Results were further corroborated by the corresponding two-dimensional HYSCORE experiments demonstrating the same shape and combination peaks from 1 to 20 molar equivalents of Cu II , strongly suggesting that highly similar binding sites were occupied. Consistent with ESEEM results, HYSCORE spectra changed substantially at higher molar equivalents of Cu II , resembling the control spectrum (Tris-/ water-coordinated Cu II ), indicating that new non histidine sites were being occupied. These sites displayed strong interaction with water protons and were thus more solvent exposed than the higher affinity sites in the HRR (Figures 4C and S11-12).