Creation of a Binuclear Purple Copper Site within a de Novo Coiled-Coil Protein

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• 作者：Daigo Shiga ; Yasuhiro Funahashi ; Hideki Masuda ; Akihiro Kikuchi ; Masanori Noda ; Susumu Uchiyama ; Kiichi Fukui ; Kenji Kanaori ; Kunihiko Tajima ; Yu Takano ; Haruki Nakamura ; Misato Kamei ; Toshiki Tanaka
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 9, 2012
• 年：2012
• 卷：51
• 期：40
• 页码：7901-7907
• 全文大小：352K
• 年卷期：v.51,no.40(October 9, 2012)
• ISSN：1520-4995

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 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鈥搗is 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 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.