Correction for 'Stability of Cu(ii) complexes with FomA protein fragments containing two His residues in the peptide chain' by Monika Katarzyna Lesiów et al., Metallomics, 2019, 11, 1518-1531, DOI: 10.1039/C9MT00131J.

Monoand dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2N(im)}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {N-im, 3N(-)} coordination modes. The complexes: CuH-1L with 3N {2N(im), N-}, CuH-2L with 3N {N-im, 2N(-)} and CuH-3L with 4N {N-im, 3N(-)} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu(2)L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{N-im, 2N-}4N{N-im, 3N(-)} and 4N{N-im, 3N(-)}4N{N-im, 3N(-)} binding sites, respectively. In the case of the Cu(2)L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{N-im, N-}4N{N-im, 3N(-)} and (Cu(OH)(4)(2-)4N{N-im, 3N(-)}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.

The coordination capacity of the copper(II) ions with peptides (fragments of the P1 protein - one of the outer membrane protein from Fusobacterium nucleatum) based on the His-Xaa-His motif was carried out using potentiometric measurements, mass spectrometry and spectroscopic techniques: UV-Vis, CD and EPR. The selected tetrapeptides (Ac-HGHE-NH2, Ac-GHEH-NH2, Ac-HEHQ-NH2 and Ac-EHEH-NH2) form both mononuclear and bis-complexes with copper(II) ions. In the case of mononuclear complexes the CuL and CuLH-2 species dominate in the solution, where the coordination sphere is create by {2 × NIm} and {2 × NIm,2 × N-amide}, respectively. The Ac-HGHE-NH2 peptide form more stable the CuLH-2 complex with the 4 N{2 × NIm,2 × N-amide} binding site compared to the other ligands. The presence of glutamic acid residue in sequence Ac-HEHQ-NH2 produced the destabilization of the CuLH-2 complex in comparison to that of the Ac-HGHE-NH2 sequence. For the CuLH-3 complex the coordination process for complexes containing a histidyl residue in the first positions (H1) proceed towards C-terminal sequence of the peptide. The bis-complexes are formed in the solution, where the metal ion is bounded by four imidazole nitrogen atoms {4 × NIm}.

The coordination of Cu(ii) ions by the Ac-KGHGNGEEGTPTVHNE-NH2 (1L) peptide - a FomA protein fragment of Fusobacterium nucleatum - and its cyclic analogue: cyclo(KGHGNGEEGTPTVHNE) (2L) was studied by potentiometric titration, spectroscopic methods (UV-Vis, CD, EPR) and mass spectrometry (MS). Both the ligands contain two histydyl residues located in the third and fourteenth position of the peptide chain. For the 1L and 2L ligands mono- and dinuclear complexes were identified and studied in an aqueous solution. At the pH range characteristic of the intestinal environment (5.5-7.5), copper(ii) complexes were identified and their formation constants were determined. The same forms of the complexes with respectively the linear peptide and the cyclic peptide show similar stability, but greater than that reported in the literature for complexes with the same coordination mode. Moreover, the 1L peptide and its complex exhibit an alpha-helix structure, whereas the 2L peptide adopts this secondary structure only after coordination with the metal ion.

Copper(ii) complex formation processes between alloferon 1 (Allo1) (H-1 GVSGH(6) GQH(9) GVH(12)G) analogues where the phenylalanine residue is introduced in the place of His residue H6F and H12F have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For the phenylalanine analogues of alloferon 1, complex speciation has been obtained for a 1 : 1, 2 : 1 and 3 : 1 metal-to-ligand molar ratio. At physiological pH and in 1 : 1 metal-to-ligand molar ratio the phenylalanine analogues of alloferon 1 form a CuL complex similar to that of alanine analogues with the 4N{NH2,N1Im,2N(Im)} coordination mode. The stability of the complexes of the phenylalanine analogues is higher in comparison to those of alanine analogues, but lower in comparison to those containing tryptophan. Injection of Allo12F into insects induced prominent apoptotic changes in all hemocytes. The presence of apoptotic bodies only in the insect hemolymph testifies to the fact that Allo12F is an extremely pro-apoptotic peptide.

