Cadmium Binding and Metal Cluster Formation in Metallothionein: A Differential Modification Study

Abstract

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH₂- and COOH-terminal half of the chain, respectively (1). The formation of these oligonuclear complexes designated as Cd₄- and Cd₃-cluster has now been monitored in MT reconstituted with varying amounts of Cd using differential modification of Cys with ¹⁴C-iodoacetamide. At ratios below 3 moles Cd/mole MT bound, no differential protection of Cys by the metal and, hence, no preferred binding is detectable. At Cd-to-protein ratios between 3 and 5 moles Cd/mole MT the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain indicating formation of the Cd₄- cluster. At still higher ratios formation of the Cd₃-cluster is initiated in the NH₂-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd₆-MT and Cd₇-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labelling of the different Cys in Cd₇-MT indicates that the ligands of the Cd₃-cluster are three times as accessible to iodoacetamide as those of the Cd₄-cluster, suggesting a greater thermodynamic stability of the latter.