Chloroperoxidase (CPO) is unique among the metabolizing heme proteins because it is a hybrid of two different families, peroxidases and cytochromes P450 (CYP450s). Unlike all other known peroxidases, with a conserved histidine as the proximal ligand of the heme iron, CPO has a cysteine ligand in common with the CYP450s. In addition, CPO can perform both peroxidase and CYP450 types of substrate oxidations. Despite these differences, the first steps of the CPO catalytic cycle are similar to those in the traditional heme peroxidases. Specifically, formation of the catalytically active Compound I species from the inactive ferric resting form proceeds via a transient peroxide complex. However, in CPO, the role of the protein environment in the distal peroxide binding site must be significantly different than that in typical peroxidases. This difference results from the presence of a unique glutamate residue instead of the two highly conserved residues, histidine and arginine, thought to be involved in Compound I formation of typical peroxidases. The goal of the computational studies presented here was to further elucidate the role of this unique glutamate residue, Glu 183, in transforming the transient peroxide intermediate to Compound I in CPO. Specifically, the proposed double role of Glu 183 in the CPO-HOOH complex first as a proton acceptor and then as a proton donor was investigated. The criteria used were the extent to which stable H bonds were formed between this unique residue and the peroxide ligand of the heme iron in the CPO-peroxide complex, during unconstrained MD simulations. Analysis of these simulations provided evidence for the double role of the Glu 183 first as proton acceptor and then as proton donor in CPO-Compound I formation from the peroxide intermediate.