Creation of a type 1 blue copper site within a de novo coiled-coil protein scaffold.
Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) 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 Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) 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 Cu(2+) 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.
Department of Material Sciences, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-chou, Nagoya 466-8555, Japan.
Daigo
D
Shiga
Daisuke
D
Nakane
Tomohiko
T
Inomata
Yasuhiro
Y
Funahashi
Hideki
H
Masuda
Akihiro
A
Kikuchi
Masayuki
M
Oda
Masanori
M
Noda
Susumu
S
Uchiyama
Kiichi
K
Fukui
Kenji
K
Kanaori
Kunihiko
K
Tajima
Yu
Y
Takano
Haruki
H
Nakamura
Toshiki
T
Tanaka
D015502:Absorptiometry, Photon; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
D002352:Carrier Proteins; C023548:copper-binding protein; D003300:Copper