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

Raquel Paul-Soto, Michael Zeppezauer, Hans Werner Adolph, Moreno Galleni, Jean Marie Frere, Andrea Carfi, Otto Dideberg, Johan Wouters, Lars Hemmingsen, Rogert Bauer

Research output: Contribution to journalArticlepeer-review

Abstract

Cd-substituted forms of the Bacillus cereus metallo-β-lactamases (BCII) were studied by perturbed angular correlation of γ-rays (PAC) spectroscopy. At very low [Cd]:[apo-β-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-β-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-β-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be 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 site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is 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) occupied 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 nitrocefin for different metal ion substitutions (Zn2E, ZnE, Cd2E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.

Original languageEnglish
Pages (from-to)16500-16506
Number of pages7
JournalBiochemistry
Volume38
Issue number50
DOIs
Publication statusPublished - 14 Dec 1999

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