One high affinity (nM) and one low affinity (M) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-␤-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of ␥-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono-and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.Two zinc binding sites in close proximity are conserved in all metallo-␤-lactamases studied so far. Only two of the metal ion ligands undergo variations between the three different subclasses of the enzyme family (1). The enzyme from Bacillus cereus 569/H/9 (BcII) 1 represents a member of subclass B1 with 3 His ligands in one site and 1 Asp, 1 Cys, and 1 His ligand in the other site (3H 1 and DCH 1 sites, respectively). Various crystal structures of BcII are available, representing mononuclear (2) and binuclear species (3, 4). It was shown earlier that both mono-and binuclear zinc enzymes from B. cereus (5) and Bacteroides fragilis (6) are catalytically active.Although catalytic mechanisms for the enzyme with either one or two zinc ions bound have been discussed (for review see Ref. 7) the respective roles of the two binding sites during catalysis are still unclear. Generally the 3H site is considered to be the primary catalytic site. However, the importance of the DCH site for catalysis became obvious from studies of the C168A mutant. When only one zinc ion is bound to this mutant, it shows a very low activity compared with the wild type, whereas wild type-like activity is almost restored when a second metal ion is bound (5).Perturbed angular correlation (PAC) of ␥-ray spectroscopy provides information on the metal ion coordination geometry through measurement of the nuclear quadrupole interaction (NQI) between the nuclear electric quadrupole moment and the electric field gradient from the surrounding charge distribution. With this method it was possible to demonstrate that the Cd(II) ions in the mononuclear wild type BcII are distributed between the two m...

Complex formation of 4‐[3,5‐bis(2‐hydroxyphenyl)‐1,2,4‐triazol‐1‐yl]benzoic acid (ICL670, H3Lx), 4‐[3,5‐bis(2‐hydroxyphenyl)‐1,2,4‐triazol‐1‐yl]benzosulfonic acid (H3Ly), and 3,5‐bis(2‐hydroxyphenyl)‐1‐phenyl‐1,2,4‐triazole (H2Lz) with Fe3+ and Fe2+ was investigated in H2O and in H2O/DMSO mixtures by potentiometry, spectrophotometry and cyclic voltammetry. ICL670 has previously been considered as a promising drug for an oral treatment of iron overload. In this paper, the stability and redox properties of the various FeII and FeIII complexes were elucidated with a particular focus on their potential involvement in the generation of oxidative stress. The overall stability constants of [FeIII(Lx)] and [FeIII(Lx)2]3− (25 °C, 0.1 M KCl in H2O) are log β1 = 22.0 and log β2 = 36.9, respectively. The affinity of these ligands for Fe2+ is remarkably poor. In particular, the 1:2 complexes [FeII(Lx)2]4− and [FeII(Ly)2]4− were found to be less stable. As a consequence, the redox chemistry of the [FeIII(Lx)]/[FeII(Lx)]− and the [FeIII(Lx)2]3−/[FeII(Lx)2]4− couples differs significantly. [FeIII(Lx)2]3− is a very weak oxidizing agent (E1/2 is approximately −0.6 V versus NHE) and reduction of [FeIII(Lx)2]3− is not anticipated under physiological conditions. The reduction potential of the [FeIII(Lx)]/[FeII(Lx)]− couple is considerably less negative and was estimated to be +0.1 V (versus NHE). The possible roles of the various Fe complexes as catalysts for the Fenton reaction in biological media are discussed. The crystal structures of H3Lx, Na[Fe(Lz)2]·4EtOH, Na[Al(Lz)2]· 4EtOH, and [Cu(Lz)(pyridine)]2 were investigated by single‐crystal X‐ray diffraction, and the possible influence of the particular steric requirements of these ligands on the stability of the metal complexes has been analyzed. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

During the past few years the results from molecular biological, biochemical, chemical, physical and theoretical approaches expanded the knowledge about metallo-beta-lactamases considerably. The main reason for the attracted interest is a persisting medical problem. Bacteria expressing metallo-beta-lactamases can be resistant to treatment with all the known beta-lactam antibiotics, and they are additionally invulnerable to combined treatment with inhibitors for the wider-spread serine-beta-lactamases. However, clinically useful inhibitors for metallo-beta-lactamases are not yet available. In spite of the rapidly expanding knowledge base a central question is still controversially discussed: is it the mononuclear, the binuclear or the metal-free state which might serve as the physiologically relevant target for inhibitor design? A summary of the present views of the roles and coordination geometries of metal ion(s) in metallo-beta-lactamases is combined with a discussion of the possibly variable metal ion content under physiological conditions.

D-and L-captopril are competitive inhibitors of metallo-␤-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of ␥-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II) 1 -BcII, the thiolate sulfur of D-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II) 1 and Co(II) 2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II) 1 -BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II) 1 -CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for D-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-␤-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.Metallo-␤-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of ␤-lactam antibiotics, including carbapenems. This relatively new form of resistance is spreading and thereby escaping the effective inhibitors developed to fight the better known serine-␤-lactamases. For all metallo-␤-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-␤-lactamase (BcII), 1 one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4), whereas the binding of 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutrylglycine)] to the enzyme from Pseudomonas aeruginosa (IMP-1) was characterized by x-ray crystallography (5). With both approaches, the inhibited binuclear zinc enzymes were studied. Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.The structural investigation of D-and L-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an ...

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C 2 H 2 ) and a tetrathiolate (C 4 ) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C 2 H 2 showed the expected Cys 2 His 2 -ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptidepeptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C 2 H 2 appeared as a simple second order process, the suggested mechanism for C 4 comprises a zinc-bridged Zn-(C 4 ) 2 species as well as a Zn-C 4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.Within the last decade the generally accepted roles of protein-bound zinc in catalysis and structure stabilization were complemented by regulatory functions. Protein binding sites have been classified as "catalytic," "co-catalytic," and "structural" zinc sites (1). A fourth type, namely "interface" was added recently (2). The possible role of a variable zinc occupancy of protein sites as a modification that provides a pathway for intracellular information transfer has been discussed (3), and the steadily growing number of known zinc proteins involved in gene regulation led to the suggestion that some classes of proteins might transduce changes in available zinc levels into changes in patterns of gene expression (4). An update of recent findings in zinc biology (5) now supports the view of zinc as a key element in cellular regulation.An important question with respect to a regulatory function, however, concerns the controversially discussed concentration of free zinc ions in different physiological environments. Cells react on a changed zinc supply by e.g. an activated transcription of metallothionein (Ref. 6 and references therein) or changes in the activity of zinc-sensing transcription activators like Zap1 (7) and MTF-1 (8). In the latter cases zinc binding to individual zinc finger motifs is supposed to induce structural changes, which in turn modify the functionality of the proteins. If the transduction ...