protein coordination process.

We have previously published data of copper and nickel bindingto the two and three histidinic 20aa and 30aa aminoacid fragments[9,10]. In these studies we showed that both nickel and copperbind to histidine independently forming, at high pH, a square pla-nar/tetragonal complex. The metal complexes involve coordina-tion, towards N-terminal, of main chain amides preceding the Hisimidazol as well as imidazolic nitrogen Nd. We have shown thatone, two Cu(II), one, two or three Ni(II) ions bind to His6, His16and His26, independently of each other at high pH.

The aim of the present study was to investigate about the struc-tural variety of complexes obtainable at low pH and to investigatefurther previous assumption that the two or three histidinic sitesare isolated each other or whether a single metal ion, Cu(II), likeNi(II) will bind to both His6 and His16 or to all the three His6,His16 and His26.

In the present paper we report data for the interaction of Cu(II)and Ni(II) with the peptide fragments in the acidic pH range ob-tained by NMR, EPR, UV–Vis and UV–Vis CD spectroscopy study.Our results reinforce the suggestion that the change in coordina-tion with pH could be the key to the pH dependence of biologicalactivities, such as the conformational changes observed in thepresence of copper which only induced aggregation of multi-his-tidinic amyloid-b peptides as the pH was lowered from physiolog-ical to 6.8 [11].

M.A. Zoroddu et al. / Journal of Inorganic Biochemistry 103 (2009) 1214–1220 1215

2. Spectroscopic measurements

NMR experiments were performed on Bruker Avance 600 MHzspectrometers equipped with inverse quadruple (QXI) resonanceprobe.

Solvent suppression for 1D experiments was achieved usingWATERGATE pulse sequence or using excitation sculpting withgradients, the best results being observed with excitation sculpt-ing. All NMR data were processed using XWINNMR (Bruker Instru-ments) software and analyzed using the Sparky 3.11 and MestReNova programs. Solutions concentrations for NMR were 5 mMand different metal to ligand molar ratios (0.002, 0.005, 0.01,0.02, 0.05, 0.1:1, Cu:30aa) at pH 5.5 were used.

X-band EPR spectra were obtained at different concentrations(from 1 to 3 mM) and different metal to ligand molar ratios (from0.05 to 3:1, Cu:20aa and 30aa) at room temperature and at 120 Kon a Varian E-9 spectrometer. In order to promote glass forma-tion during freezing, some drops of glycol were added. Spectrawere calibrated against diphenylpycrylhydrazyl (dpph; g =2.0036). Estimated errors are for A||Cu = ± 3.0 � 10�4 cm�1andg|| = ± 0.003, A\N = ± 1.0 � 10�4 cm�1. WINEPR-System 2.11 (Bru-ker) has been used. Absorption and Visible CD spectra were re-corded using concentrations from 2 mM to 0.7 mM on a VarianCary 50 Scan spectrophotometer and J-810 Jasco (Jasco Inc., Ea-ton, MD) equipped with a temperature controller. The tempera-ture of the experiments was fixed at 25 �C and the spectrawere recorded over a pH range 3.0–11.0 at steps of 0.5 unitsby addition of NaOH.

3. Computational methods

Molecular mechanics geometry optimisation was made byusing AMBER force field method which was implemented inHyperChemTM 8.0.3 (Hypercube, Inc., Gainesville, FL, 2007) molecu-lar modeling software. The Polak Ribiere (conjugate gradient) algo-rithm was used to find the minimum energy within theHyperChem package.

Cu(II) binding site was modelled on the basis of the geometry ofanalogous multi-histidinic copper-complexes obtained from dif-fractometric data of selected X-ray crystal structures (i.e. PDB1aq8 and PDB 1a4b).

Models of the most likely coordination spheres of Cu(II) ‘‘lowpH” multi-histidinic species were generated by using ViewerPro4.2 (Accelerys, Inc., 2001) program.

Fig. 1. Comparison of visible CD for Ni(II)–20aa 1:1 and Ni(II)–30aa 2:1 and 3:1pH10–11.

