The resolution of N SHF coupling gradually decreased upon addition of Cu II , in agreement with earlier data. 17 This can either be caused by spectral broadening by interaction between Cu II ions in close proximity, or by a variety of sites being occupied (but not merely 'diluting out' the SHF signal, see SI for more detailed discussion). To further investigate the appearance of additional, lower-affinity metal ion binding sites, and determine the maximum binding capacity of HRG, a set of samples with high Cu II loading (from 50 to 400 molar equivalents) was assessed (Figure ). Here, the protein concentration was varied (from 6.25 M to 50 M) while the Cu II concentration was kept constant (at 2.5 mM), yielding a constant double-integral of the resulting CW signal, within the confidence estimate (Figure ). Importantly, while the 50-equivalent Cu II sample showed mainly broadening in the CW spectrum compared to the 5-equivalent Cu II sample, the higher Cu II equivalent samples clearly demonstrated the appearance of additional Cu II species by stark changes in the spectral lineshapes (Figure ).