Metallo-␤-lactamases catalyze the hydrolysis of most ␤-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus ␤-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (⌬G 0 ) of 32 ؎ 2 kJ⅐mol ؊1. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ⌬G 0 value of 65 ؎ 1.4 kJ⅐mol ؊1 .These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.␤-Lactamases catalyze hydrolysis of the ␤-lactam ring of antibiotics belonging to the penicillin family (1-3). Synthesis of these enzymes represents the major cause of bacterial resistance to ␤-lactam antibiotics (4 -7). They are classified into two structural superfamilies (8), namely the active site serine enzymes (both ␤-lactamases and penicillin-binding proteins) and the metallo-␤-lactamases (MBLs). 4 The latter, also referred to as class B ␤-lactamases, require one or two zinc ions for activity (9 -11). They show no structural similarity with the active site serine ␤-lactamases, and they are part of a remarkable set of enzymes (i.e. the zinc metallo-hydrolase family of the ␤-lactamase fold or, more simply, the MBL superfamily (10 -16)) that exhibit a wide variety of functions related to hydrolysis and redox reactions and DNA and RNA metabolism. Seventeen groups of enzymes have been identified on the basis of their biological functions (13), which all share a novel ␣␤/␤␣ fold. Most of the three-dimensional structures reveal a binuclear center with metal ligands located on loops connecting secondary structure elements (15, 17).Zinc ␤-lactamases have been found in many bacterial species, including pathogenic strains (18, 19). Most of them are able to hydrolyze almost all ␤-lactam antibiotics (20, 21), including carbapenems (i.e. a family of last resort ␤-lactams that generally escape the activity of the most widespread serine ␤-lactamases), and they are not sensitive to the classical inactivators of serine ␤-lactamases, such as clavulanate, sulbactam, and tazobactam (22,23). Furt...