Metallo-β-lactamases (MBLs) are a group of enzymes responsible for a significant proportion of bacterial resistance to β-lactam antibiotics by catalysing the hydrolysis of the β-lactam. The MBL from B. cereus BcII (569/H/9) forms both mono- and bi-nuclear species with native zinc and also with cobalt and cadmium ions. Which species is formed is controlled by the pH because some of the protein ligands used for metal-ion binding must lose a proton to form the metallo-enzyme from the apo-enzyme. The protonation states of the ligands that are used to bind various metal-ions in BcII have been determined using isothermal titration calorimetry (ITC) at different pH's maintained by different buffers. If a single equilibrium is established, by positive cooperative binding, between two metal-ions and the enzyme to form the bi-nuclear enzyme it requires the solution of a cubic equation to model the ITC data. At pH 5.2 and 5.6 the dominant species for all three metals is the mono-nuclear MBL, but only with the native mono-nuclear ZnBcII is the metal-bound water ionised. Compared with ZnBcII, fewer protons are released upon formation of mono-nuclear CoBcII and CdBcII which probably involves metal ion binding to the DCH site. For all three metal-ions, the number of metal-ions binding per molecule of enzyme increases from one to two as the pH is increased from pH 5.20 to 7.20. Only cadmium shows distinct sequential binding in the ITC outputs and the two cadmium ions bind independently and non-cooperatively, showing very distinct and different binding parameters. The apparent single ITC titration curves when two zinc- or cobalt-ions are bound to the β-lactamase are, in fact, not indicative of strong cooperative binding, but are best modelled by two sequential binding steps. The binuclear Cd2BcII is formed with the release of only two protons from the apo-enzyme and, at pH 7.2, probably has an unionised water bridging the two metal-ions, whereas in Zn2BcII and Co2BcII it is a bridged hydroxide-ion, giving rise to the release of three protons on formation of the binuclear enzyme.