Together, ITC and heparin binding data suggested that Cu II is a promising proxy for Zn II for performing EPR spectroscopic studies of the local environment of metal ion binding sites and their spatial distribution in HRG. For details on sample preparation and experimental procedures see supplementary information (SI). For EPR investigations of HRG, a 'pseudo-titration' series (each titration point is a discrete sample) was prepared, keeping HRG concentration constant (125 M final protein concentration) and adding Cu II in the range from 1 to 20 molar equivalents (125 M to 2.5 mM). Frozen solution CW EPR spectra were recorded for each sample to observe the Cu II species present (Figure , Figures ). The continuous, almost linear increase in the Cu II signal from 1 to 20 molar equivalents of Cu II (Figure ) indicated that the Cu II observed in the spectrum accounted for virtually all Cu II added, excluding potential precipitation or anti-ferromagnetic interaction which would have resulted in a reduced Cu II signal. Resolved SHF couplings could be assigned to directly ligating nitrogen atoms. These 14 N SHF splittings remained clearly visible from the initial addition of 1 up to ~10-12 molar equivalents of Cu II (Figure ). This suggested the absence of coordinatively distinct metal ion binding sites with substantially different SHF coupling patterns. Thus, CW EPR data nicely complemented the presented ITC results. While ITC showed the presence of 10-12 high-affinity Cu II binding sites in HRG, CW EPR demonstrated that the first 10-12 Cu II ions were bound to nitrogen ligands. In the context of HRG these sites are tentatively assigned to histidine sidechains in the HRR.