Stoichiometry of complex formation between copper(I) and the N-terminal domain of the Menkes protein

The inherent cellular toxicity of copper ions demands that their concentrat ion be carefully controlled. The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the int racellular copper concentration through the N-terminus of the enzyme, compr ising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif. Based on the proposal that bi nding of copper to these modules regulates the Menkes ATPase cellular locat ion by promoting changes in the tertiary structure of the enzyme, we have e xpressed the entire N-terminal domain (MNKr) and the second metal-binding m odule (MNKr2) of the Menkes protein in E. coli and purified them to homogen eity. Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoich iometry of one metal ion per module. However, the array of six modules, MNK r, 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 solv ent 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-bindi ng domains.