Next, we apply Bader's Atoms-in-Molecules (AIM) approach to elucidate the complex hydrogen bonding to the vanadate cofactor. The AIM approach has been increasingly used to determine noncovalent interactions. In the literature on VCPO, almost all knowledge of the hydrogen bonding is based on distance considerations, but distance considerations can be misleading. The AIM approach involves a topological analysis of the probability density function. Atoms that are interacting (bonding or secondary interactions) have a single line of locally maximum electron density, and this line is referred to as a bond path. A point on the bond path between two interacting atoms with the lowest value of the electron density is called the bond critical point, and we report herein the values of the electron density, ρ, at the bond critical points in units of electrons/bohr 3 . The bond critical point along a covalent bond, will typically have ρ= 0.2 electrons/bohr 3 , but for a hydrogen bond the ρ value at the bond critical point will be less than 0.1 electrons/bohr 3 . Knowing the precise hydrogen bonding in VCPO will aid the study of the mechanism, which has so far been limited to the study of the cofactor in isolation or at most by taking into consideration the hydrogen bonding between Lys353 and vanadate. In addition to this AIM study, we report a natural bond orbital (NBO) study of the tri-and quadprotonated states of vanadate to gain a better understanding of their electronic structure. Figure . The active site of vanadium chloroperoxidase (VCPO) has a vanadate cofactor which is shown here in an unprotonated state. The vanadate is surrounded by a sphere of residues which hydrogen bond to it. (Three crystallographic water molecules are hidden for clarity.) Figure . The mono-, di-, tri-, quad-protonated states of VCPO, 1-23, which are the input structures for this QM/MM study. The actual QM region used in this QM/MM study is shown in Figures . Structure 1 shows some of the surrounding amino acid residues so that the stereochemistry is clear in all of the structures. (His496, which is bonded to V, is omitted for simplicity.)