Attila Jancsó

Heterodinuclear Zinc(II)−Iron(III) Complexes and Dinuclear Zinc Complexes as Models for Zinc-Containing Phosphatases

Five new dinuclear model complexes for zinc-containing phosphatases with dinucleating ligands have been prepared and characterized by single-crystal X-ray crystallography. The heterodinuclear, μ-alkoxo-bridged zinc(II)−iron(III) complexes 1−3 contain the symmetric ligands N,N,N′,N′-tetrakis[2-(5,6-dimethyl)benzimidazolylmethyl]-1,3-diamino-2propanol (Htdmbpo) and N,N,N′,N′-tetrakis{2-[N′′-(2hydroxyethyl)]benzimidazolylmethyl}-1,3-diamino-2-propanol (Hthebpo), and the asymmetric ligand N,N-bis[2-(4,5-dimethyl)benzimidazolylmethyl]-N′,N′-bis(2-pyridylmethyl)-1,3-diamino-2-propanol (Hbdmbbppo), respectively. X-ray crystallography revealed that the zinc center exhibits a trigonal-bipyramidal coordination, while the octahedral coordination sphere of the iron center is completed by a solvent molecule. In contrast, the zinc complexes 4 and 5, which also have (alkoxo)(cacodylato)dimetal cores with the dinucleating ligands used in 1 and 3, exhibit both metal centers in a trigonal-bipyramidal environment. Additionally, the solution speciation of the zinc(II) complexes formed with Htdmbpo and Hbdmbbppo were determined and the activity of the in situ prepared zinc complexes towards the transesterification of the RNA model substrate 2-(hydroxypropyl)-4-nitrophenyl phosphate (hpnp) was investigated. The dinuclear [Zn2LH−1(OH)]2+ complex of both ligands efficiently promotes the transesterification. The kinetic data indicated a higher activity for the complex of the asymmetric ligand Hbdmbbppo, as a result of its stronger substrate binding ability. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase.

The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H(2)N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine epsilon-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K(D,Zn)=1.6x10(-9)M and K(D,Cu)=5.0x10(-12)M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K(D) values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.

N-terminal fragment of the anti-angiogenic human endostatin binds copper(II) with very high affinity

A histidine-rich peptide HSHRDFQPVLHL-NH(2) (L), identical with the N-terminal fragment of the anti-angiogenic human endostatin has been synthesized. Endostatin is a recently identified broad spectrum angiogenesis inhibitor, which inhibits 65 different tumor types. The N-terminal 25-mer peptide fragment of human endostatin has the same antitumor effect as the entire protein. The zinc(II) binding is crucial for the antitumor effect in both cases. Our peptide may provide all critical interactions for zinc(II) binding present in the N-terminal 25-mer peptide fragment. In addition, the N-terminus of human endostatin has a supposedly high affinity binding site for copper(II), similar to human serum albumin. Since copper(II) is intimately involved in angiogenesis, this may have biological relevance. In order to determine the metal binding properties of the N-terminal fragment of endostatin, we performed equilibrium, UV-visible (UV-vis), CD, EPR and NMR studies on the zinc(II) and copper(II) complexes of L. In the presence of zinc(II) the formation of a stable [NH(2),3N(im),COO(-)] coordinated complex was detected in the neutral pH-range. This coordination mode is probably identical to that present in the zinc(II) complex of the above mentioned N-terminal 25-mer peptide fragment of human endostatin. Moreover, L has extremely high copper(II) binding affinity, close to those of copper-containing metalloenzymes, and forms albumin-like [NH(2),N(-),N(-),N(im)] coordinated copper(II) complex in the neutral pH-range, which may suggest that copper(II) binding is involved in the biological activity of endostatin.

Copper(II), nickel(II) and zinc(II) complexes of N-acetyl-His-Pro-His-His-NH2: equilibria, solution structure and enzyme mimicking.

