Is there a bridging ligand in metal-substituted beta-lactamases : A spectroscopic and theoretical answer

L ESTIU, G; RASIA, R M; CRICCO, J A; VILA, AJ

INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY

JOHN WILEY & SONS INC

Año: 2002 vol. 88 p. 118 - 132

0020-7608

The spectral features of Co(II)-substituted metallo-β-lactamases were analyzed. Nuclear magnetic resonance (NMR) evidence is provided that shows no magnetic coupling between the two metal ions in Co(II),Co(II)-βLII (β-lactamase II from Bacillus cereus), suggesting that no bridging ligand connects them. Ultraviolet?visible (UV?vis) data are rationalized on the basis of semiempirical quantum chemical calculations of the intermediate neglect of differential overlap type (ZINDO/S). The binuclear active sites of the enzymes from B. cereus (βLII) and B. fragilis (CcrA) were modeled from the protein data bank (PDB) coordinates, and calculations were performed at the multireference configuration interaction level. The active site geometries of these enzymes were examined using the experimental electronic spectra as a guide. The model which best fits to the spectroscopic data lacks a bridging solvent molecule, in agreement with the NMR data. The model could also reproduce spectral changes seen in the mixed Zn(II),Co(II) adduct. The bands in the visible range are associated with ligand field transitions at the high-affinity site, whereas the UV-calculated features originate in ligand-to-metal charge transfer to the open shell Co(II) in the low-affinity site. These results indirectly support the hypothesis that the Co(II),Co(II) derivative of CcrA is also unbridged, in contrast with the structure of the native zinc enzyme. These results indicate that the existence of a bridging ligand is not necessary for metallo-β-lactamase activity, and that the second zinc ion is not essential for lowering the pKa of the bound water.