The question whether the gradual loss of SHF resolution in the CW spectra was due to inhomogeneous line broadening (while having the same underpinning SHF coupling) was investigated using hyperfine spectroscopy. In the presence of HRG, EDNMR spectra displayed additional peaks compared to Cu II in buffer (Figures and, Table ), confirming coordination by at least two imino nitrogen nuclei of imidazole rings to Cu II . These were tentatively assigned to histidine residues forming the Cu II binding sites in the HRR. The hyperfine structure was virtually unchanged from 1 up to 15-20 molar equivalents of Cu II . Relating these results to the frozen CW data, the disappearance of the 14 N SHF concomitant with line broadening could not be explained by the appearance of additional couplings, which would have given rise to additional peaks in the EDNMR. Instead, it could be concluded that dipolar line broadening because of electron-electron interactions caused by close proximity of binding sites led to the gradual disappearance of the SHF. Furthermore, higher Cu II loading (≥ 50 molar equivalents) led to the complete loss of the additional peaks in the EDNMR spectra, suggesting that other (non-histidine) binding sites have become dominant, which must be of lower affinity due to their occupation only at higher Cu II concentrations.