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Dancs A,University of Szeged | Dancs A,CNRS Structure and Reactivity of Complex Molecular Systems | May N.V.,Research Center for Natural science | Selmeczi K.,CNRS Structure and Reactivity of Complex Molecular Systems | And 5 more authors.
New Journal of Chemistry | Year: 2017

Two new tripodal peptides containing non-protected N-terminal (L1, tren3his) and C-terminal (L2, nta3his) histidines have been synthesized in order to combine the structuring effect of tripodal scaffolds and the strong metal binding properties of histidine moieties. In the present work the copper(ii) complexes of these ligands have been studied by combined pH-metric, UV-Vis, CD, EPR and MS methods. At a 1:1 metal-to-ligand ratio the two ligands behave as the corresponding dipeptides containing N/C-terminal histidines, but above pH 9 the participation of the tertiary amine in the fused chelate rings results in unique binding modes in the case of both ligands. Besides, the formation of oligonuclear complexes also confirms the positive influence of tripodal platforms on metal coordination, and provides the potential to be efficient functional models of oxidase enzymes. Accordingly, the oligonuclear complexes of both ligands exhibit considerable catecholase-like activity. The oxidation of 3,5-di-tert-butyl-catechol proceeds with the participation of separated Cu2+ centers in the presence of L1 complexes. However, the proximity of the two metal ions in the dinuclear complexes of L2 allows their cooperation along the catalytic cycle. Substrate binding modes, effects of reactants, intermediate and side product formation have also been studied, allowing us to propose a plausible catalytic mechanism for each copper(ii)-ligand system. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.


Szorcsik A.,MTA SZTE Bioinorganic Chemistry Research Group | Matyuska F.,University of Szeged | Benyei A.,Debrecen University | Nagy N.V.,Institute of Organic Chemistry | And 4 more authors.
Dalton Transactions | Year: 2016

Copper(ii) complexes of a polydentate tripodal ligand L × 3HCl (L = N,N′,N″-tris(5-pyrazolylmethyl)-cis,cis-1,3,5-triaminocyclohexane) were characterized in both solution and solid states. Combined evaluation of potentiometric, UV-VIS, and EPR data indicated the formation of two mononuclear (CuHL, CuL) and three trinuclear (Cu3H-xL2, x = 2, 3, 4) complexes. The high stability and spectroscopic properties of the CuL species indicate a coordination of two pyrazole rings in addition to the three secondary amino groups of L in a square pyramidal geometry. In parallel with the formation of trinuclear species, intense charge transfer bands appear at around 400-500 nm, which indicate the formation of pyrazolate-bridged complexes. The crystal structure of [Cu3H-4L2](ClO4)2·5H2O (1) reveals the formation of a unique trinuclear complex that features a tetra(pyrazolate)-bridged linear tricopper(ii) core. The Cu⋯Cu interatomic distances are around 3.8 Å. The two peripheral copper(ii) ions have a slightly distorted square pyramidal geometry. The four pyrazole rings bound to the peripheral copper(ii) ions are deprotonated and create a flattened tetrahedral environment for the central copper(ii), i.e. the formation of the trinuclear complexes is under the allosteric control of the two peripheral copper(ii) ions. The triply deprotonated trinuclear complex is an efficient catechol oxidase mimic with a surprisingly low pH optimum at pH = 5.6. Since the mononuclear CuL species is not able to promote the oxidation of 3,5-di-tert-butylcatechol, we assume that the central copper(ii) ion of the trinuclear complex with an unsaturated coordination sphere has a fundamental role in the binding and oxidation of the substrate. The experimental and structural details were further elaborated by a series of hybrid density functional theory calculations that support the presence of an antiferromagnetically coupled ground state. However, the magnitude and the pattern of spin coupling are dependent on the composition of the functionals. The optimized theoretical structures highlight the role of the crystal packing effects in inducing asymmetry between the two peripheral copper(ii) sites. © 2016 The Royal Society of Chemistry.


Nemeth E.,MTA SZTE Bioinorganic Chemistry Research Group | Kozisek M.,Gilead Sciences | Schilli G.K.,University of Szeged | Gyurcsik B.,MTA SZTE Bioinorganic Chemistry Research Group | Gyurcsik B.,University of Szeged
Journal of Inorganic Biochemistry | Year: 2015

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn2 +-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn2 +-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn2 +-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site - existing in the wild-type enzyme but not in the W464 mutants - was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization. © 2015 Elsevier Inc.All rights reserved.


PubMed | Gilead Sciences, University of Szeged and MTA SZTE Bioinorganic Chemistry Research Group
Type: | Journal: Journal of inorganic biochemistry | Year: 2015

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn(2+)-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn(2+)-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn(2+)-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site--existing in the wild-type enzyme but not in the W464 mutants--was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.