4. Results and discussion

Starting from pH 4, below physiological pH, many nickel andcopper species have been evidenced by previous potentiometricmeasurements [9,10]. Depending on the metal to ligand molar ra-tios, NiHL and NiL for the 20-, Ni2L for the 20- and 30- and Ni3L forthe 30-aminoacid fragments have been obtained. CuHL, CuL,Cu2H2L for the 20- and in addition to CuL and Cu2L, three proton-ated species CuH3L, CuH2L, CuHL for the 30-aminoacid fragment,have been evidenced within physiological pH.

In all these species the amide deprotonation and coordination isan unfavourable process. All these complexes cannot be unambig-uously characterized because of the overlap and the free metalpresent. Recent results by Viles et al. [12,14] showed that visibleCD is a very potent probe to study Cu(II) and Ni(II) interactionswith peptides and, together with NMR and EPR spectroscopic tech-niques, strongly helps to characterize various metal bindingmodes.

4.1. Visible CD and UV–Visible (UV–Vis) absorption studies on Ni(II)binding

Visible CD spectra are very sensitive to the relative position ofcoordinating ligands with changes in the intensity and sign of CDbands. Individual transitions can be resolved as separate bands,particularly where the CD bands are of opposite sign; they are onlyobtained when a metal ion is in a chiral environment and the freemetal ion d–d transitions are typically CD silent. Relatively strongCD bands are often observed for d–d transitions of tetragonal com-plexes involving backbone amide and histidine coordination viaimidazole ring. The vicinal effects, responsible for CD activity, arisewhen the chiral a carbon is held in a chelate ring between two do-nor atoms, typically adjacent main chain amides [12], while multi-ple histidines binding mode is CD silent.

Nickel or copper binding to histidine containing proteins orpeptides forms, at high pH, 4N complexes which result in a CDspectrum with a positive band to shorter wavelength than that ofthe absorption maximum and a negative band to longer one.

Copper and nickel–20aa- and –30aa-bound species show pHdependent CD curves as a consequence of the different complexspecies which form in solution.

Visible CD spectra for the 20aa:Ni 1:1 and 1:2 and for the30aa:Ni 1:1, 1:2 and 1:3 have, at pH 9.5–11, a positive ellipticityat 514 nm which appears to longer wavelength than that of theabsorption maximum (kmax = 438 nm for Ni-bound) and a negativeone at 420 nm to shorter wavelength than that of the absorptionmaximum. The band at about 280 nm, clearly visible at pH 9 (Sup-plementary material, inset Fig. S1), is attributed to the chargetransfer transition N�amide–Ni(II).

The curves of 1:1 molar ratio overlay identically showing thatthe mode of nickel coordination has not changed for the largerfragment.

Fig. 1 shows comparison of visible CD for Ni(II) at 1 mol equivadded onto the 20aa and 2 and 3 mol equiv added onto the 30aafragment at pH 10–11.

The shape of the spectrum is the same with the two and threeNi(II) bound-30aa, showing that Ni(II) ions bind independently toeither His6, His16 or His26.

From UV–Vis spectra obtained for 1:1, 1:2 and 1:3 ligand to me-tal molar ratios (Fig. 2a), the abs at 438 nm, kmax characteristic of4N–Ni species, increases as a function of the nickel added at pH10. This behaviour shows that the three Ni atoms bind tightly tothe three histine residues by forming Ni–30aa, Ni2–30aa and Ni3–30aa at the three histidine anchoring sites His6, His16 and His26.

Fig. 2. (a) UV–Visible spectra of Ni–30aa, Ni2–30aa, Ni3–30aa, pH 10 and (b) UV–Visible spectra of Cu–30aa, Cu2–30aa, Cu3–30aa, pH 11.

Fig. 3. Visible CD copper dependent for the 20aa fragment at pH 10.

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No significant CD activity was measured for Ni(II)–20aa at lowpH. A visible CD is not apparent until the pH is raised up to 8,but some species have clearly been potentiometrically evidenced.The lack of appreciable d–d transition CD bands suggests minimalvicinal effects [12] implying that there is no main-chain coordina-tion at physiological pH and below, thus suggesting the involve-ment of imidazol donor atoms in macrochelate systems.

At pH 8 the profound change in the visible CD spectra suggeststhe change in coordinating ligands around the nickel atom.

