Thiolate Spin Population of Type I Copper in Azurin Derived from 33S Hyperfine Coupling

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper–sulfur (Cu–S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron–electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from whi...