Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Abstract

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...

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

“…The metal distribution found for Cd(II) 1 -BcII (70% of Cd(II) are located in the H-site; 30% of Cd(II) in the DCH site) compares very well to the preference for the H-site suggested for cobalt but differs from the distribution found for the mono-zinc species where both sites are equally populated (17). The EX-AFS spectrum from CphA in the presence of 0.8 eq of Cd(II) relative to the enzyme, here denoted Cd(II) 1 -CphA, can be fitted with only the DCH site occupied, and the fit gives one additional oxygen ligand very similar to the one found in Cd(II) 1 -BcII.…”

“…Besides this difference, the d-d regions are almost identical in shape and intensity at low and high stoichiometry of Co(II) relative to the enzyme. A similar H-site occupancy at low and high stoichiometry reflects a strong preference of Co(II) for the H-site in Co(II) 1 -BcII (17). Binding of D-captopril to Co(II)-BcII leads to changes both in the charge transfer region and in the d-d region, indicating the binding of an additional ligand and likely changes in the coordination geometry of both sites.…”

“…In general, the intensity of the ligand-to-metal charge transfer bands is mainly due to Cys (sulfur)-Co(II) interaction, whereas the d-d transitions are caused by the His-Co(II) interaction (17).…”

“…The dissociation constants for a first (K mono ) and second (K bi ) cadmium ion bound to BcII in presence of 25 M D-captopril and 0.1 M NaCl were obtained from competition experiments with the chromophoric chelator Mag-fura-2 (Molecular Probes, Eugene, OR) in 15 mM HEPES, pH 7.0, as described previously (17,18).…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…Considerable information exists regarding the B1 and B3 enzymes, including X-ray diffraction (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), spectroscopic (9,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), mechanistic (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59), and computational studies (60)(61)(62)(63)(64)…”