Uwe Heinz

4‐[3,5‐Bis(2‐hydroxyphenyl)‐1,2,4‐triazol‐1‐yl]‐ benzoic Acid: A Novel Efficient and Selective Iron(III) Complexing Agent

An exceptionally stable 1:2 complex [FeL2]3− is formed from the ligand H3L and FeIII. In contrast, the affinity of this ligand for other biometals is relatively small. These properties make H3L a highly promising candidate for medical applications (e.g. for the treatment of iron overload).

Kinetic and spectroscopic characterization of native and metal-substituted β-lactamase from Aeromonas hydrophila AE036

Two metal ion binding sites are conserved in metallo‐β‐lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur‐to‐metal charge transfer bands are observed for all mono‐ and di‐metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non‐competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Zinc Ion-induced Domain Organization in Metallo-β-lactamases A FLEXIBLE “ZINC ARM” FOR RAPID METAL ION TRANSFER?

The reversible unfolding of metallo-beta-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger alpha-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-beta-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of gamma-rays spectroscopic data. We suggest that this flexible zinc arm motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an activation on demand regulation of enzyme activity.The reversible unfolding of metallo-β-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger α-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-β-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of γ-rays spectroscopic data. We suggest that this flexible “zinc arm” motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an “activation on demand” regulation of enzyme activity.

Studies on ternary metallo-β lactamase-inhibitor complexes using electrospray ionization mass spectrometry

Metallo-β-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to β-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.

4-[3,5-Bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoesäure: ein neuartiger, effizienter und selektiver Eisen(III)-Komplexbildner

Einen auserordentlich stabilen 1:2-Komplex [FeL2]3− bildet der Ligand H3L (siehe Formel) mit FeIII. Seine Affinitat fur andere Biometalle ist dagegen vergleichsweise klein. Diese Eigenschaften machen ihn zu einem vielversprechenden Kandidaten fur medizinische Anwendungen (z.u2009B. zur Therapie der Eisenuberladung).

3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazole based ligands — protonation and metal complex formation

Abstract3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazole (H2La) and 4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid (H3Lb) have been prepared, and crystal structure of the intermediate 2-(2-hydroxyphenyl)-4H-1,3-benzoxazin-4-one has been determined. Temperature dependent 1H NMR spectroscopic measurements of H2La indicated dynamic behavior with the equilibrium between the two asymmetric tautomers. For H3Lb, pD-dependent 1H NMR spectroscopic measurements showed small but characteristic shifts in the range of 0 ≤ pD ≤ 1, indicative of a triazole nitrogen atom protonation; the corresponding pKa of 0.98 ± 0.04 was determined by spectrophotometric titrations. (H2O, 26°C, 1 M KCl/HCl). Formation of [FeIII(La)]+ (pH 2.5) and [FeIII(La)2]− (pH > 6) was verified by UV-Vis spectroscopy. Complex formation of H3Lb with Al3+ and VO2+ was investigated by 1H NMR spectroscopic titration and cyclic voltammetry, respectively. Single crystals of the phenoxo bridged [VVO(HLb)(EtO)]2·2EtOH were characterized by X-ray structural analysis.

Crystal structure of N,N,N-tris[2-(salicylideneaminato)ethyl]aminemanganese( III), Mn[N(C9H9NO)3]

C 27 H 27 MnN 4 O 3 , monoclinic, P12 1 /n1 (no. 14), a = 7.906(2) A, b = 25.609(5) A, c = 11.736(2) A, β = 96.55(3)°, V = 2360.6 A 3 , Z = 4, R gt (F) = 0.050, wR ref (F 2 ) = 0.127, T = 293 K.