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Updated: May 18, 2026

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Published on: December 16, 2013
Creation of a binuclear purple copper site within a de novo coiled-coil protein
Daigo Shiga1, Yasuhiro Funahashi, Hideki Masuda
1Department of Material Sciences, Graduate School of Engineering, Nagoya Institute of Technology , Gokiso-chou, Nagoya 466-8555, Japan.
Abstract:
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-vis 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 A(∼480)/A(∼530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.
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