Enhancing ferryl accumulation in H₂O₂-dependent cytochrome P450s

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Cytochrome P450s (CYPs) are crucial enzymes involved in various biological processes.
• The CYP152 family, dependent on hydrogen peroxide (H2O2), plays a role in oxidative catalysis.
• Ferryl (iron(IV)-oxo) species are key intermediates in CYP enzymatic mechanisms.

Purpose of the Study:

• To develop a facile strategy for enhancing ferryl species accumulation in H2O2-dependent CYPs.
• To characterize a highly chemoselective CYP decarboxylase, OleT_SA, from Staphylococcus aureus.
• To investigate the relationship between resting spin-state equilibrium and Compound I (CpdI) accumulation.

Main Methods:

• Characterization of Staphylococcus aureus CYP decarboxylase (OleT_SA).
• Examination of OleT_SA Compound I (CpdI) accumulation with various fatty acid substrates.
• Targeted mutagenesis of the proximal pocket to alter spin-state equilibrium.

Main Results:

• OleT_SA was found to be soluble at high concentrations.
• Ferryl species accumulation demonstrated a dependence on resting spin-state equilibrium.
• Mutagenesis favoring the high-spin form significantly enhanced CpdI accumulation to near-stoichiometric yields.

Conclusions:

• A novel strategy effectively enhances ferryl species accumulation in H2O2-dependent CYPs.
• Targeting spin-state equilibrium is a viable approach to optimize CYP catalytic intermediates.
• This work provides insights into the mechanism and engineering of CYP decarboxylases for improved efficiency.