The justification for choosing the protonation states of the various amino acid residues in the QM region of the input structures for the QM/MM calculations was the following. In a study of the kinetics of VCPO, van Schijndel et al. observed that at pH<4 H2O2 will not bind to VCPO and chloride inhibits VCPO. From this observation, they argued that a histidine within the active site must be monoprotonated at pH>4. Zhang and Gascon argued from this observation that His404 is becoming diprotonated at low pH, and diprotonation of His404 leads to the enzyme being inhibited by halide at low pH. As a result, in their QM/MM study of VCPO, Zhang and Gascon kept His404 in the monoprotonated state, HIE, as shown in Figure .