Electrochemically Activated Catalytic Pathways of Human Metabolic Cytochrome P450s in Ultrathin Films

Electrochemical understanding of metalloenzyme biocatalysts can guide in the development of green bioreactors for stereoselective syntheses and tailoring of efficient bio-inspired catalysts for large-scale applications. In addition, if the enzymes themselves can be stabilized into viable catalytic materials such as films or nanoparticles, valuable and unique stereo- and regiospecific reactions can be catalyzed in simple aqueous medium. In particular, biological electrocatalysis is an emerging area that is expected to play a significant role in fine chemical and drug synthesis, niche electronic devices, sustainable energy, and biomedical fields. Our research has specifically been inspired by the broad stereoselective biocatalytic properties of human cytochrome P450 (cyt P450) enzymes. Cyt P450s are a large family of heme iron monooxygenases with high expression in liver and other human organs that serve as the major oxidative catalysts in human metabolism. Electrochemical bioreactors featuring cyt P450s have the potential to complement and accelerate drug development processes by facilitating candidate identification and toxicity evaluation of metabolites. Challenging aspects associated with electrochemical studies of cyt P450s in thin films include enzyme stability, electronic connectivity between cyt P450-heme cofactor and electrodes, density of immobilized electrocatalytically active enzyme molecules, and bioactive cyt P450 conformations in films on electrodes. To achieve these features, various electrode surface environments have been explored. We designed ultrathin bioactive films of cyt P450 enzymes with polyions or insoluble surfactants that demonstrated the first direct electron transfer and biocatalytic applications of this class of enzymes on electrodes. Subsequently, we constructed films of genetically engineered microsomes, rat and human liver microsomes, and cyt P450s assembled with microsomal cyt P450 reductase (CPR) that enabled a bioelectrocatalytic pathway that closely mimicked the in vivo cyt P450 biocatalytic mechanism. These systems allowed us to develop a clearer understanding of cyt P450 electron transfer and enabled uses in microfluidic toxicity screening arrays. In this review, we provide an overview of different catalytic pathways that are accessed and driven electrochemically using pure human cyt P450 enzymes and those present in membrane-bound forms along with CPR.