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

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 alpha-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(II) 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 angstrom, Cu-S(Cys) at 2.3 angstrom, and a long Cu-CI at a 2.66 angstrom, which are also characteristic of the natural type 1 blue copper proteins.