A new model for cobalt binding to the metallo-beta-lactamase BcII

LLARRULL, L.I.; TIONI, M.F.; BENNETT, B.; VILA, A.J.

Rosario

Congreso; XXXV Reunión Anual Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

BcII from Bacillus cereus presents two Zn(II) ions bound in the active site. BcII was first crystallized with only one Zn ion tetrahedrally coordinated to three histidine residues (His116, His118 and His196) and a bridging H2O/OH- molecule, in the so-called 3H-site.[1,4] Spectroscopic experiments and a second crystal structure later indicated that BcII was also capable of binding a second metal ion, which is trigonal bipyramidally coordinated to Asp120, Cys221, His263, the bridging H2O/OH- and an additional water molecule, and comprises the DCH-site.[2,3,4] Great controversy still exists on the metal content in the catalytically relevant species of BcII and on the mechanism of action of this enzyme. Many kinetic experiments cannot be accounted for by the metal bound species derived from the currently accepted model of Co(II) binding to BcII. Hence, we decided to revise the metal-binding events by monitoring BcII Co(II) uptake using UV-Vis, EPR and paramagnetic NMR spectroscopy. Our results indicate that di-Co(II) BcII accumulates at metal/enzyme ratios < 0.5, contrary to the existent model which suggests that di-Co(II) BcII accumulates at metal/enzyme ratios > 1. Parallel-mode EPR experiments detect a spin coupled di-Co(II) BcII species at 0.6 equivalents of added Co(II), and the titration followed by paramagnetic NMR spectroscopy shows that signals from ligands at both metal-binding sites appear at substoichiometric metal concentrations. Two equivalents of cobalt are bound sequentially, with macroscopic binding constants KD1 = 0.12 ± 0.06 microM for the first cobalt bound to apo-BcII and KD2 = 0.16 ± 0.04 microM for the second equilibrium, as determined through competition experiments with the chromophoric metal ligand Mag-fura-2. An exchange of the metal ion between the 3H and the DCH-sites seems to occur on mono-Co(II) BcII [4], and hence mono-Co(II) BcII seems not to be a system with one occupied and one free site. Besides, the UV-Vis titration indicates binding of a third cobalt ion with lower affinity (KD3 = 94 microM, in a site that affects the cobalt (DCH-site) to Cys221 charge-transfer band.