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 Zn2+ 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 1.23 H+ for each Cd2+ binding. These H+ effects are consistent with a coupled deprotonation process upon binding of Zn2+ and Cd2+. 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).