Characterization of the metal-binding sites of the β-lactamase from Bacteroides fragilis

In an effort to better understand the structure and function of the metallo-β-lactamase from Bacteroides fragilis, spectroscopic and metal- binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy wits used to confirm that tile Co(II)-substituted β-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are high-spin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (ε ≃ 30 M^-1·cm^-1) and an intense feature at 320 nm (ε ≃ 1570 M^-1·cm^-1). The latter is assigned to a cysteine sulfur to Co(II) ligand- to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of it Co(II) substituted C168S mutant. ¹H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp 183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on k(cat). These results are discussed in light of the recent crystal structure of the metallo-β- lactamase from B. cereus.