Cu + distribution in metallothionein fragments Maria T. Salgado and Martin J. Stillman * Department of Chemistry, University of Western Ontario, London, Ont., Canada N6A 5B7 Received 18 March 2004 Abstract The differential distribution of Cu þ between separate a and b domains of metallothionein (the isolated peptide fragments) and the rate of transfer of Cu þ between the two domains using copper–thiolate specific emission spectroscopy are reported. Kinetic data show the rate of transfer of Cu þ from the Cu 6 a to the Cd 3 b domain is 2  10 1 s 1 while the transfer from Cu 6 b to the Cd 4 a domain is much slower at 8  10 3 s 1 , indicating the greater binding affinity of Cu þ for the MT b domain. We report that the emission intensity of Cu 6 b is 0.45 the emission intensity of Cu 6 a–MT. k max is shown to be a probe of the environment of the Cu þ . A series of copper-containing domain intermediates to the formation of the filled Cu 6 S 9 -b and Cu 6 S 11 -a-clusters are identified. A mechanism is proposed for the formation of Cu 12 (ba)–MT that involves metal exchange reactions of Cu þ ions from the a to the b domain with initial formation of a Cu 4 b-cluster. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Emission spectroscopy; Copper–thiolate clusters; a and b domains; Metallothionein; Copper transport; Kinetic analysis Copper is an essential trace element required for the normal growth and development of all organisms, however, excess concentrations of copper can become harmful [1] when not bound to appropriate biological chelators [1,2]. Copper–metallothionein (Cu–MT) has been proposed as one of the major metal-transport systems that newly synthesized proteins use as their source of copper [3]. Metallothioneins (MT) are a un- ique class of low molecular weight metalloproteins found in vertebrates, invertebrates, fungi, and yeast, characterized by a high cysteine content and an absence of both aromatic residues and disulfide bonds [4]. Ex- posure of an organism to Zn 2þ , Cu þ , Cd 2þ , and Hg 2þ ions results in induction at the transcriptional level of rapid de novo synthesis of metallothionein, to which these metals bind [5–9]. Binding of copper to MT serves as a protective role by suppressing copper toxicity be- cause Cu–MT decreases the ability of the metal ions to participate in harmful intracellular interactions [2], and so MT has been linked to play a key role in the storage, transport or detoxification of essential and nonessential metal ions [2,4]. Therefore, it is of great importance to understand the mechanisms by which MT sequesters and transfers metals such as Cu þ , due to the wide range of copper-dependent proteins present in biological sys- tems that play a crucial part in the proper functioning of biological systems [1–3]. Metal binding in mammalian MT takes place through metal–thiolate bonds that cross-link the protein into its final three-dimensional structure that involves two dis- tinctive metal–thiolate clusters [2–10], the N-terminal, b domain and the C-terminal, a domain. The three-di- mensional structure depends on the metal identity, co- ordination preference, and number of metals bound to the protein. Seven divalent Cd 2þ metal ions bind with tetrahedral coordination geometry to MT, whereas, monovalent Cu þ metal ions bind with trigonal or diag- onal coordination with the cysteine sulfurs of the pro- tein and exhibit saturation up to 12 metals [2,4]. Fig. 1 shows the binding stoichiometry and cluster arrange- ment in mammalian MT for both tetrahedral Cd 2þ (CdS 4 ) and trigonal Cu þ (CuS 3 ) metal ions [11], creating domain stoichiometries of Cd 3 S 9 -b and Cd 4 S 11 -a [10,12,13] for Cd 2þ ions and Cu 6 S 9 -b and Cu 6 S 11 -a for Cu þ ions [11,14]. The conformational differences im- parted by these two metals are significant and the metal- dependent, three-dimensional structure may be a key * Corresponding author. Fax: +1-519-661-3022. E-mail address: Martin.Stillman@uwo.ca (M.J. Stillman). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.183 Biochemical and Biophysical Research Communications 318 (2004) 73–80 BBRC www.elsevier.com/locate/ybbrc