This paper investigates the enzyme chloroperoxidase (CPO) by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. The effects of anionic substrate, protein environment, and proximal ligand mutation on the high-valent iron-oxo species, compound I (Cpd I), and the ferric hydroperoxide complex, compound 0 (Cpd 0), are studied. The results indicate that the presence of an anionic substrate (acetate) and the protonation state of one critical residue (Glu104) have a considerable impact on the relative stabilities of Cpd I and Cpd 0. In the absence of the substrate or when the substrate is protonated, Cpd I is considerably more stable, and its formation barrier is smaller than in the case where the substrate is in its anionic state and when Glu104 is deprotonated. This trend, which is shown to be a simple manifestation of the Hammond principle, reproduces the experimental observation that the working pH of the enzyme is acidic. Furthermore, in the absence of substrate (or when it is protonated), the relative Cpd 0/Cpd I energies are found to be a good index of Cpd I stability in heme enzymes and to follow the experimental order: horseradish peroxidase (HRP) > CPO > P450. In silico mutation of the proximal ligand from cysteine to selenocysteine was found to have no effect at all on the properties of Cpd I (e.g., spin density on the chalcogen, Mossbauer parameters, etc.) and its relative stability to Cpd 0 or on the corresponding barrier for formation. This surprising finding shows that the polar CPO pocket applies a leveling effect that stabilizes the anionic forms of the proximal ligands (CysS(-) and CysSe(-)). This in turn means that the Se-Cpd I of the mutant CPO is observable.