4.2. NMR studies on Ni(II) binding at ‘‘low pH”

From the differential broadening of the NMR resonances ob-tained at ‘‘low” pH, the direction of the coordination occurs to-wards C-terminus in the form of less stable macrocycles. Theyare favoured also because of the possible metal interaction withside chain residues as carboxylate group from glutammic acid inthe 9th position, two residues following the histidine anchoringbinding site towards C-terminus. This is also in agreement withthe deprotonation of glutammic acid at low pH (pKa in the range4–4.7 or 4–4.9 for the 20aa and the 30aa ligand, respectively) [9].

From the intensity of signals from 1D NMR integrals, as a com-parison between the 30aa free ligand and at 1:1 Ni–30aa-bound atpH 6.5, it is clear that the only protons affected by metal addition,are those of the residues from His6+n towards C-terminus, thusindicating the direction of coordination from the anchoring site(Supplementary material, Fig. S2).

At high pH, when amide main-chain coordination with the for-mation of 6, 5 and 5 membered rings is a favoured process, Nd ofhistidine is involved in nickel coordination, as we previously foundby NMR studies [13].

From NMR spectra obtained at low pH and for sub-stoichiome-tric amount of nickel, though the overlap of aromatic signals (Sup-plementary material, Fig. S4a and b) makes the interpretation

difficult, His He1 and His Hd2 signals simultaneously disappearedupon nickel addition, pointing to Ne as the anchoring site. Thus,we can suggest that, tautomeric effect, i.e. binding either by HisNd or Ne as a function of the pH, could be possible for nickelbinding.

4.3. Visible CD and UV–Visible (UV–Vis) absorption studies on Cu(II)binding

Visible CD spectra for the 20aa and for the 30aa–Cu interactionhave, at pH 10, a positive band at about 640 nm which appears tolonger wavelength than that of the absorption maximum(kmax = 540 nm for Cu-bound) and a negative at 510 nm to shorterwavelength than that of the absorption maximum. The band atabout 310 nm, which appears above pH 8, is attributed to theN�amide–Cu(II) and at 360 nm to the Nimidazol–Cu(II) charge transfertransition [15].

Fig. 3 shows the Visible CD copper dependence for the 20aafragment.

The shape of the spectrum is the same for the 20aa- and for the30aa-, showing that, also for copper, the mode of coordination hasnot changed at high pH, for the larger fragment. The shapes of CDspectra obtained are similar to those reported for copper and nickelbound to prion protein fragments containing a single histidine res-idue at position 111 or 96 or, for copper and nickel bound to modelpeptides at high pH in order to ensure 4N coordination [12,14].Due to the similarity, we can suggest that the shape could be indic-ative of 4N square planar species with the involvement of His asthe anchoring binding site and amide main-chain coordinationwith the formation of 6, 5 and 5 membered rings.

From UV–Vis spectra obtained for 1:1, 1:2 and 1:3 ligand to me-tal molar ratios (Fig. 2b), the abs at 540 nm, kmax characteristic of4N–Cu species, proportionally increases as a function of the copperadded at pH 11. This behaviour shows that the three Cu atoms bindtightly to the three histidine residues by forming Cu–30aa, Cu2–30aa and Cu3–30aa at the three histidine anchoring sites His6,His16 and His26.

Cu3–30aa species was not previously evidenced by potentio-metric measurements because precipitation occurred at the condi-tion used. The increase in the abs, at 540 nm for copper and at438 nm for nickel by adding 1, 2 or 3 mol equiv, suggests thatthe longer fragment is loaded with the 3 mol equiv of metal atpH 11 and 10, respectively.

Fig. 5. Visible CD Cu–30aa 1:1 pH range 3.5–11.5.

M.A. Zoroddu et al. / Journal of Inorganic Biochemistry 103 (2009) 1214–1220 1217

There is a clear transition in the visible CD spectra at pH 7.7 sug-gesting that at this pH a change in the coordinating ligands aroundthe copper ion, occurs.

For the 20aa and the 30aa fragments with Cu at 1:1, 1:2 and1:3 M ratios below pH 6, no significant visible CD activity was mea-sured, although several species have clearly been potentiometrical-ly evidenced, thus providing a suggestion for the involvement ofimidazol atoms in macrochelate systems (Fig. 4).

20aa–Cu 1:1 visible CD measurements at pH 6.5 result in aspectrum having a sigmoidal shape which hints the presence ofspecies where multiple histidine residues coordinate a single cop-per ion [2]. In addition the band at 290 nm can be attributed to theNimidazole p2–Cu(II) charge transfer transition [15]. At pH 8, mixedspecies involving also amide deprotonation from the backbone,can be observed.