The copper(II), nickel(II) and zinc(II) binding ability of the multi-histidine peptide N-acetyl-His-Pro-His-His-NH(2) has been studied by combined pH-potentiometry and visible, CD and EPR spectroscopies. The internal proline residue, preventing the metal ion induced successive amide deprotonations, resulted in the shift of this process toward higher pH values as compared to other peptides. The metal ions in the parent [ML](2+) complexes are exclusively bound by the three imidazole side chains. In [CuH(-1)L](+), formed between pH 6-8, the side chains of the two adjacent histidines and the peptide nitrogen between them are involved in metal ion binding. The next deprotonation results in the proton loss of the coordinated water molecule (CuH(-1)L(OH)). The latter two species exert polyfunctional catalytic activity, since they possess superoxide dismutase-, catecholase- (the oxidation of 3,5-di-tert-butylcatechol) and phosphatase-like (transesterification of the activated phosphoester 2-hydroxypropyl-4-nitrophenyl phosphate) properties. On further increase of the pH rearrangement of the coordination sphere takes place leading to the [CuH(-3)L](-) species, the deprotonated amide nitrogen displaces a coordinated imidazole nitrogen from the equatorial position of the metal ion. The shapes of the visible and CD spectra reflect a distorted arrangement of the donor atoms around the metal ion. In presence of zinc(II) the species [ZnL](2+) forms only above pH 6, which is shortly followed by precipitation. On the other hand, the [NiL](2+) complex is stable over a wide pH range, its deprotonation takes place only above pH 8. At pH 10 an octahedral NiH(-2)L species is present at first, which transforms slowly to a yellow square planar complex.

Probing the Cu2+ and Zn2+ binding affinity of histidine-rich glycoprotein

The zinc(II) and copper(II) binding ability of two oligopeptide fragments, Ac-HHPHG-NH(2) and Ac-HHPHGHHPHG-NH(2), derived from the repeat-region of the His-Pro-rich domain of histidine-rich glycoprotein (HRG) and the structure of the formed complexes have been investigated by potentiometry, NMR-, UV-visible-, CD-, SRCD- and EPR spectroscopy. Exclusive coordination of the side-chain imidazoles of the peptides has been observed with both metal ions in the acidic and neutral pH range. While the three His units of the pentapeptide resulted in a modest stability of the ML complexes, the decapeptide with its increased number of His residues offered a high-affinity metal binding site for both metal ions with the participation of at least four nitrogen donors. Due to the high number of potential donor groups, the formation of binding isomers of the protonated and parent complexes is very likely. Both peptides show a synchrotron radiation (SR) CD-pattern resembling to that of the polyproline II structure, similarly to that of the His-Pro-rich domain of the HRG protein. The longer sequence was shown to bind a second metal ion in the slightly acidic pH-range. The determined stability data suggest a remarkable extra stabilization emerging in the decapeptide for the coordination of the second metal ions, as compared to the ML complexes of the pentapeptide. Whether the observed cooperativity has similarities to the cooperative metal binding feature of HRG or the two phenomena have different sources is a question yet to be clarified.

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH(2), L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH(2)L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K(D,Cu)=7.4×10(-13)M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH(2)L is coordinated by four nitrogens {NH(2),3N(im)} in the equatorial plane. In ZnH(2)L the peptide binds to zinc(II) through a {NH(2),2N(im),COO(-)} donor set, and its zinc binding affinity is relatively modest (K(D,Zn)=4.8×10(-7)M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH(2)L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH(2)L complexes has been verified by semiempirical quantum chemical calculations (PM6), too.

Approaching the minimal metal ion binding peptide for structural and functional metalloenzyme mimicking

The peptides Ac-His-Pro-His-Pro-His-NH(2) (L1) and Ac-Lys-His-Pro-His-Pro-His-Gln-NH(2) (L2) have been prepared and the equilibria of their proton, copper(II) and zinc(II) complexes in aqueous solution have been studied by the combination of pH-potentiometric titrations, UV/visible and circular dichroism (CD) spectroscopy. The latter methods also provided information on solution structure of the complexes formed under different conditions. Both ligands formed complexes with three imidazole nitrogens around the metal ion at pH ~7. In the L1 containing system precipitation of either copper(II) or zinc(II) complexes occurred upon slight increase of the pH. The re-titration of the filtered and acidified precipitates revealed that the insoluble materials were neutral complexes rather than metal-hydroxides. Indeed, by attaching amino acids with polar side-chains to the His-Pro-His-Pro-His template in L2 we could prevent any precipitation, and the soluble complexes around pH ~7 exerted three imidazole nitrogens and a (deprotonated) water molecule around the metal ions. To our knowledge L2 provides the first example of a short peptide preventing both the amide nitrogen coordination in copper(II) and the formation of copper(II) and zinc(II) hydroxides. The zinc(II) and copper(II) complexes at pH ~7 having similar structure to the natural hydrolytic and redox enzymes, respectively, showed considerable activity in hydrolytic cleavage assays with a model substrate (2-hydroxypropyl-4-nitrophenyl phosphate), as well as with native plasmid DNA, and in a superoxide dismutase-like reaction.