Neb-colloostatin was discovered during the isolation of Neb-TMOF from the ovary of the grey fleshfly Neobellieria bullata. Its amino acid sequence was determined as SIVP4LGLP8VP10IGP13IVVGP18R. The analogues with point mutation P4H and P8H were synthesized and their copper(II) complexes were studied by potentiometry, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation 1:1 and 2:1 metal-to-ligand molar ratios for both peptides were studied. At physiological pH (7.4), both peptides form mononuclear the CuH-1L complex with 3N{NH2, N−, NIm} coordination mode. For the P4H and P8H peptides at high pH (∼11) the binding sites of copper(II) ions are quite different. For the CuH-4L complex of the P4H peptide the 4N{4N−} coordination mode is dominante, while for the P8H peptide the 4N{NIm, 3N−} binding site towards N-termini is formed with (6,5,5) chelate ring. At high pH the P8H peptide cannot form the 4N {NH2, 3N−} complex because of the presence of the Pro4 residue in the amino acid sequence (a break point in the metal coordination). At pH 7.4 dinuclear the Cu2H-4L complexes dominate with suggested the 3N{NH2, 2N−}3N{NIm, 2N−}binding sites where the 3N{NIm, 2N−} coordination mode for the P4H peptide towards C-termini, and for P8H peptide towards N-termini are formed. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The systems studied have lower apoptotic properties compared to those of Neb-colloostatin and its copper(II) complex at pH 7.4.

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV–visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH−1L and/or CuH−2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH−1L, CuH−2L and CuH−3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

The nineteenth amino acid peptide Neb-colloostatin SIVPLGLPVPIGPIVVGPR and its analogue with point mutation (P4A) were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. The amine-N nitrogen atoms of serine residues were found to be the primary metal binding sites of both peptides. These binding modes provide the deprotonation and coordination of two amide nitrogens of the Neb-colloostatin and the formation of the 3N {NH2,2N−,CO} complex at pH 7.5–8. The proline residue P4 is the breakpoint in the coordination of copper(II) ions and the 4N {NH2,3N−} complex was not observed. At pH 8 the analogue (P4A) of Neb-colloostatin forms the copper(II) complex with 4N {NH2,3N−} binding site. The potentiometric and spectroscopic results did not reveal the stabilizing role of the oxygen donors of side chains of seryl residues (S1) in copper(II) complexes. At pH 8 the copper(II) ions change the secondary structures of both peptides in comparison to those without copper(II) ions. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The copper(II) complexes of Neb-colloostatin and its analogue exert stronger pro-apoptotic activity on insect hemocytes in comparison to those of peptides without metal ions.

Mononuclear and polynuclear copper(II) complexes of alloferon 1 with point mutations, H1A/H12A H2N-A(1)GVSGH(6)GQH(9)GVA(12)G-COOH, H1A/H9A H2N-A(1)GVSGH(6)GQA(9)GVH(12)G-COOH, and H1A/H6A H2N-A(1)GVSGA(6)GQH(9)GVH(12)G-COOH, have been studied by potentiometric, UV-visible, CD, and EPR spectroscopy, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand molar ratios ranging from 1 : 1 to 3 : 1 was obtained. Over a wide 6-8 pH range, including physiological pH 7.4, and a 1 : 1 metal-to-ligand molar ratio, the peptides studied formed a CuH-1L complex with the 4N{NH2,N(-),2NIm} coordination mode. The presence of the 4N binding site for the CuH-1L complexes prevented the deprotonation and coordination of the second amide nitrogen atom to copper(II) ions (pK-1/-2 7.83-8.07) compared to that of pentaGly (6.81). The amine nitrogen donor and two imidazole nitrogen atoms (H(6)H(9), H(6)H(12) and H(9)H(12)) can be considered to be independent metal-binding sites in the species formed. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2 : 1 and 3 : 1 molar ratios dominate in the solution, respectively. For the Cu(II)-H1A/H9A and Cu(II)-H1A/H12A systems, the Cu3H-9L complexes are likely formed by the coordination of amide nitrogen atoms towards C-termini with ring sizes (7,5,5).

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A1GVSGH6GQH9GVH12G (Allo1A) and (H9A) H1GVSGH6GQA9GVH12G (Allo9A) have been studied by potentiometric, UV–visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H6,H12) leads to the formation of the CuL complex with 4N {NH2,NIm–H1,NIm–H6,NIm–H12} binding site in wide pH range (5–8). For the Cu(II)–Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH−1L consists of the 3N {NH2,N−,CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)–Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His1 and His6 residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