Fig. 5 shows visible CD spectra for the 30aa with 1 mol equiv ofCu(II) ion at pH values between 3.5 and 11.5. Below pH 7 very lowCD activity is detected, in agreement with multi-histidinic involve-ment in coordinating metal; however, the accompanying CD bandat 290 nm assigned as N-imidazol-to-Cu(II) charge transfer isobserved.

At pH 7, a weak broad negative band at 610 nm can be seen and,together with the band at 310 nm, suggest the presence of mixedspecies at physiological pH.

4.4. EPR results for Cu(II) interaction with the 20aa and the 30aafragment

EPR spectroscopy has been used to investigate about the Cu-sitetransition between the two coordination geometries evidencedgoing from mildly acidic condition to high pH.

For all the spectra, at both 120 K and room temperature, axial ornearly axial parameters are obtained with g|| > g\ > 2.040, suggest-ing a dx2�y2 or, less commonly, a dxy ground state in a square planar,square pyramidal or tetragonal elongated octahedral stereochem-istry [16,17]. Parameters obtained at room temperature show nosignificant differences from those inferred from low temperaturespectra. Addition of the second and the third equivalent of copperdoes not modify EPR spectrum of the first copper atom. Thus, thethree sites can be considered independent (Fig. 6). In addition, inthe pH range 10–11 and at 1:1, 1:2 and 1:3 ligand to metal molarratios, no free copper has been evidenced and the only bound

Fig. 4. Visible CD Cu–20aa 1:1, pH range 3.5–8.

Fig. 6. EPR spectra of Cu–30aa, Cu–20aa 1:1 and 2:1 metal to ligand molar ratio, atpH 10.5; inset 2nd derivative on the perpendicular region.

4N–30aa species is present, thus indicating the formation of Cu3–30aa species.

In basic solution deprotonation of amide groups leads to asquare planar four coordination as previous observed [10], A||

(Cu–30aa, 3N) = 172.7 � 10�4 cm�1 and g|| = 2.232, A|| (Cu–30aa,4N) = 196.7 � 10�4 cm�1 and g|| = 2.195 and A|| (Cu–20aa, 4N) =190.3 � 10�4 cm�1 and g|| = 2.203. From a careful inspection onthe g\ region and from the 2nd derivative on the perpendicular sig-nal, it was possible to recognize the 7- and 9-lines shf pattern for3N and 4N coordination with A\N = 12.4 � 10�4 cm�1 and13.2 � 10�4 cm�1, respectively (Figs. 6 and 7). In fact, the shift tosmaller g|| values, observed at high pH, indicates increased delocal-ization of the unpaired electron away from the copper nucleus andhas often been interpreted in terms of increased covalency in themetal–ligand bond [18].

Correlations between g|| and A|| arising from various factorshave been noted for the Cu–30aa and –20aa species obtained

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below physiological pH (Fig. 8 and Supplementary materialFig. S3). The marked reduction of A|| (Cu) = 152.1 � 10�4 cm�1

and 159.2 � 10�4 cm�1 with the simultaneous increase of

Fig. 7. EPR spectra of Cu–20aa 2:1 pH 8; inset, 2nd derivative on the perpendicularregion.

Fig. 8. EPR spectra of Cu–30aa 1:1 pH 5.5 and pH 6.

Fig. 9. Models of the most likely coordination sphe

g|| = 2.343, 2.324 (pH 5.5, for the Cu–20aa and –30aa, respectively)and A|| (Cu) = 161.4 � 10�4 cm�1 with g|| = 2.302 (pH 6, Cu–30aa),point to coordination with uncharged aza-aromatic ligands asimidazole residues instead of peptide charged N� [19]. Absorptionsalso are in agreement with 2N-aza or 3N-aza coordination,kmax � 680, 690 nm (e � 60, 90 M�1 cm�1, respectively).

