Mechanism of Cu⁺-Transporting ATPases: Soluble Cu⁺ Chaperones Directly Transfer Cu⁺ to Transmembrane Transport Sites

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu⁺-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu⁺ does not access Cu⁺-ATPases in a free (hydrated) form but is bound to a chaperone protein. Cu⁺ transfer from Cu⁺ chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent Cu⁺ movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious Cu⁺ transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of Cu⁺ by Archaeoglobus fulgidus Cu⁺ chaperone CopZ to the corresponding Cu⁺-ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. Cu⁺-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu⁺-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu⁺. Furthermore, under nonturnover conditions, CopZ transferred Cu⁺ to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers Cu⁺ directly to transmembrane transport sites of Cu⁺-ATPases.