Enhancing ferryl accumulation in H2O2-dependent cytochrome P450s

A facile strategy is presented to enhance the accumulation of ferryl (iron(IV)-oxo) species in H2O2 dependent cytochrome P450s (CYPs) of the CYP152 family. We report the characterization of a highly chemoselective CYP decarboxylase from Staphylococcus aureus (OleTSA) that is soluble at high concentrations. Examination of OleTSA Compound I (CpdI) accumulation with a variety of fatty acid substrates reveals a dependence on resting spin-state equilibrium. Alteration of this equilibrium through targeted mutagenesis of the proximal pocket favors the high-spin form, and as a result, enhances Cpd-I accumulation to nearly stoichiometric yields. Alteration of the spin-state equilibrium is leveraged as a tool to optimize the accumulation of the ferryl cytochrome P450 intermediate Compound I. [Display omitted] •A highly soluble and chemoselective CYP152 from Staphylococcus aureus was characterized.•Alteration of the “cys-pocket” alters the spin-state equilibrium.•Compound I can be generated using H2O2 at stoichiometric concentrations.