There is a major controversy in cytochrome P450 chemistry regarding the nature of the active oxidant responsible
for substrate monoxygenation. Part of this controversy originates from the fact that the later stages in the catalytic
cycle of P450 enzymes proceed so fast that little experimental evidence is available. Early studies suggested an iron(IV)-
oxo heme cation radical ([heme(+•)-FeIV=O] or Compound I) as the active species able to abstract a hydrogen atom from a
substrate and rebind the hydroxyl group to form an alcohol product. Such simplistic early models involving a single active
species have subsequently been invalidated by several experimental studies which clearly indicates that there must be at
least two active species of some description. Based on these and other data, a two-oxidant hypothesis was put forward
where Compound I and its precursor in the catalytic cycle ([heme-FeIII-OOH]– or Compound 0) are competitive oxidants.
Density functional theory studies, however, suggest an alternative hypothesis involving a two-state-reactivity scenario
where Compound I has two close-lying spin states that react differently with substrates and masquerade as two distinct
oxidants. These theoretical studies show that the two spin states of Compound I react with substrates via aliphatic and
aromatic C–H hydroxylation, C=C epoxidation and sulfoxidation reactions, and explain experimentally observed product
distributions and kinetic isotope effects. This review will give an overview of recent studies on the two-oxidant versus
two-state-reactivity hypotheses and how theory contributes to the understanding of enzymatic reaction processes.
Keywords: P450 enzymes, compound I, compound 0, density functional theory, enzyme modeling, enzyme function, iron-oxo
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