Does the proline residue affect the coordination of divalent metal ions?

The aim of the present work is to investigate the interaction of metal ions with short model peptides containing proline residues next to the metal binding site. In these peptides, the metal binding site is represented by a histidine imidazole, an effective anchoring site for metal ions such as Cu(II), Zn(II) and Fe(II), already at acidic pH. In the case of Cu(II), increasing the pH value of the solution, the binding to backbone N-amides can occur towards both the N- or C-terminal direction, but with a different level of affinity.1 On the other hand, the occurrence of this coordination behaviour for Zn(II) and Fe(II) is controversial.2,3 The present study concerns the terminally protected short peptide Ac-AAAHAAA-NH2 and two mutants: Ac-AAPHAAA-NH2 and Ac-AAPHPAA-NH2. In the former peptide, the Ala residue in position 3 has been replaced with a proline, while, in the latter one, the two alanines adjacent to the histidine residue (position 3 and 5) have been replaced with prolines. The investigation allowed to better understand the coordination behaviour of the three metal ions under different pH condition, shedding light on the effect caused by the presence of proline residues close to the metal binding site. In fact, proline residues do provide greater structural rigidity to the peptide chain, hampering conformational changes and rearrangements around the metal center and making unavailable for the binding their corresponding backbone amide. The thermodynamic and spectroscopic characterization of the metal complex-formation equilibria has been achieved by means of potentiometry, UV-Vis spectrophotometry, circular dichroism, electron paramagnetic resonance and mass spectrometry. Financial support of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1-NextGenerationEU (PRIN PNRR 2022-P2022EMY52) is gratefully acknowledge.