title: Creation of a binuclear purple copper site within a de novo coiled-coil protein.
abstract: 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(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.
authors.affiliation: Department of Material Sciences, Graduate School of Engineering,  Nagoya Institute of Technology , Gokiso-chou, Nagoya 466-8555, Japan.
authors.forename: Daigo
authors.initials: D
authors.lastname: Shiga
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Funahashi
authors.forename: Hideki
authors.initials: H
authors.lastname: Masuda
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Masanori
authors.initials: M
authors.lastname: Noda
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
authors.forename: Kenji
authors.initials: K
authors.lastname: Kanaori
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Tajima
authors.forename: Yu
authors.initials: Y
authors.lastname: Takano
authors.forename: Haruki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Misato
authors.initials: M
authors.lastname: Kamei
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D015502:Absorptiometry, Photon; D000595:Amino Acid Sequence; D001665:Binding Sites; D003116:Color; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D003300:Copper; D003576:Electron Transport Complex IV