However, the substitution of peptide charged N�-donors withaza-aromatic N-donors should shift significantly to higher g|| andslightly to smaller A|| values. This fact together with the shift ofthe quotient f = g||/A||, which can be considered an empirical indexof distortion, from 111.6, or 115 cm (pH 10, 4N species, Cu–20aaand –30aa, respectively) to 154.1 cm (pH 5.5, Cu–20aa) and146.5 cm (pH 5.5, Cu–30aa), points to a geometrical distortion ofthe planar moiety towards a tetrahedral arrangement. From theg|| and the angle between chelate ring planes x relation [18,19],which is well established for all the donor atom types from CuN4

to CuO4, it is possible to calculate the extent of distortion. Forthe ‘‘low” pH Cu-bound species with two coordinated aza-aromaticN-donors, calculated x is approximately 70�, thus, in favour of apseudo-tetrahedral structure. Distortion arrangement determinessmaller nitrogen shf coupling as the overlap of the copper unpairedelectron is reduced. The shf coupling constants are also affected byhybridized states of nitrogen orbitals; the nitrogens with planarconformations, such as deprotonated amide nitrogens, have largercoupling constants than those for nitrogens with tetrahedral con-formations. The spin density on sp3 state is slightly less than thaton the nitrogens in sp2 state. The inspection of the perpendicularregion of the EPR spectra at low pH (5.5) together with the 2ndderivative calculation and simulation aid, allowed us to tentativelydetermine A\N = 11.1 � 10�4 cm�1 for the Cu–30aa species.

Also, replacement of ‘‘soft” donor atoms by ‘‘hard” donor atomstends to shift towards bigger g|| and smaller A||. Thus, involvementof oxygen donors in the macrocycles formation could not be ex-cluded [20].

In addition, giso and Aiso change similarly in an antiparallel fash-ion from high to low pH.

All these observations taken together point to an increase ofaromatic N-donors and a distortion from the planarity to tetrahe-dral geometry for the macrochelate species. The proposed modelsat ‘‘low” pH, which can be dynamically in equilibrium among them,are {2Nim}, {2Nim, O�carbox}, {2Nim, 2O�carbox}, {3Nim}, {3Nim, O�carbox}(Fig. 9).

res of Cu(II) ‘‘low pH” multi-histidinic species.

M.A. Zoroddu et al. / Journal of Inorganic Biochemistry 103 (2009) 1214–1220 1219

A general structural distortion from a possible apical interactionmay be excluded because an additional ligand, which reduces thesymmetry of the CuX4 system, should decrease A|| and g|| also.

4.5. NMR studies on Cu(II) binding at ‘‘low pH”

Paramagnetic Cu(II) broadens 1H NMR signals when in closeproximity to metal or on donor atoms directly coordinated to me-tal [21,22]. Fig. 10 shows the effect of the addition of Cu(II) ion onthe appearance of the 1H NMR spectra obtained at pH 5.5 and atsub-stoichiometric Cu:30aa molar ratios (from 0.002 to 0.1:1). Aselective and simultaneous disappearance of both Hd2 and He1

can be clearly observed. This result strongly points to Ne as theanchoring site and to its involvement in poly-imidazol macrocyclescomplexes formation at low pH. The almost complete lack of sig-nificant broadening of all the other resonances, but only a dropin intensity of His6 Hb1, Glu9 Hc, Hb2 and Thr7 NH indicates thedirection of coordination towards C-terminal, in agreement withthe formation of macrochelate systems.

Fig. 10. (a) 1H NMR aromatic region and (b) 1H NMR aliphatic region of

4.6. Geometry optimisation

An optimisation procedure of the structural features of Cu(II)–{2Nim, 2O�carbox} and Cu(II)–{3Nim, O�carbox} systems has beenperformed.

Cu(II) binding site was modelled on the basis of the geometry ofanalogous multi-histidinic copper-complexes obtained from dif-fractometric data of selected X-ray crystal structures (i.e. PDB1aq8 and PDB 1a4b) [23,24] which were used as a template. Thespatial position and the distances between the metal ion and itsdonor (N, O) atoms were constrained to the values found in the se-lected crystal structures (usually from 1.9 to 2.2 Å for Cu–N andfrom 2.3 to 2.7 Å for Cu–O distances). The geometry of the rest ofthe peptide was optimised without any constraints allowing allthe atoms, bonds and dihedral angles to change simultaneouslyto reach the lowest overall energy.

Models of the most likely coordination spheres of Cu(II) ‘‘lowpH” multi-histidinic species, generated by using ViewerPro 4.2(Accelerys, Inc., 2001) program, are reported in Fig. 9.

