The cytochromes P450 are a versatile range of mono-oxygenase enzymes that catalyze a variety of different chemical reactions, of which the key reactions include aliphatic hydroxylation and C=C double bond epoxidation. To establish the fundamental factors that govern substrate epoxidation by these enzymes we have done a systematic density functional theory study on substrate epoxidation by the active species of P450 enzymes, namely the iron(IV)-oxo porphyrin cation radical oxidant or Compound I. We show here, for the first time, that the rate constant of substrate epoxidation, and hence the activation energy, correlates with the ionization potential of the substrate as well as with intrinsic electronic properties of the active oxidant such as the polarizability volume. To explain these findings we present an electron-transfer model for the reaction mechanism that explains the factors that determine the barrier heights and developed a valence bond (VB) curve crossing mechanism to rationalize the observed trends. In addition, we have found a correlation for substrate epoxidation reactions catalyzed by a range of heme and nonheme iron(IV)-oxo oxidants with the strength of the O-H bond in the iron-hydroxo complex, i.e. BDEOH, which is supported by the VB model. Finally, the fundamental factors that determine the regioselectivity change between substrate hydroxylation and epoxidation are discussed. It is shown that the regioselectivity of aliphatic hydroxylation versus double bond epoxidation is not influenced by the choice of the oxidant but is purely substrate dependent. © 2010 American Chemical Society.