Coordination of copper(ii) ions by the fragments of neuropeptide gamma containing D1, H9, H12 residues and products of copper-catalyzed oxidation

Abstract

A potentiometric, spectroscopic (UV-Vis, CD and EPR) and mass spectrometric (ESI-MS) study of Cu(II) binding to the (1-2,7-21)NPG, Asp¹-Ala-Ile⁷-Ser-His⁹-Lys-Arg-His¹²-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met²¹-NH₂, and Ac-(1-2,7-21)NPG, Ac-Asp¹-Ala-Ile⁷-Ser-His⁹-Lys-Arg-His¹²-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met²¹-NH₂, fragments of neuropeptide gamma were carried out. The results clearly indicate the stabilization of the 1 N {NH₂, β-COO⁻}, 2 N {NH₂, β-COO⁻, N_Im} and 3 N {NH₂, β-COO⁻, 2N_Im} complexes by the coordination of the β-carboxylate group of the D¹ residue. For the (1-2,7-21)NPG the CuH₂L complex with 3 N {NH₂, β-COO⁻, 2N_Im}, the binding mode dominates in a wide pH range of 4–8.5. With the sequential increase of pH, deprotonated amide nitrogens are involved in copper coordination. For the Ac-(1-2,7-21)NPG peptide the imidazole nitrogen atoms are the primary metal binding sites forming macrochelates in the pH range 4 to 7. The CuHL complex with 4 N {N_Im, N⁻, N⁻, N_Im} coordination mode is formed in pH range 6–9. Deprotonation and co-ordination of the third amide nitrogen were detected at pH ∼8.6. Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. To elucidate the products of the copper(II)-catalyzed oxidation of the (1-2,7-21)NPG and Ac-(1-2,7-21)NPG, the liquid chromatography-mass spectrometry (LC-MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. In the presence of hydrogen peroxide with 1 : 4 peptide–H₂O₂ molar ratio for the Ac-(1-2,7-21)NPG peptide the oxidation of the methionine residue to methionine sulfoxide and for (1-2,7-21)NPG to sulfone was observed. For the Cu(II)–peptide–hydrogen peroxide in 1 : 1 : 4 molar ratio systems, oxidation of the histidine residues to 2-oxohistidines was detected. Under experimental conditions the (1-2,7-21)NPG and Ac-(1-2,7-21)NPG undergo fragmentations by cleavage of the S⁸-H⁹, H⁹-K¹⁰, R¹¹-H¹² and H¹²-K¹³ peptide bonds supporting the participation of the H⁹ and H¹² residues in the coordination of copper(II) ions. For the (1-2,7-21)NPG peptide chain the involvement of the D¹ residue in the coordination of metal ions is supported by the alkoxyl radical modification of this amino acid residue.