The copper(II) and zinc(II) coordination mode of HExxH and HxxEH motif in small peptides: The role of carboxylate location and hydrogen bonding network Giuseppe Grasso a , Antonio Magrì b , Francesco Bellia b , Adriana Pietropaolo c, ⁎, Diego La Mendola d, ⁎⁎, Enrico Rizzarelli a a Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy b Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche (CNR) Catania, Viale A. Doria 6, 95125 Catania, Italy c Dipartimento di Scienze della Salute, Università di Catanzaro, Viale Europa, 88100 Catanzaro, Italy d Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126 Pisa Italy abstract article info Article history: Received 5 August 2013 Received in revised form 30 September 2013 Accepted 30 September 2013 Available online 9 October 2013 Keywords: Peptide Copper Zinc Metallopeptidases Histidine Copper(II) and zinc(II) complexes with two hexapeptides encompassing HExxH and HxxEH motif were charac- terized by means of a combined experimental and theoretical approach. Parallel tempering and density function- al theory (DFT) investigations show the presence of different hydrogen bonding networks between the copper(II) and zinc(II) complexes with the two peptides, suggesting a significant contribution of these non- covalent interactions to the stability constant values. The glutamate carboxylate group has a direct role in metal ion binding. The location of this amino acid along the sequence of the investigated peptides is critical to de- termine thermodynamic and spectroscopic features of the copper(II) complex species, whereas is less relevant in the zinc(II) complexes formation. Electrospray ionization mass spectrometry (ESI-MS) characterization of the zinc(II) complex species show that in the [ZnH −2 L] two deprotonated amide nitrogen atoms are involved in the metal coordination environment, an uncommon behavior in zinc(II) complexes for multi-histidine ligands. © 2013 Elsevier Inc. All rights reserved. 1. Introduction The high frequency of occurrence of a single amino acid in pro- teins characterizes the so-called “Xaa-rich” proteins (Xaa here refers to any type of amino acids). The physiological and pathological roles of cysteine- [1,2], glutamine- [3,4], glycine- [5,6], leucine- [7,8] and proline-rich [9–11] proteins with their sequence repeats have been reported. Although the average frequency of occurrence of histidine in all proteins is relatively low [12], the histidine-rich proteins are probably the most noticeable ones among the “Xaa-rich” proteins [13–19]. The versatility of histidine coordination favors transition metal bindings and many studies have been focused on characteriz- ing the metal interactions with sequentially histidine-rich motifs/ proteins. However, proteins with inconsecutive histidines in primary sequences are also able to form local histidine-rich environments as consequence of the folding process, so that the metal ions can be coordinated [20–24]. Among the large quantity of metalloproteins which employ histidine residues for metal coordination, almost all copper-binding histidine rich proteins comprise only histidines as part- ners in metal interactions [12]. Copper complexes of linear and cyclic multi-histidine peptides have been the focus of numerous studies with the aims to understand the sta- bilizing role of histidyl residues in the active center of copper proteins [25–36]. The general features of the complex formation reactions and the coordination mode of this metal ion with His-containing peptides have been reviewed by several authors [37–39]. For multi-histidine peptides with protecting groups at the N- and C-termini, there is general agreement that: i) the metal ion speciation and the coordination modes are driven by the number and the position of the His residues in the peptide sequences; ii) the coordination modes of the major species are generally described by the formation of 5-, 6- or 7-membered chelates including the imidazole and deprotonated amide nitrogens; iii) the metal binding of deprotonated amide nitrogens starts from the internal histidine anchoring residue and is followed by the subsequent amino acids towards the N-terminus favoring the (6,5,6)-membered chelate formation; iv) the increase in the number of histidine residues favors the formation of macrochelates in slightly acidic and/or neutral solution as found in large peptide fragments of natural proteins includ- ing prions, histones and amyloid peptides [40–44]; v) both thermody- namic and spectroscopic data indicate the existence of coordination isomers in solution. Journal of Inorganic Biochemistry 130 (2014) 92–102 ⁎ Corresponding author. Tel.: +39 0 961 391 157; fax: +39 0 961 391 490. ⁎⁎ Corresponding author. Tel.: +39 0 50 2219500; fax: +39 0 50 2219605. E-mail addresses: apietropaolo@unicz.it (A. Pietropaolo), lamendola@farm.unipi.it (D. La Mendola). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.09.021 Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio