Gauging the relative oxidative powers of compound I, ferric-hydroperoxide, and the ferric-hydrogen peroxide species of cytochrome P450 toward C-H hydroxylation of a radical clock substrate

Density functional calculations were performed in response to the controversies regarding the identity of the oxidant species in cytochrome P450. The calculations were used to gauge the relative C-H hydroxylation reactivity of three potential oxidant species of the enzyme, the high-valent oxo-iron species Compound I (Cpd I), the ferric hydroperoxide Compound O (Cpd O), and the ferric-hydrogen peroxide complex Fe(H₂O₂). The results for the hydroxylation of a radical probe substrate, 1, show the following trends: (a) Cpd I is the most reactive species; in its presence the other two reagents will be silent, (b) In the absence of Cpd I, substrate oxidation by Cpd O and Fe(H₂O₂) will take place via a stepwise mechanism that involves initial O-O homolysis followed by H-abstraction from 1. (c) Cpd O will undergo mostly porphyrin hydroxylation and only ∼15% of substrate oxidation producing mostly the rearranged alcohol, 3 (Scheme 2). (d) Fe(H ₂O₂) will generate mostly free hydrogen peroxide (uncoupling). A small fraction will perform substrate oxidation and lead mostly to 3. Reactivity probes for these reagents are kinetic isotope effect (KIE) and the product ratio of unrearranged to rearranged alcohols, [2/3], Thus, for substrate oxidation by Cpd O or Fe(H₂O₂) KIE will be small, ∼2, while Cpd I will have large KIE values. Typically both Cpd O and Fe(H₂O₂) will lead to a [2/3] ratio < 1, while Cpd I will lead to ratios > 1. In addition, the product isotope effect (KIE ₂/KIE₃ ≠ 1) is expected from the reactivity of Cpd I.