Cu⁺-specific CopB transporter: Revising P_1B-type ATPase classification.

Abstract

The copper-transporting P_1B-ATPases, which play a key role in cellular copper homeostasis, have been divided traditionally into two subfamilies, the P_1B-1-ATPases or CopAs and the P_1B-3-ATPases or CopBs. CopAs selectively export Cu⁺ whereas previous studies and bioinformatic analyses have suggested that CopBs are specific for Cu²⁺ export. Biochemical and spectroscopic characterization of <i>Sphaerobacter thermophilus</i> CopB (<i>St</i>CopB) show that, while it does bind Cu²⁺, the binding site is not the prototypical P_1B-ATPase transmembrane site and does not involve sulfur coordination as proposed previously. Most important, <i>St</i>CopB exhibits metal-stimulated ATPase activity in response to Cu⁺, but not Cu²⁺, indicating that it is actually a Cu⁺ transporter. X-ray absorption spectroscopic studies indicate that Cu⁺ is coordinated by four sulfur ligands, likely derived from conserved cysteine and methionine residues. The histidine-rich N-terminal region of <i>St</i>CopB is required for maximal activity, but is inhibitory in the presence of divalent metal ions. Finally, reconsideration of the P_1B-ATPase classification scheme suggests that the P_1B-1- and P_1B-3-ATPase subfamilies both comprise Cu⁺ transporters. These results are completely consistent with the known presence of only Cu⁺ within the reducing environment of the cytoplasm, which should eliminate the need for a Cu²⁺ P_1B-ATPase.