Thermodynamic Studies of the Mechanism of Metal Binding to the Escherichia coli Zinc Transporter YiiP

Sequence homology of the Escherichia coli YiiP places it within the family of cation diffusion facilitators, a family of membrane transporters that play a central role in regulating cellular zinc homeostasis. Here we describe the first thermodynamic and mechanistic studies of metal binding to a cation diffusion facilitator. Isothermal titration calorimetric analyses of the purified YiiP and binding competitions among Zn²⁺, Cd²⁺, and Hg²⁺ revealed a mutually competitive binding site common to three metal ions and a set of noncompetitive binding sites, including one Cd²⁺ site, one Hg²⁺ site, and at least one Zn ²⁺ site, to which the binding of Zn²⁺ exhibited partial inhibitions of both Cd²⁺ and Hg²⁺ bindings. Lowering the pH from 7.0 to 5.5 inhibited binding of Zn²⁺ and Cd²⁺ to the common site. Further, the enthalpy change of the Cd²⁺ binding to the common site was found to be related linearly to the ionization enthalpy of the pH buffer with a slope corresponding to the release of 1.28 H⁺ for each Cd²⁺ binding. These H⁺ effects are consistent with a coupled deprotonation process upon binding of Zn²⁺ and Cd ²⁺. Modification of histidine residues by diethyl pyrocarbonate specifically inhibited Zn²⁺ binding to the common binding site, indicating that the mechanism of binding-deprotonation coupling involves a histidine residue(s).