Results from hyperfine spectroscopy strongly suggested the presence of two or more histidine residues coordinating the metal ion per binding site up to 10-15 molar equivalents of Cu II , with spectroscopic profiles indicating that all high affinity sites were of similar geometry and populated statistically. Furthermore, EPR data of high-equivalent Cu II samples (50 or more molar equivalents) suggested that, once the higher affinity sites were occupied, additional metal ion binding occurred at lower affinity sites that did not involve histidine residues. These data are consistent with the presence of 25 histidine residues within the HRR, which could offer 2 residues per metal ion for up to 12 ions bound. The majority of the HRR histidine residues in HRG are part of a series of tandem repeats of the consensus sequence G[H/P][H/P]PH (see Figure ). In rabbit HRG, a further 18 histidine residues are spread over the HRR, flanking regions including PRR1 and PRR2, bringing the total number to 43 histidine residues within the disordered PRR1-HRR-PRR2 stretch. This is in line with the observation that beyond 50 molar equivalents of Cu II , lower-affinity non-histidine binding sites were occupied.