Electronic structure of the ground and excited states of the CuA site by NMR spectroscpy

The electronic properties of Thermus thermophilus CuA in the oxidized form were studied by 1H and 13C NMR spectroscopy. All of the 1H and 13C resonances from cysteine and imidazole ligands were observed and assigned in a sequence-specific fashion. The detection of net electron spin density on a peptide moiety is attributed to the presence of a H-bond to a coordinating sulfur atom. This hydrogen bond is conserved in all natural CuA variants and plays an important role for maintaining the electronic structure of the metal site, rendering the two Cys ligands nonequivalent. The anomalous temperature dependence of the chemical shifts is explained by the presence of a low-lying excited state located about 600 cm-1 above the ground state. The room-temperature shifts can be described as the thermal average of a σu* ground state and a πu excited state. These results provide a detailed description of the electronic structure of the CuA site at atomic resolution in solution at physiologically relevant temperature.