Investigating the electronic structure of the Atox1 copper(I) transfer mechanism with density functional theory

To maintain correct copper homeostasis, the body relies on ion binding metallochaperones, cuprophilic ligands, and proteins to move copper around as a complexed metal. The most common binding site for Cu(I) proteins is the CX1X2C motif, where X1 and X2 are nonconserved residues. Although this binding site motif is well established, the mechanistic and electronic details for the transfer of Cu(I) between two binding sites have not been fully established, in particular, whether the transfer is dissociative or associative or if the electron-rich Cu(I)-Cys interactions influence the transfer. In this work, we investigated the electronic structure of the Cu(I)-S interactions during the copper transfer between Atox1 and a metal binding domain on the ATP7A or ATP7B protein. Initially, three Cu(I) methylthiolate complexes, [Cu(SCH3)2](-1), [Cu(SCH3)3](-2), [Cu(SCH3)4](-3), were investigated with density functional theory (DFT) to fully elucidate the electronic structure and bonding between Cu(I) and thiolate species. The two-coordinate, linear species with a C-S-S-C dihedral angle of ∼90° is the lowest energy conformation because the filled π antibonding orbitals are stabilized in this geometry. The importance of π-overlap is also seen with the trigonal planar, three-coordinate Cu(I) complex, which is similarly stabilized. A corresponding four-coordinate species could not be consistently optimized, so it was concluded that tetrahedral coordination was not likely to be stable. The transfer of Cu(I) from the Atox1 metallochaperone to a metal binding domain of the ATP7A or ATP7B protein was then modeled by using the CGGC Atox1 binding site for the donor model and the dithiotreitol ligand (DTT) for the acceptor model. The two- and three-coordinate intermediates calculated along the five-step transfer mechanism converged to near optimal Cu-S π-overlap for the respective geometries, which demonstrates that the electronic structure in this electron-rich environment influences the intermediate's geometries in the transfer mechanism.