Spectroscopic characterization of a binuclear metal site in Bacillus cereus β-lactamase II

The zinc metalloenzyme beta-lactamase II (beta LII) from Bacillus cereus has been overexpressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties of recombinant beta LII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements. The apoenzyme is able to bind two metal ion equivalents, which confer on beta LII its maximum enzymatic efficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixed Zn(LT)Co(II) derivative of beta LII were obtained and probed by electronic and paramagnetic NMR spectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule, adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinity metal site, together with two or three solvent molecules. This coordination polyhedron resembles the binuclear metal site of the Bacteroides fragilis beta-lactamase [Concha, N., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duee, E., Paul-Soto, R., Galleni, M., Frere, J. M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from the X-ray study of beta LII [Carfi, A., Fares, S., Duee, E., Galleni, M., Duet, C., Frere, J. M., and Dideberg, O. (1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a general feature of metallo-beta-lactamases.