These data indicated that the different binding sites may have different relaxation behaviour, leading to the hypothesis that Cu II bound to high-affinity sites relaxes faster and thus vanishes from the signal once lower-affinity sites are being occupied (see SI for more detailed discussion). Vertical lines indicate the different frequencies to be expected in presence of two histidine residues (NQI: ν0, ν+, ν-, DQ: νdq); these were employed for quantitative analysis. C) (+,+) HYSCORE spectra for 1 (I) and 5 (II), 20 (III), 50 (IV) and 400 (V) molar equivalents of Cu II and control (VI; 1 eq. Cu II in Tris buffer), obtained at X-band frequencies (9.5 GHz) and on the maximum of the field-swept spectra.