ABSTRACT
The inherent cellular toxicity of copper ions demands that their concentration be carefully controlled. The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the intracellular copper concentration through the N-terminus of the enzyme, comprising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif. Based on the proposal that binding of copper to these modules regulates the Menkes ATPase cellular location by promoting changes in the tertiary structure of the enzyme, we have expressed the entire N-terminal domain (MNKr) and the second metal-binding module (MNKr2) of the Menkes protein in E. coli and purified them to homogeneity. Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoichiometry of one metal ion per module. However, the array of six modules, MNKr, binds Cu(I) to produce a homogeneous conformer with 4 mol equiv of metal ion. The metal ions are bound in an environment that is shielded from solvent molecules. We suggest a model of the Menkes protein in which the Cu(I) binding induces tertiary changes in the organization of the six metal-binding domains.
