Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

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Abstract
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+byArchaeoglobus fulgidusCu+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.