Copper is a meticulously regulated redox-active micronutrient found in a number of important enzymes, including cytochrome c oxidase and superoxide dismutase. Because free or excess intracellular copper can cause oxidative damage, both prokaryotes and eukaryotes have developed specific copper trafficking and transport pathways (1, 2). Deficiencies in these processes are linked to human diseases, including Wilson and Menkes disease. In Wilson disease, accumulation of copper in the liver and brain leads to cirrhosis and neurodegeneration, and in Menkes disease, copper transport across the small intestine is impaired, leading to copper deficiency in peripheral tissues (3, 4). Both disorders are caused by mutations in Cu ϩ -transporting P 1B -type ATPases (5-7), enzymes that are found in most organisms and function in the cellular localization and/or export of cytosolic copper (8, 9).The Cu ϩ -ATPases include eight transmembrane (TM) 4 helices, of which three (TM6, TM7, and TM8) contribute invariant residues to form the transmembrane metal-binding site, a cytosolic ATP binding domain linking TM6 and TM7, an actuator domain between TM4 and TM5, and cytosolic metal binding domains (MBDs) of ϳ60 -70 amino acids that bind Cu ϩ (8, 10). Whereas prokaryotic Cu ϩ -ATPases typically have one or two MBDs, eukaryotic homologs have up to six such domains. Each MBD contains a highly conserved CXXC consensus sequence for binding Cu ϩ and adopts a ␤␣␤␤␣␤ fold (11-14) nearly identical to that of the Atx1-like cytosolic copper chaperones, including yeast Atx1, human Atox1, and bacterial . These chaperones also contain a CXXC motif and deliver Cu ϩ to one or all of the MBDs (20 -26). It is not clear how Cu ϩ reaches the transmembrane metal-binding site and how the cytosolic chaperones participate in this process. The hyperthermophilic Cu ϩ -ATPase CopA from Archaeoglobus fulgidus is readily expressed in fully active recombinant form, is highly stable, and contains all of the essential structural elements for copper transfer, including one N-terminal and one C-terminal MBD (27)(28)(29). CopA is therefore an excellent model system both for investigating the mechanisms of P 1B -type ATPases and for studying interactions between a cytosolic chaperone and its intact partner Cu ϩ -ATPase. The only potential copper chaperone protein in the A. fulgidus genome, which we have designated A. fulgidus CopZ, differs from all other * This work was supported in part by National Institutes of Health Grant GM58518 (to A. C. R.), National Science Foundation Grant MCM-0235165 (to J. M. A.), and National Institutes of Health Grants HL13531 (to B. M. H.) and DK068139 (to T. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.