文摘
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 CuA site, has two copper ions bridged by two Cys residues. We constructed the CuA 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鈥搗is spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g region, which was similar to the native or engineered CuA site with an A480/A530 > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu2S2 core structure, with two typical N(His)鈥揅u bonds per core at 1.90 脜, two S (Cys)鈥揅u bonds at 2.21 脜, and the Cu鈥揅u bond at 2.51 脜, which are also characteristic structures of a purple copper site.
