To test the presence of a single water/hydroxide molecule and the protonation state of the phosphonate group of the ligand, we performed QM/MM simulations starting from the MM equilibrated structures using DFTB3 in Amber. The QM region was defined by the active site of VIM-2, i.e. the two zinc ions, the ligand, the single water molecule, and the zinccoordinating residues up to their β-carbons. In addition, we performed a QM/MM simulation by replacing the active site water with a hydroxide ion to study its stability. The total charge of the QM region was 0 and -1 for the water and hydroxide systems, respectively. The spin multiplicity was 1 in both cases. Each system was initially minimized for a total of 1000 steps, followed by a 10 ps NPT relaxation and 100 ps production at 300 K using Langevin dynamics, implemented in sander.MPI from Amber22. To evaluate the stability of the zinc coordination, we analyzed zinc coordination distances and performed visual inspections of the active sites.