Creation of a Type 1 Blue Copper Site within a de Novo Coiled-Coil Protein Scaffold

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• 作者：Daigo Shiga ; Daisuke Nakane ; Tomohiko Inomata ; Yasuhiro Funahashi ; Hideki Masuda ; Akihiro Kikuchi ; Masayuki Oda ; Masanori Noda ; Susumu Uchiyama ; Kiichi Fukui ; Kenji Kanaori ; Kunihiko Tajima ; Yu Takano
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：December 29, 2010
• 年：2010
• 卷：132
• 期：51
• 页码：18191-18198
• 全文大小：319K
• 年卷期：v.132,no.51(December 29, 2010)
• ISSN：1520-5126

文摘

Type 1 blue copper proteins uniquely coordinate Cu²⁺ in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu²⁺ coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu²⁺ in the hydrophobic core of the scaffold. The protein bound Cu²⁺, displayed a blue color, and exhibited UV−vis spectra with a maximum of 602−616 nm, arising from the thiolate−Cu²⁺ ligand to metal charge transfer, depending on the exogenous axial ligand, Cl⁻ or HPO₄²⁻. The protein−Cu²⁺ complex also showed unresolved small A values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu²⁺ coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu−N(His) at around 1.9−2.0 Å, Cu−S(Cys) at 2.3 Å, and a long Cu−Cl at a 2.66 Å, which are also characteristic of the natural type 1 blue copper proteins.