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),Cdn−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),Cdn−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.