Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment -TYTEHA- of histone H4.

In order to reveal more information about the toxicity caused by metals and furthermore their influence to the physiological metabolism of the cell, the hexapeptide model Ac-ThrTyrThrGluHisAla-am representing the C-terminal 71-76 fragment of histone H4 which lies into the nucleosome core, was synthesized. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked hexapeptide, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5 and pH 7 for Cu(II) and Ni(II) ions respectively. In basic solutions a major square-planar 4 N Ni(II)-complex, adopting a {N(Im), 3N(-)} coordination mode, was formed. In the case of Cu(II) ions, a 3 N complex, involving the imidazole nitrogen of histidine and two deprotonated amide nitrogens of the backbone of the peptide, at pH 7 and a series of 4 N complexes starting at pH 6.5, were suggested. In addition Ni(II)-mediated hydrolysis of the peptide bond-Tyr-Thr was evident following our experimental data.

New perspectives on thiamine catalysis: from enzymic to biomimetic catalysis.

This paper is a brief review of the detailed mechanism of action of thiamine enzymes, based on metal complexes of bivalent transition and post-transition metals of model compounds, thiamine derivatives, synthesized and characterized with spectroscopic techniques and X-ray crystal structure determinations. It is proposed that the enzymatic reaction is initiated with a V conformation of thiamine pyrophosphate, imposed by the enzymic environment. Thiamine pyrophosphate is linked with the proteinic substrate through its pyrophosphate oxygens. In the course of the reaction, the formation of the "active aldehyde" intermediate imposes the S conformation to thiamine, while a bivalent metal ion may be linked through the N1' site of the molecule, at this stage. Finally, the immobilization of thiamine and derivatives on silica has a dramatic effect on the decarboxylation of pyruvic acid, reducing the time of its conversion to acetaldehyde from 330 minutes for the homogeneous system to less than 5 minutes in the heterogenous system.

Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment -ELAKHA- of histone H2B.

The coordination properties of the peptide Ac-GluLeuAlaLysHisAla-amide, the C-terminal 102-107 fragment of histone H2B towards Cu(II) and Ni(II) ions were studied by means of potentiometry and spectroscopic techniques (UV/Vis, CD, EPR and NMR). It was found that the peptide has a unique ability to bind Cu(II) ions at physiological pH values at a Cu(II): peptide molar ratio 1:2, which is really surprising for blocked hexapeptides containing one His residue above position 3. At physiological pH values the studied hexapeptide forms a CuL(2) complex {N(Im),2N(-)}, while in acidic and basic pH values the equimolar mode is preferred. In basic solutions Ac-GluLeuAlaLysHisAla-amide may bound through a {4N(-)} mode forming a square-planar complex, in which the imidazole ring is not any more coordinated or it has been removed in an axial position. On the contrary, Ni(II) ions form only equimolar complexes, starting from a distorted octahedral complex at about neutral pH values to a planar complex, where hexapeptide is bound through a {N(Im),3N(-)} mode in equatorial plane. The results may be of importance in order to reveal more information about the toxicity caused by metals and furthermore their influence to the physiologic metabolism of the cell.

Interaction of Cu(2+) with His-Val-His and of Zn(2+) with His-Val-Gly-Asp, two peptides surrounding metal ions in Cu,Zn-superoxide dismutase enzyme.

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). The interactions of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II) have been studied by using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. The binding modes of the species [CuAH](2+) and [CuA](+) were characterized by histamine type of coordination. [CuA](+) is further stabilized by the formation of a macrochelate with the involvement of the imidazole of the C-terminal histidine. The existence of macrochelate results in a slight distortion of the coordination geometry providing good base for the development of enzyme models. The enhanced stability of the macrochelate suppresses the formation of bis-complexes as well as the amide deprotonation. This process, however, takes place at higher pH resulting in the formation of the 4 N(-) coordinated [NH(2),N(-),N(-),N(im)] species [CuAH(2-)](-). On the other hand, in the case of the Zn(II)-His-Val-Gly-Asp system, coordination takes place at the terminal carboxylate in species [ZnBH(2)](2+). Monodentate binding occurs via the N-terminal imidazole in [ZnBH](+) while histamine type of coordination is possible in [ZnB], [ZnB(2)H](-) and [ZnB(2)](2-) species. Amide deprotonation does not take place in the case of Zn(2+), hydroxo-complexes are formed instead.

