Considering that hyperfine spectroscopy is strongly impacted by fast relaxation these findings explained our observations from EDNMR and ESEEM/HYSCORE, where the substantial changes seen at above 20 molar equivalents of Cu II could not be explained by a mere addition to the spectral signatures visible at lower concentrations. Thus, the slower relaxing Cu II sites dominated the hyperfine spectra in excellent agreement with the relaxation data. Figure . A) Two-pulse decay for selected samples (zoom-in, for full x-axis see Figure ). B) Waterfall plot of background-corrected PELDOR traces (for raw traces see Figure ) obtained from the pseudo-titration series of HRG and Cu II . Samples were either prepared from the initial batch of purified HRG (black lines) or from the second batch of HRG (blue lines), see C). C) Corresponding mean modulation depths (± 2 ×  error) as obtained from the pseudo-titration series of HRG and Cu II . Samples containing 1 to 10 molar equivalents of Cu II (black scatter) were prepared from the initial batch of purified HRG. A second batch of HRG was purified to further increase the equivalents of Cu II (blue scatter) to observe saturation of the high-affinity binding sites. Solid lines show simulations of PELDOR modulation depths based on the speciation model corresponding to values of n = [2, 4, 6, 8, 10, 12, 14, 16] (Simulations were performed with the following other parameters: [P]0 = 1.25 x 10 -4 M, [M]0 = [0.125, 0.250, 0.375, 0.500, 0.675, 0.750, 0.875, 1.000, 1.125, 1.250, 1.500, 1.875, 2.500] x 10 -3 M, KD1 = 5 x 10 -8 , m = 3, KD2 = 10 x 10 -6 and λ = 0.015. For further details on the model see SI). and S14-17, Tables ) further revealed an almost linear increase in modulation depth () with Cu II loading from 1 to ~12 molar equivalents and plateauing thereafter (up to 20 molar equivalents of Cu II ). In agreement with the above hypothesis, there was a marked change for the higher equivalent Cu II samples, with virtually no modulation depth observed from 50 molar equivalents of Cu II (Figure ). Thus, PDS results were in excellent agreement with the hyperfine experiments, demonstrating once more substantial changes in the spectral signature beyond 20 molar equivalents of Cu II , which completely dominated PDS traces and eliminated any modulation depth. We concluded that the high-affinity sites relax fast, while lower-affinity sites that were occupied at higher Cu II loading relax slower. Together, this showed why a simple 'additive' assumption to simulate spectra with increasing Cu II loading was not feasible.