To obtain the information about the influence of the serine residues (S8,S16) on the acid–base properties of the neuropeptide gamma, the peptide with point mutations (S8,16A) and its N-acetyl derivative were synthesized. Any additional deprotonations were not observed. It means that the presence of serine residues is necessary in the amino acid sequence of the neuropeptide gamma to have its acid–base properties. The stability constants, stoichiometry and solution structures of copper(II) complexes of the neuropeptide gamma mutants D1AGH4GQIA8H9KRH12KTDA16FVGLM21-NH2 (S8,16A) 2ANPG and its N-acetyl derivative Ac-2ANPG were determined in aqueous solution. The equilibrium and structural properties of copper(II) complexes have been characterized by pH-metric, spectroscopic (UV–visible, CD, EPR) and mass spectrometric (MS) methods. At physiological pH7.4 the 2ANPG forms the CuH2L and CuHL complexes in equilibrium with 3N {NH2,βCOO−-D1,2NIm} and 4N {NH2,N−,2NIm} binding sites, respectively. The exchange Ser on Ala residues does not alter the coordination mode of the peptide. To elucidate the products of the copper(II)-catalyzed oxidation of 2ANPG and Ac-2ANPG 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 sulphoxide was observed. For the 1:1:4 Cu(II)–2ANPG–H2O2 system oxidation of two His residues and cleavage of the G3H4 peptide bond was observed, while for the 1:1:4 Cu(II)–Ac-2ANPG–H2O2 system oxidation of three histidine residues to 2-oxohistidines was also observed.

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

The point mutation in the sequence of neuropeptide gamma (H4A, H4ANPG, Asp1-Ala-Gly-Ala4-Gly-Gln-Ile-Ser-His9-Lys-Arg-His12-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met21-NH2) was introduced to observe the influence of histidine H4 residue on the acid–base properties of the peptide and coordination abilities to copper(II) ions. The potentiometric studies of neuropeptide gamma were only performed in pH 2.5–7.4 pH range. At higher pH than 7.4 additional deprotonations of the neuropeptide gamma were observed. The potentiometric measurements for both peptides (H4ANPG and Ac-H4ANPG, Ac-Asp1-Ala-Gly-Ala4-Gly-Gln-Ile-Ser-His9-Lys-Arg-His12-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met21-NH2) without copper(II) ions were performed in whole 2.5–10.5 pH range. It means that H4 residue in neuropeptide gamma has influence on its acid–base properties and it’s most likely that this residue takes place in hydrogen bond formation. The acetylation of the N-terminal amino group changes the acid–base properties of the H4ANPG. The potentiometric study for the Ac-H4ANPG peptide suggests in the 5.7–7 pH range four additional deprotonations. It may support the tend to amyloid-like fibril formation by tachykinin neuropeptides and as it is suggested the presence of the hydrogen bonds (labile protons) in tiny fibrils. The potentiometric, spectroscopic (UV–Vis, CD and EPR) and mass spectrometric (ESI-MS) results for the Cu(II)–H4ANPG system clearly indicate the stabilization of the 2N {NH2,β-COO−–Asp1,NIm–H9,OH–S8} complex by the coordination of the amine and β-carboxylate groups of the D1 residue and most likely by the OH and imidazole nitrogen of the Ser8 and His9 residues, respectively. The additional deprotonations of the Ac-H4ANPG do not have any influence on its coordination abilities in comparison to those containing two histidine residues in the peptide chain. To elucidate the products of the copper(II)-catalyzed oxidation of the H4ANPG and Ac-H4ANPG, the liquid chromatography–mass spectrometry (LC–MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. For both peptides in the Cu(II)–peptide–hydrogen peroxide in 1:1:4M ratio systems, oxidation of two histidine (H9,H12) residues to 2-oxohistidines was observed. Under experimental conditions the metal-catalyzed oxidation both peptides undergo fragmentations by cleavage of the S8–H9, H9–K10, H12–K13 peptide bonds supporting the participation of the H9 and H12 residues in the coordination of copper(II) ions.

