Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereusbeta-lactamase II: A perturbed angular correlation of gamma-rays spectroscopic study

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectrosc opy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole i nteractions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios betwe en 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadm ium ions can be bound per molecule of apoenzyme. These results show the exi stence of two interacting Cd-binding sites in BCII. The relative population s of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1 :3 ratio for the microscopic dissociation constants of the two different me tal sites (when only one cadmium ion is bound). X-ray diffraction data at p H 7.5 demonstrate that also for Zn(II) two binding sites exist, which may b e bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the si te with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This ass ignment shows that cadmium ions preferentially bind to the Cys site. This i s in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme , where an analysis of the corresponding PAC spectrum showed that Cd(II) oc cupied the Cys site, whereby Zn(II) occupied the site with three histidines . The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of niaocefin for different metal ion substitutions(Zn2E, ZnE, Cd2E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.