Fig. 2: Structural characterization of H4pep-Cu²⁺ species.

A, B UV-visible (A, [H4pep] = 0.4 mM, pH 5.6) and UV-CD spectra (B, [H4pep] = 0.1 mM, pH 5.6) of H4pep-Cu²⁺ complexes at increasing Cu²⁺ equivalents, ranging from 1:0 to 1:2 H4pep:Cu²⁺ ratios. Inset in (A) ([H4pep] = 2.35 mM), focuses on the Cu²⁺ d-d band in the range from 500 to 800 nm. Solid lines in (B) highlight significant samples in determining the stoichiometry of the complexes. C EPR spectra of Cu²⁺ and H4pep-Cu²⁺ species at different H4pep:Cu²⁺ ratios (acetate buffer, pH 5.6). Spectra were recorded in presence of a fixed Cu²⁺ concentration (0.1 mM) and increasing concentrations of H4pep. D Speciation diagrams obtained by spectral fitting of UV-visible data and spectral deconvolution of CD and EPR data. 1Cu2Pep and 2Cu2Pep species are assigned according to the “Stoichiometry of H4pep-Cu²⁺ complexes” section.