Electron-transfer rates govern product distribution in electrochemically-driven P450-catalyzed dioxygen reduction

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Electrochemistry

Background:

• Cytochrome P450 enzymes are crucial for selective oxidations.
• Developing efficient electrode-driven biocatalytic systems requires optimized electron transfer (ET).
• Achieving native-like turnover rates in P450 systems is a key challenge.

Purpose of the Study:

• To correlate heme reduction rates with ET pathways and product distributions in P450 systems.
• To investigate the relationship between ET rates and catalytic efficiency.
• To engineer P450 enzymes for improved biocatalytic performance.

Main Methods:

• Utilized single-surface cysteine mutants of Bacillus megaterium P450 heme domain.
• Modified protein thiols with N-(1-pyrene)-iodoacetamide for bonding to graphite electrodes.
• Formed electroactive monolayers and analyzed electron transfer rates (k(s)(o)) and heme-cysteine distances.
• Employed rotated-disk electrode voltammetry to determine P450-catalyzed dioxygen reduction products.

Main Results:

• Cysteine mutants at positions 62, 383, and 387 formed electroactive monolayers with similar redox potentials.
• Electron transfer rates (k(s)(o)) and heme-cysteine distances varied significantly (e.g., 50 s⁻¹ at 16 Å, 650 s⁻¹ at 19 Å).
• A strong correlation was observed between ET rates and product distributions, with higher k(s)(o) values indicating more electrons transferred per dioxygen.

Conclusions:

• Electron transfer rates directly influence the catalytic outcomes of P450 biocatalysts.
• Engineering ET pathways in P450 enzymes is critical for enhancing selective oxidation efficiency.
• This work provides a framework for designing improved electrode-driven biocatalytic systems.