Mononuclear and polynuclear complexes of the (1-4,18-36)NPK, Asp(1)-Ala-Asp-Ser(4)-Gly(18)-His(19)-Gly-Gln-Ile-Ser-His(24)-Lys-Arg-His(27)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(36)-NH(2), and mononuclear complexes of its acethyl derivative Ac-Asp(1)-Ala-Asp-Ser(4)-Gly(18)-His(19)-Gly-Gln-Ile-Ser-His(24)-Lys-Arg-His(27)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(36)-NH(2) have been studied by potentiometric, UV-vis, CD, EPR spectroscopic, and mass spectrometry (MS) methods. As it was observed for other tachykinins (neurokinin A, neuropeptide gamma and its fragments) containing the same C-terminal sequence His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2), also for the fragments of neuropeptide K the additional deprotonation most likely on the serine OH group was observed. It is likely that tachykinin peptides contain catalytic Ser/His/Asp triad or dyads Ser/Lys and the serine protease activity. The high water solubility of the resulting metal complexes allowed us to obtain complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1 for (1-4,18-36)NPK, while only the 1:1 molar ratio was studied for Cu(II)-Ac-(1-4,18-36)NPK because of precipitation. For the metal-to-ligand 1:1 molar ratio the (1-4,18-36)NPK forms in a wide 6.5-10.5 pH range the CuHL complex with a 3N {NH(2),2N(-),β-COO(-)-Asp(3)} binding site. For a metal-to-ligand 1:1 molar ratio at higher pH than 9.5 the dimeric species dominate. For the Ac-(1-4,18-36)NPK peptide the imidazole nitrogen atoms are the primary metal-binding sites forming macrochelates in the pH 4-7.5.

A potentiometric, spectroscopic (UV-visible, CD and EPR) and electrospray ionization mass spectrometric (ESI-MS) study of Cu(II) binding to the neurokinin A with point mutation (S5A) (ANKA), His-Lys-Thr-Asp-Ala(5)-Phe-Val-Gly-Leu-Met-NH2 and its N-acethyl derivative (Ac-ANKA), Ac-His-Lys-Thr-Asp-Ala(5)-Phe-Val-Gly-Leu-Met-NH2 were carried out. For the ANKA and Ac-ANKA the additional deprotonation was not observed. It suggests that for the tachykinin peptides with C-terminal sequence of neurokinin A for the additional deprotonation the presence of the serine residue is necessary. For the Cu(II)-ANKA 1:2 system at physiological pH 7.4 the CuH2L2 species is present with histamine-like 4N, 2×{NH2,NIm} coordination mode. With increasing pH the deprotonation and coordination of amide nitrogen atoms occur and the CuH-2L, CuH-3L complexes are formed. In pH range 4.5 - 9.5 the dimeric Cu2HL2, Cu2L2 and Cu2H-1L2 species in solution are also present. To elucidate the products of the copper(II)- catalyzed oxidation of the ANKA and Ac-ANKA, 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 1:1 peptide-H2O2 molar ratio for both peptides the oxidation of the methionine residue to methionine sulfoxide was observed. For the Cu(II)-peptide-hydrogen peroxide in 1:2:2 molar ratio systems oxidations of the histidine residues to 2-oxo-histidines and methionine sulfoxide to methionine sulfone were detected.

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)-NH2, 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)-NH2, fragments of neuropeptide gamma were carried out. The results clearly indicate the stabilization of the 1 N {NH2, beta-COO-}, 2 N {NH2, beta-COO-, N-Im} and 3 N {NH2, beta-COO-, 2N(Im)} complexes by the coordination of the beta-carboxylate group of the D-1 residue. For the (1-2,7-21)NPG the CuH2L complex with 3 N {NH2, beta-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 similar to 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 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.

Mononuclear and polynuclear complexes of the alloferon I with point mutation (HI K) Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (AlloK) and its acetylated derivative Ac-Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9-)Gly-Val-His(12)-Gly (Ac-AlloK) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1 for AlloK while to 3:1 for Ac-AlloK At physiological pH 7.4 and the metal-to-ligand 1:1 molar ratio the AlloK peptide forms the CuL complex with the 4N (NH2, N-, 2N(1m)) binding mode. In the Cu(II)-AlloK 4:1 system in wide pH 6.5-10 range the Cu(4)H(-)7L complex dominates with the 3N (NH2,2 N-} 3 x (N-1m,2 N-) coordination mode. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites of Ac-AlloK. For Ac-AlloK and 1:1 metal-to-ligand molar ratio the CuHL complex with the 3N {3N(1m)} binding sites in pH 4.5-7.5 range is present in solution. The amine nitrogen donor and all of the histidine residues can be considered to be independent metal-binding sites in the species formed in the systems studied. As a consequence, tri- (for the Ac-AlloK) and tetra-nuclear (for the AlloK peptide) complexes for the metal-to-ligand 3:1 and 4:1 molar ratios, respectively, are present in the solution. (C) 2012 Elsevier Inc. All rights reserved.

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)-NH(2)) 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)-NH(2)) 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 {NH(2),β-COO(-)}, 2N {NH(2),β-COO(-),N(Im)}, and 3N {NH(2),β-COO(-),2N(Im)} coordination modes as the result of coordination through the β-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)/H(2)O(2) 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-H(2)O(2) 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.