Mechanism of Cadmium Ion Substitution in Mammalian Zinc Metallothionein and Metallothionein α Domain: Kinetic and Structural Studies

Cellular metallothionein (MT) protects against Cd²⁺ exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn₇-MT-2 with Cd²⁺ was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn₄-α domain revealed that only the fastest step involved the Zn₃-β domain. Each step of the Zn₄-α domain reaction with Cd²⁺ displayed hyperbolic dependence of the observed rate constant on Cd²⁺ concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd²⁺ to the Zn_(4−n),Cd_n−thiolate cluster (n = 0−3) of the α domain and the first order rate constant for the exchange of Cd²⁺ for Zn²⁺ in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn_(4−n),Cd_n−thiolate clusters (n = 0−3) was prepared by titration of the Zn₄-α domain with ¹¹³Cd²⁺. The products were analyzed by one-dimensional ¹¹³Cd²⁺ NMR spectroscopy to define the distribution of ¹¹³Cd²⁺ among the four cluster binding sites. Each of these species was also reacted with Cd²⁺. The properties of these reactions were similar to those extracted from the reaction of Cd²⁺ with the overall domain. Thus, the kinetic results were linked to ¹¹³Cd²⁺ occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd²⁺ with the Zn₄-α domain in relation to the α domain cluster structure.