Regulation of ferryl reactivity by the cytochrome P450 decarboxylase OleT

The cytochrome P450 OleT catalyzes the decarboxylation of long-chain fatty acid substrates to produce terminal alkenes using hydrogen peroxide as a co-substrate. The facile activation of peroxide to form Compound I in the first step of the reaction, and subsequent CC bond cleavage mediated by Compound II, provides a unique opportunity to visualize both ferryl intermediates using transient kinetic approaches. Analysis of the Arrhenius behavior yields activation barriers of ∼6 kcal/mol and ∼ 18 kcal/mol for the decay of Compound I and Compound II respectively. The influence of the secondary coordination sphere, probed through site-directed mutagenesis approaches, suggests that restriction of the donor-acceptor distance contributes to the reactivity of Compound I. The reactivity of Compound II was further probed using kinetic solvent isotope effect approaches, confirming that the large barrier owes to a proton-gated mechanism in the decarboxylation reaction coordinate. Hydrogen-bonding to an active-site histidine (H85) in the distal pocket plays a key role in this process.