0.002, 0.005, 0.01, 0.02, 0.05, 0.1:1, Cu to 30aa molar ratio, pH 5.5.

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The models obtained by using the computational methods [25],show a copper ion in a distorted tetrahedral arrangement (mediumdistances Nim–Cu(II) = 2.1 Å, O�carbox–Cu(II) = 2.6 Å; angles Nim–Cu(II)–Nim = 97�, Nim–Cu(II)–O�carbox = 115�). It may be observedthat the models obtained can be considered in agreement withour experimental results regarding the pseudo-tetrahedral geome-try of the copper coordination site obtained through EPR spectros-copy results.

5. Conclusions

Metal complexes with peptides containing multi-histidine resi-dues have striking coordination abilities and can mimic the struc-tures of various multi-histidine metal binding sites in protein. Thepresence of two or three imidazoles within the peptide sequenceallows metal ions to form poly-imidazole macrocycles bindingmode, which dominates at low metal concentration and at physio-logical pH and below.

Below pH 7 and from sub-stoichiometric to stoichiometric me-tal to ligand molar ratios, binding of copper is dynamic and severalcoordination modes could be in rapid exchange. From all the spec-troscopic results, the most populated states which appear at ‘‘low”pH are in agreement with {2Nim}, {2Nim, O�carbox}, {2Nim, 2O�carbox},{3Nim}, {3Nim, O�carbox} states. By raising the pH a concomitant mod-ification in the binding mode of His residue from Ne to Nd with theformation of six membered metallocycle with Nd of His and the Hismain chain deprotonated amide is likely the driving force of suchinduced conformational change.

The formation of macrochelates with copper binding to sidechain of imidazole functions has been reported for several histidinecontaining peptides such as prion protein and amyloid peptides in-volved in neurodegenerative diseases [26,27].

His-rich protein domains may play a critical role in copperhomeostasis and antioxidant activity [1], especially in the proteinscontaining His residues in the very flexible or unstructureddomains.

At high pH, peptides may bind up to three Cu(II) or Ni(II) ionsstrongly increasing rigidity and structural features of unstructuredregions of proteins.

As reported in the literature, the binding of three metal ions tothe repeat region may be critical for the metal transport inside thecells via endocytosis mechanism [27]; by the decrease of pH as inthe endosome, metal ions can be released and then reduced.

The transition between the two coordination modes could bethe critical point for a dual mechanism operating in multi-histidineproteins, e.g. metal transport in a redox-inactive state throughindividual multiple anchoring sites, protecting against deleteriouseffects, plus antioxidant activity from macrochelate and multi-his-tidinic metal coordination. In fact, it has been reported that multi-imidazole coordination mode might be critical for the biologicalimplications, in particular for the redox activity [28,29]. FromEPR parameters, obtained at room and low temperature, a distor-tion from planar to tetrahedral arrangement was clearly evidenced,particularly for the Cu–20aa macrochelate systems. As reported inthe literature [30,31], SOD activity is better developed for com-plexes in a distorted environment. In fact, in this case, the molec-ular orbital scheme has a Cu-dxy atomic orbital, which is bettersuited to allow dp–pp overlap with O�2 anion. Otherwise, in thecase of planar complexes, interaction is not allowed by the symme-try of the HMSOs of the two species. The multi-histidinic coordina-tion mode facilitates Cu+–Cu++ redox cycle and dismutation ofsuperoxide radical becomes active. In conclusion, the copper trans-port and the antioxidant activity could be the major biologicalfunction of this kind of sites in proteins.

We believe that our study could give an additional clue tounderstanding the role of copper binding in crucial multi-histidinicprotein, such as copper containing oxidases in catalytic centres[32] or prion protein or amyloid precursor protein involved in neu-rodegenerative disorders.

Acknowledgements

This work was supported by the Regione Autonoma Sardegna‘‘Master and Back” program and the Fondazione Banco di Sardegna,Sassari, Sardegna, Italy. CERM (University of Florence) and Prof.Ivano Bertini are gratefully acknowledged for the use of spectro-scopic facilities. Porto Conte Ricerche, Tramariglio, Alghero isgratefully acknowledged for the use of NMR facilities.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jinorgbio.2009.06.008.

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