title: 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, Asp(1)-Ala-Ile(7)-Ser-His(9)-Lys-Arg-His(12)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(21)-NH(2), and Ac-(1-2,7-21)NPG, Ac-Asp(1)-Ala-Ile(7)-Ser-His(9)-Lys-Arg-His(12)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(21)-NH(2), fragments of neuropeptide gamma were carried out. The results clearly indicate the stabilization of the 1 N {NH(2), β-COO(-)}, 2 N {NH(2), β-COO(-), N(Im)} and 3 N {NH(2), β-COO(-), 2N(Im)} complexes by the coordination of the β-carboxylate group of the D(1) residue. For the (1-2,7-21)NPG the CuH(2)L complex with 3 N {NH(2), 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-H(2)O(2) 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 S(8)-H(9), H(9)-K(10), R(11)-H(12) and H(12)-K(13) peptide bonds supporting the participation of the H(9) and H(12) residues in the coordination of copper(II) ions. For the (1-2,7-21)NPG peptide chain the involvement of the D(1) residue in the coordination of metal ions is supported by the alkoxyl radical modification of this amino acid residue.
authors.affiliation: Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952, Gdansk, Poland.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Jankowska
authors.forename: Marta
authors.initials: M
authors.lastname: Pietruszka
authors.forename: Teresa
authors.initials: T
authors.lastname: Kowalik-Jankowska
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D056831:Coordination Complexes / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry*; D011199:Potentiometry; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization; D013057:Spectrum Analysis; D015320:Tachykinins / Q000737:chemistry*
chemical_list: D002413:Cations, Divalent; D056831:Coordination Complexes; D010446:Peptide Fragments; D011522:Protons; D015320:Tachykinins; C059905:tachykinin neuropeptide gamma; D003300:Copper