Szunyogh D.,MTA SZTE Bioinorganic Chemistry Research Group | Szokolai H.,University of Szeged | Thulstrup P.W.,Copenhagen University | Larsen F.H.,Copenhagen University | And 6 more authors.
Angewandte Chemie - International Edition | Year: 2015

Metal-ion-responsive transcriptional regulators within the MerR family effectively discriminate between mono- and divalent metal ions. Herein we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study AgI, ZnII, and HgII binding to model systems encompassing the metal-ion-binding loop of CueR from E. coli and V. cholerae. In the presence of AgI, a conserved cysteine residue displays a pKa value for deprotonation of the thiol that is close to the physiological pH value. This property is only observed with the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys 112 protonated demonstrate that the conserved Ser 77 backbone carbonyl oxygen atom from the other monomer of the homodimer is "pulled" towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR, the mechanism relies on the protonation of Cys 112. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


Czene A.,MTA SZTE Bioinorganic Chemistry Research Group | Toth E.,University of Szeged | Gyurcsik B.,MTA SZTE Bioinorganic Chemistry Research Group | Gyurcsik B.,University of Szeged | And 6 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2013

The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444-576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013), J. Biol. Inorg. Chem. 18, 309-321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C*of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress. © 2013.


PubMed | MTA SZTE Bioinorganic Chemistry Research Group
Type: Journal Article | Journal: Dalton transactions (Cambridge, England : 2003) | Year: 2016

Copper(ii) complexes of a polydentate tripodal ligand L 3HCl (L = N,N,N-tris(5-pyrazolylmethyl)-cis,cis-1,3,5-triaminocyclohexane) were characterized in both solution and solid states. Combined evaluation of potentiometric, UV-VIS, and EPR data indicated the formation of two mononuclear (CuHL, CuL) and three trinuclear (Cu3H-xL2, x = 2, 3, 4) complexes. The high stability and spectroscopic properties of the CuL species indicate a coordination of two pyrazole rings in addition to the three secondary amino groups of L in a square pyramidal geometry. In parallel with the formation of trinuclear species, intense charge transfer bands appear at around 400-500 nm, which indicate the formation of pyrazolate-bridged complexes. The crystal structure of [Cu3H-4L2](ClO4)25H2O (1) reveals the formation of a unique trinuclear complex that features a tetra(pyrazolate)-bridged linear tricopper(ii) core. The CuCu interatomic distances are around 3.8 . The two peripheral copper(ii) ions have a slightly distorted square pyramidal geometry. The four pyrazole rings bound to the peripheral copper(ii) ions are deprotonated and create a flattened tetrahedral environment for the central copper(ii), i.e. the formation of the trinuclear complexes is under the allosteric control of the two peripheral copper(ii) ions. The triply deprotonated trinuclear complex is an efficient catechol oxidase mimic with a surprisingly low pH optimum at pH = 5.6. Since the mononuclear CuL species is not able to promote the oxidation of 3,5-di-tert-butylcatechol, we assume that the central copper(ii) ion of the trinuclear complex with an unsaturated coordination sphere has a fundamental role in the binding and oxidation of the substrate. The experimental and structural details were further elaborated by a series of hybrid density functional theory calculations that support the presence of an antiferromagnetically coupled ground state. However, the magnitude and the pattern of spin coupling are dependent on the composition of the functionals. The optimized theoretical structures highlight the role of the crystal packing effects in inducing asymmetry between the two peripheral copper(ii) sites.


PubMed | MTA SZTE Bioinorganic Chemistry Research Group
Type: Journal Article | Journal: Dalton transactions (Cambridge, England : 2003) | Year: 2015

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.


PubMed | MTA SZTE Bioinorganic Chemistry Research Group
Type: Journal Article | Journal: Metallomics : integrated biometal science | Year: 2014

The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant N4-NColE7-C* - where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag - is catalytically inactive. The crystal structure of this mutant demonstrates that its overall fold is very similar to that of the native NColE7 structure. This implicates the stabilizing effect of the remaining N-terminal sequence on the structure of the C-terminal catalytic site and the essential role of the deleted residues in the mechanism of the catalyzed reaction. Complementary QM/MM calculations on the protein-DNA complexes support the less favourable cleavage by the mutant protein than by NColE7. Furthermore, a water molecule as a possible ligand for the Zn(2+)-ion is proposed to play a role in the catalytic process. These results suggest that the mechanism of the Zn(2+)-containing HNH nucleases needs to be further studied and discussed.


PubMed | CERN, University of Szeged, MTA SZTE Bioinorganic Chemistry Research Group and Copenhagen University
Type: Journal Article | Journal: Angewandte Chemie (International ed. in English) | Year: 2016

Metal-ion-responsive transcriptional regulators within the MerR family effectively discriminate between mono- and divalent metal ions. Herein we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study Ag(I) , Zn(II) , and Hg(II) binding to model systems encompassing the metal-ion-binding loop of CueR from E.coli and V.cholerae. In the presence of Ag(I) , a conserved cysteine residue displays a pKa value for deprotonation of the thiol that is close to the physiological pHvalue. This property is only observed with the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys112 protonated demonstrate that the conserved Ser77 backbone carbonyl oxygen atom from the other monomer of the homodimer is pulled towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR, the mechanism relies on the protonation of Cys112.

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