Complexation Abilities of Neuropeptide Gamma toward Copper(II) Ions and Products of Metal-Catalyzed Oxidation

The stability constants, stoichiometry, and solution structures of copper(II) complexes of neuropeptide gamma (NPG) (D-1-A-G-H-4-G-Q-I-S-H-9-K-R-H-12-K-T-D-S-F-V-G-L-M-21-NH2) and acethyl-neuropeptide gamma (Ac-D-1-A-G-H-4-G-Q-I-S-H-9-K-R-H-12-K-T-D-S-F-V-G-L-M-21-NH2) were determined in aqueous solution. For both peptides the additional deprotonations were observed; therefore, the potentiometric data calculations for NPG were only made in 2.5-7.4 pH range. For Ac-NPG one additional deprotonation was observed, likely hydroxy group of Ser residue, and the potentiometric data calculations in the 2.5-10.5 pH range may be performed. The potentiometric and spectroscopic data (UV-vis, CD, EPR) for the neuropeptide gamma show that a D-1 residue stabilizes significantly the copper(II) complexes with 1N {NH2,beta-COO-}, 2N {NH2,beta-COO-,N-lm}, and 3N {NH2,beta-COO-,2N(lm)} coordination modes as the result of coordination through the beta-carboxylate group. The Ac-NPG forms with the copper(II) ions the 3N {3N(Im)} complex in a wide 4.5-7.5 pH range. At higher pH deprotonation and coordination of the sequential amide nitrogens occur. Metal-catalyzed oxidation of proteins is mainly a site-specific process in which amino acids at metal-binding sites to the protein are preferentially oxidized. To elucidate the products of the copper(II)-catalyzed oxidation of NPG and Ac-NPG the liquid chromatography-mass spectrometry method (LC-MS) and the Cu(II)/H2O2 as a model oxidizing system were employed. For solutions containing a 1:4 peptide-hydrogen peroxide molar ratio oxidation of the methionine residue to methionine sulphone was observed. For the 1:1:4 Cu(II)-NPG-H2O2 system oxidation of two His residues and cleavage of the G(3)-H-4 and R-11-H-12 peptide bonds were detected, supporting involvement of His(4) and His(12) in binding of the copper(II) ions. Oxidations of three histidine residues to 2-oxohistidines and fragmentations of Ac-NPG near the His (H-4, H-9,H-12) residues support participation of the histidyl-imidazole nitrogen atoms in coordination of the metal ions.