Manganese and cobalt binding in a multi-histidinic fragment.

The binding of Mn(II) and Co(II) ions to a multi-histidinic peptide, the three repeats (T1R2S3R4S5H6T7S8E9G10)3 portion of Cap43 protein, has been studied. Potentiometric measurements have been used to investigate the protonation equilibria and stoichiometry of the species obtained in a wide range of pH and at a 1 : 1 ligand-to-metal molar ratio. NMR, UV-visible and EPR spectroscopy techniques have been used to investigate the role of multi-histidinic and glutamate sites in coordinating metal ions. (1)H-(1)H TOCSY, (1)H-(13)C HSQC multidimensional NMR techniques were performed to understand the details of metal binding sites and the conformational behaviour of the peptide. The effects of the peptide titration with the two metals have been followed by paramagnetic selective line-broadening in the 1D NMR spectra and the signals' disappearance in the 2D (1)H-(13)C HSQC and (1)H-(1)H TOCSY. Both ions showed common binding donor atoms: the main manganese and cobalt binding centre of the peptide fragment is associated with histidine and glutamate residues. The specific perturbation of NMR resonances indicated that the coordination involves imidazole Nε of histidine and carboxyl γ-O of glutamate residue. All the three imidazole Nε of His6, His16 and His26, as well as carboxyl γ-O of Glu9, Glu19 and Glu29, in an octahedral arrangement are involved in the coordination in the physiological pH range. The involvement of hydroxyl γ-O from the threonine (or serine) side chain can also be observed. Manganese and cobalt complexation induces important structural changes within the C-terminal portion of the ligand, constraining it to leave its disordered conformation. A model of the structure of manganese and cobalt species can be obtained from our data.