Stray Cu(II) may cause oxidative damage when coordinated to the -TESHHK- sequence derived from the C-terminal tail of histone H2A.

CH(3)CO-Thr-Glu-Ser-His-His-Lys-NH(2), a hexapeptide representing the 120-125 sequence of histone H2A, coordinates Cu(II) ions efficiently. Monomeric complexes are formed. In the major complex at physiological pH, CuH(-1)L, Cu(II) is coordinated equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, and axially through the imidazole nitrogen of the His-5 residue. This complex reacts with H(2)O(2) and the resulting reactive oxygen intermediate efficiently oxidizes 2'-deoxyguanosine. The underlying mechanism involves the formation of Cu(III) and a metal-bound hydroxyl radical species.

Complexation of Cu(2+) by HETPP and the pentapeptide Asp-Asp-Asn-Lys-Ile: a structural model of the active site of thiamin-dependent enzymes in solution.

To obtain structural information on the active site of thiamin-dependent enzymes in solution, we have studied the interactions of Cu(2+) ions with 2-(alpha-hydroxyethyl)thiamin pyrophosphate (HETPP), the pentapeptide Asp-Asp-Asn-Lys-Ile surrounding the thiamin pyrophosphate moiety in the transketolase enzyme, and the tertiary Cu(2+)-pentapeptide-HETPP system in aqueous solutions at various pH values. In the binary Cu(2+)-pentapeptide system around physiological pH, the bonding sites were the terminal NH2 group, the aspartate beta-carboxylates, and a deprotonated peptide nitrogen, while, in the Cu(2+)-HETPP system at the same pH, the Cu(II) was coordinated to the pyrophosphate group and to the pyrimidine N(1') atom. It is found that, in the tertiary system at physiological pH, the peptide bone offers three coordination sites to the metal ion, and the coordination sphere is completed by two additional phosphate oxygens and the nitrogen N(1') of the thiamin coenzyme. The stability constants in the tertiary system are higher than those in the simpler Cu(2+)-HETPP and Cu(2+)-peptide systems. The present data show that the coenzyme adopts the so-called S conformation in solution. The importance of our findings concerning the N(1') coordination and the S conformation in the tertiary system is discussed in conjunction with the role of HETPP as an intermediate of thiamin catalysis.

On the mechanism of action of thiamin enzymes in the presence of bivalent metal ions.

Results on the interactions between the bivalent metal ions Zn2+, Cd2+, Hg2+, Co2+, Ni2+ and 'active aldehyde' thiamin derivatives are reviewed. The techniques used in these studies include spectroscopic methods, i.e., IR-Raman, UV-Vis, multidimensional and multinuclear NMR in solution and in solid state, and X-ray crystal structure determinations. More recently, potentiometric studies on thiamin pyrophosphate and 2-(alpha-hydroxyethyl)thiamin in combination with NMR and EPR techniques were also undertaken. All these studies lead to useful conclusions on the mechanism of action of thiamin enzymes in the presence of bivalent metal ions.

Zinc(II) and cadmium(II) metal complexes of thiamine pyrophosphate and 2-(alpha-hydroxyethyl)thiamine pyrophosphate: models for activation of pyruvate decarboxylase.

Metal complexes of thiamine pyrophosphate (TPP) of the general formula [M2(TPPH)2Cl2]x4H2O (M = Zn2+, Cd2+) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(alpha-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed.