Abstract
It is unclear how the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson and Menkes disease proteins in the cytoplasm. To begin to address this problem, we have characterized Cu(I) release from wild-type Atox1 and two point mutants (Met(10)Ala and Lys(60)Ala). The dynamics of Cu(I) displacement from holo-Atox1 were measured by using the Cu(I) chelator bicinchonic acid (BCA) as a metal acceptor. BCA removes Cu(I) from Atox1 in a three-step process involving the bimolecular formation of an initial Atox1-Cu-BCA complex followed by dissociation of Atox1 and the binding of a second BCA to generate apo-Atox1 and Cu-BCA(2). Both mutants lose Cu(I) more readily than wild-type Atox1 because of more rapid and facile displacement of the protein from the Atox1-Cu-BCA intermediate by the second BCA. Remarkably, Cu(I) uptake from solution by BCA is much slower than the transfer from holo-Atox1, presumably because of slow dissociation of DTT-Cu complexes. These results suggest that Cu chaperones play a key role in making Cu(I) rapidly accessible to substrates and that the activated protein-metal-chelator complex may kinetically mimic the ternary chaperone-metal-target complex involved in Cu(I) transfer in vivo.