Reinvestigation of the copper(II)–carcinine equilibrium system: ‘‘two-dimensional’’ EPR simulation and NMR relaxation studies for determining the formation constants and coordination modes Zsuzsanna A ´ rkosi a , Zolta ´n Paksi b , La ´szlo ´ Korecz a , Tama ´s Gajda b, * , Bernard Henry c, * , Antal Rockenbauer a, * a Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, H-1525 Budapest, Hungary b Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary c Laboratoire de Chimie Physique Organique et Colloidale, UMR SRSMC CNRS no. 7565, Universite ´ Henri Poincare ´ – Nancy I., B.P. 239, F-54506 Vandoeuvre-le `s-Nancy Cedex, France Received 30 April 2004; received in revised form 27 August 2004; accepted 2 September 2004 Available online 8 October 2004 Abstract The equilibria and solution structure of complexes formed between copper(II) and carcinine (b-alanyl-histamine) at 2 6 pH 6 11.2 have been studied by EPR and NMR relaxation methods. Beside the species that have already been described in the literature from pH-potentiometric measurements, several new complexes have been identified and/or structurally characterized. The singlet on the EPR spectrum detected in equimolar solutions at pH 7, indicates the formation of an oligomerized ðCuLÞ 2nþ n com- plex, with {NH 2 ,N im } coordination. The oligomerization is probably associated with the low stability of the ten-membered macr- ochelate ring, which would form in the mononuclear complex CuL 2+ . In presence of moderate excess of ligand the formation of four new bis-complexes (CuL 2 H 2þn n , n = 2,1 and 0/1) was detected with {N im }{N im }, {NH 2 ,N im }{N im } and {NH 2 ,N  ,N im }{N im } type co-ordination modes, respectively. At higher excess of ligand ([L]/[Cu 2+ ] > 10) and at pH  7, the predominant species is CuL 4 H 4þ 2 . The 1 H and 13 C relaxation measurements of carcinine solutions (0.6 M) in presence of 0 mM 6 [Cu 2+ ] tot 6 5 mM at pH = 6.8, allowed us to extract the carbon-to-metal distances, the electronic relaxation and tumbling correlation times, as well as the ligand exchange rate for the species CuL 4 H 4þ 2 . According to these results, the metal ion is {4N im } co-ordinated in the equatorial plane, while the neutral amino groups are unbounded. Since naturally occurring carcinine shows in vivo antioxidant property, the SOD-like activity of the copper(II)–carcinine system has also been investigated and the complex CuLH 1 was found to be highly active. Ó 2004 Elsevier Inc. All rights reserved. 1. Introduction Carnosine (b-alanyl-histidine) is one of the most abundant nitrogenous compounds (0.5–20 mM) present in the non-protein fraction of vertebratesÕ tissues [1]. Carnosine is postulated to act as intracellular buffer in skeletal muscle, antiglycating agent, hydrophilic antioxi- dant, free radical and metal ion scavenger [1–3]. The putative role of carnosine as biological metal ion chela- tor, has inspired a number of investigations dealing with its copper(II) and zinc(II) complexes [4–9]. Several nat- ural derivatives of carnosine are known, such as anser- ine, ophidine and carcinine (b-alanyl-histamine, Scheme 1). The antioxidant, buffering and metal chelat- ing ability of these derivatives are well documented, but their biological role is not understood in details. 0162-0134/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.09.002 * Corresponding authors. Tel.: +36 62544054; fax: +36 62420505. E-mail address: gajda@chem.u-szeged.hu (T. Gajda). www.elsevier.com/locate/jinorgbio Journal of Inorganic Biochemistry 98 (2004) 1995–2005 JOURNAL OF Inorganic Biochemistry