ESEEM spectra up to 20 molar equivalents of Cu II showed very similar frequencies (within experimental uncertainty) typically observed for remote nitrogen atoms at 9-10 GHz electron Larmor frequency and were assigned to nuclear quadrupole interactions (NQI, several narrow lines in the range of ~0.5-2 MHz) and to the 14 N double quantum transition (DQ, a broader line around 4 MHz) (Figures and). These results were in line with the EDNMR data, indicating that the involved histidine binding sites were very similar (or the same sites were filled gradually). Importantly, while favourable cases may allow extraction of information on tensors and bond lengths and angles, 67 this approach is not feasible for HRG due to its putative structural heterogeneity and the large number of equivalent but non-identical binding sites. Quantitative analysis of ESEEM experiments provided further support for our EDNMR data (Figures and, Table ). Here, changes in the DQ peak integral, which directly depends on the number of histidine residues involved in the binding site, suggested coordination of the Cu II by at least two imidazole rings for up to 15 molar equivalents of Cu II , and less histidine residues per Cu II available for binding at higher Cu II loading (see SI for more detailed discussion). Importantly, ESEEM dropped off significantly at higher Cu II loading (≥ 50 molar equivalents), which was consistent with EDNMR data.