Potentiometric and spectroscopic evidence for co-ordination of dimethyltin(IV) to phosphate groups of DNA fragments and related ligands

The co-ordination of dimethyltin(IV) to 5′-GMP, 5′-ATP and 5′-d(CGCGCG)2 and to their sugar constituents (D-ribose and 2-deoxy-D-ribose) was investigated in aqueous solution by means of potentiometric titration and 1H and 31P NMR spectroscopic methods. The results showed that in acidic media the phosphate groups can provide suitable sites for metal ion co-ordination, while the hydroxy groups of the sugars or the sugar moieties of the two nucleotides play a role in this process at higher pH. The base moieties of 5′-GMP and 5′-ATP were not co-ordinated to dimethyltin(IV). The stability constants of the complexes formed in the above systems were determined by pH-metric titration. The data revealed a stronger co-ordination ability of the triphosphate as compared with that of the monophosphate. The comparison of the stability constants of the D-ribose and 2-deoxy-D-ribose complexes showed that more stable species were formed when neighbouring alcoholic hydroxy groups were available for co-ordination. The observed chemical shift changes of the 31P NMR resonances, as compared with those measured for the metal-free systems, demonstrated that the phosphate groups of the DNA fragment 5′-d(CGCGCG)2 chains act as binding sites for dimethyltin(IV) between pH 4.5 and 7. The 1- and 2-D 1H NMR spectra indicated that the base and sugar moieties do not participate in the co-ordination process under these conditions.

Zn2+ Complexes of Di‐ and Tri‐nucleating Azacrown Ligands as Base‐Moiety‐Selective Cleaving Agents of RNA 3′,5′‐Phosphodiester Bonds: Binding to Guanine Base

The ability of the dinuclear Zn2+ complex of 1,4‐bis[(1,5,9‐triazacyclododecan‐3‐yloxy)methyl]benzene (L1) to promote the cleavage of the phosphodiester bond of dinucleoside‐3′,5′‐monophosphates that contain a guanine base has been studied over a narrow pH range from pH 5.8 to 7.2 at 90 °C. Comparative measurements have been carried out by using the trinuclear Zn2+ complex of 1,3,5‐tris[(1,5,9‐triazacyclododecan‐3‐yloxy)methyl]benzene (L2) as a cleaving agent and guanylyl‐3′,5′‐guanosine (5′‐GpG‐3′) as a substrate. The strength of the interaction between the cleaving agent and the starting material has been elucidated by UV spectrophotometric titrations. The speciation and binding mode have been clarified by potentiometric titrations with hydrolytically stable 2′‐O‐methylguanylyl‐3′,5′‐guanosine and 1H NMR spectroscopic measurements with guanylyl‐3′,5′‐guanosine. The results show that the guanine base is able to serve as a site for anchoring for the Zn2+–azacrown moieties of the cleaving agents L1 and L2, analogously to uracil base. The interaction is, however, weaker than with the uracil base and, hence, only the 5′‐GpG‐3′ site (in addition to 5′‐GpU‐3′ and 5′‐UpG‐3′ sites) is able to markedly modulate the phosphodiester cleavage by the Zn2+ complexes of di‐ and trinucleating azacrown ligands containing an ether oxygen as a potential H‐bond‐acceptor site.

The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative

Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu(2)H(-2)L(2) species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH(-2)L and, above pH~10, CuH(-3)L. Experimental data supports a {NH(2), 2 × N(amide),N(im)} macrochelate structure in CuH(-2)L whereas a {NH(2), 3 × N(amide)} coordination environment in CuH(-3)L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH(-2)L and above pH~9, NiH(-3)L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N(amide),N(im)} donor set, and in parallel the re-dissolving of precipitate. Above pH~11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands.