Comparing gII and AII values retrieved from the CW spectra (Table ) to Peisach-Blumberg correlations and recent reports on nitrogen (histidine/imidazole)-coordinated Cu II , our data suggested multiple Cu II coordination modes. Initially, Cu II was bound by as many as four nitrogen ligands (histidine residues; Tris buffer, see SI chapter 'EPR sample preparation' for discussion), while higher excess of Cu II led to coordination involving both nitrogen and oxygen atoms (2N2O; 3N1O), as well as the appearance of free Cu II (see Cu II in water spectrum), indicating that the maximum binding capacity of HRG was exceeded. These findings have a direct structural impact for HRG, as they demonstrated a Cu II concentration-dependent shift in Cu II coordination, implying that induced structural changes in HRG are required for, and depend on, metal loading.