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 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 HPO₄^2-. 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.