Creation of a binuclear purple copper site within a de novo coiled-coil protein

摘要

Although various kinds of metal binding proteins have been constructed by de novo design, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the Cu A site, has two copper ions bridged by two Cys residues. We constructed the Cu _A site consisting of two Cys and two His residues in a de novo designed four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV-vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g ∥ region, which was similar to the native or engineered Cu _A site with an A _(～480)/A _(～530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu _2S _2 core structure, with two typical N(His)-Cu bonds per core at 1.90 ?, two S (Cys)-Cu bonds at 2.21 ?, and the Cu-Cu bond at 2.51 ?, which are also characteristic structures of a purple copper site.