Spectroscopic evidence for metal-thiolate clusters in complexes of Cd(II) with dithiol hexapeptides and in Cd-Metallothionein

Abstract

Metallothioneins constitute a novel family of widely occurring metal-thiolate proteins which are engaged in the metabolism and the detoxification of posttransitional metal ions (Zn(II), Cd(II), Cu(I), etc.). All mammalian forms characterized are single chain proteins of 61 amino acids residues with binding sites for 7 metal ions [1]. The large metal-binding capacity is conditioned by the presence of 20 cysteine residues which provide thiolate ligands for the formation of two adamantane-like metal-thiolate cluster structures containing 3 and 4 metal ions, respectively [2,3]. The unique occurrence of these clusters in metallothionein is believed to be related to the presence of 7-Cys-X-Cys-sequences in the polypeptide chain where X stands for an amino acid residue other than Cys. To explore the metal complexing features of such dithiol sequences, we have now chemically synthesized by the Merrifield procedure the hexapeptides Ser-Cys-Val-Cys-Ala-Ala, Ala-Cys-Lys-Cys-Ala-Ala and Ala-Cys-Ser-Cys-Ala-Ala and have examined the spectroscopic features of their complexes with Cd(L) and related metal ions. Stepwise addition of Cd(II) to aqueous solutions of these peptides yields complexes of 1:2, 2:3 and 1:1 metal-to-peptide stoichiometry (Fig. 1(a)). The first two complexes display features ▪ typical of tetrahedral tetrathiolate coordination also seen in Cd(II)-metallothionein [4] (Fig. 1(c)). The 2:3 complex is thought to be a binuclear cluster complex composed of two tetrahedral cadmium-thiolate units connected via two bridging thiolate ligands. It differs from the mononuclear 1:2 complex by a slight red shift of the absorption envelope which can be attributed to the greater polarization of the bridging sulfur ligands by the metal. In addition, its formation is accompanied by a nearly complete loss of the of the strong circular dichroism denoting the greater symmetry its structure. The spectral shift signalling the formation of the ligand-bridged binuclear complex is most clearly indicated by the emergence of a maximum near 260 nm in the difference-difference absorption spectrum (Fig. 1(b)). Spectral changes entirely analogous to those accompanying the successive formation of the mononuclear and binuclear tetrahedral Cd(II)-peptide complex also occur in the course of reconstituting Cd(II)-metallothionein from Cd(II) and apometallothionein (Figs. 1(c) and (d)). Under the conditions employed (pH 8), Cd(II) binds at first to separate tetrathiolate sites. However, with all thiolate ligands becoming occupied, a red shift develops signifying the change-over to the clustered structure containing 40% bridging ligands. Interestingly, these spectral changes are much less pronounced when Cd(II) is incorporated at lower pH. Below pH, the binding of successive equivalents of Cd(II) is, in fact, accompanied by a blue shift of the Cd(II)-thiolate absorption envelope indicating an initial preferential formation of thiolate-bridged structures. Hence, it would appear that depending on pH the building-up of the metal-thiolate clusters in metallothionein proceeds through different pathways.