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 Cu(2+) export. Biochemical and spectroscopic characterization of Sphaerobacter thermophilus CopB (StCopB) show that, while it does bind Cu(2+), the binding site is not the prototypical P(1B)-ATPase transmembrane site and does not involve sulfur coordination as proposed previously. Most important, StCopB exhibits metal-stimulated ATPase activity in response to Cu(+), but not Cu(2+), 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 StCopB 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 Cu(2+) P(1B)-ATPase.