The electrochemical properties of cytochrome P450 2C9 (CYP2C9) and polymorphic modifications P450 2C9*2 (CYP2C9*2) and P450 2C9*3 (CYP2C9*3) were studied. To analyze the comparative electrochemical and electrocatalytic activity, the enzymes were immobilized on electrodes modified with a membrane-like synthetic surfactant (didodecyldimethylammonium bromide (DDAB)). An adequate choice of the type of modified electrode was confirmed by cyclic voltammetry of cytochromes P450 under anaerobic conditions, demonstrating well-defined peaks of reduction and oxidation of the heme iron. The midpoint potential, Emid, of cytochrome P450 2C9 is −0.318 ± 0.01 V, and Emid = −0.324 ± 0.01 V, and Emid = −0.318 ± 0.03 V for allelic variant 2C9*2 and allelic variant 2C9*3, respectively. In the presence of substrate diclofenac under aerobic conditions, cytochrome P450 2C9 and its polymorphic modifications P450 2C9*2 and P450 2C9*3 exhibit catalytic properties. Stimulation of the metabolism of diclofenac by cytochrome P450 2C9 in the presence of antioxidant medications mexidol and taurine was shown.

In the present work, screen-printed electrodes (SPE) modified with a synthetic surfactant, didodecyldimethylammonium bromide (DDAB) and streptolysin O (SLO) were prepared for cytochrome P450 3A4 (CYP3A4) immobilization, direct non-catalytic and catalytic electrochemistry. The immobilized CYP3A4 demonstrated a pair of redox peaks with a formal potential of −0.325 ± 0.024 V (vs. the Ag/AgCl reference electrode). The electron transfer process showed a surface-controlled mechanism (“protein film voltammetry”) with an electron transfer rate constant (k_s) of 0.203 ± 0.038 s⁻¹. Electrochemical CYP3A4-mediated reaction of N-demethylation of erythromycin was explored with the following parameters: an applied potential of −0.5 V and a duration time of 20 min. The system with DDAB/SLO as the electrode modifier showed conversion of erythromycin with an efficiency higher than the electrode modified with DDAB only. Confining CYP3A4 inside the protein frame of SLO accelerated the enzymatic reaction. The increases in product formation in the reaction of the electrochemical N-demethylation of erythromycin for SPE/DDAB/CYP3A4 and SPE/DDAB/SLO/CYP3A4 were equal to 100 ± 22% and 297 ± 7%, respectively. As revealed by AFM images, the SPE/DDAB/SLO possessed a more developed surface with protein cavities in comparison with SPE/DDAB for the effective immobilization of the CYP3A4 enzyme.

The functional significance of cytochrome P450s (CYP) enzymes is their ability to catalyze the biotransformation of xenobiotics and endogenous compounds. P450 enzymes catalyze regio- and stereoselective oxidations of C-C and C-H bonds in the presence of oxygen as a cosubstrate. Initiation of cytochrome P450 catalytic cycle needs an electron donor (NADPH, NADH cofactor) in nature or alternative artificial electron donors such as electrodes, peroxides, photo reduction, and construction of enzymatic “galvanic couple”. In our review paper, we described alternative “handmade” electron sources to support cytochrome P450 catalysis. Physical-chemical methods in relation to biomolecules are possible to convert from laboratory to industry and construct P450-bioreactors for practical application. We analyzed electrochemical reactions using modified electrodes as electron donors. Electrode/P450 systems are the most analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Comparative analysis of flat 2D and nanopore 3D electrode modifiers is discussed. Solar-powered photobiocatalysis for CYP systems with photocurrents providing electrons to heme iron of CYP and photoelectrochemical biosensors are also promising alternative light-driven systems. Several examples of artificial “galvanic element” construction using Zn as an electron source for the reduction of Fe³⁺ ion of heme demonstrated potential application. The characteristics, performance, and potential applications of P450 electrochemical systems are also discussed.

The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

The activity of cytochrome P450 enzymes decreases in older adults, which can lead to toxic effects from polypharmacy. Cytochromes P450 are the most significant enzymes involved in the metabolism of foreign compounds, including pharmaceutical substances. Vitamin B2, or riboflavin (RF), is a potent antioxidant that is vital for the body and participates in numerous enzyme-catalyzed redox reactions. RF is phosphorylated intracellularly to form flavin mononucleotide (FMN), which is further metabolized into flavin adenine dinucleotide (FAD). The active site of the NADPH-dependent cytochrome P450 reductase (CPR), a redox partner of CYP enzymes, is necessary for the catalytic functions of cytochromes P450. The active site of reductase is a complex formed by two types of vitamin B2, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). In our study, we investigated the impact of the phosphorylated form of vitamin B2, FAD, and FMN on the catalytic activity of cytochrome P450 2C9 (CYP2C9) towards non-steroidal anti-inflammatory medications diclofenac and naproxen. It was shown that FAD significantly enhanced the catalytic efficiency of CYP2C9. The 4-hydroxylation of diclofenac was enhanced by 148 ± 10%. The O-demethylation of naproxen showed an increase of 120 ± 14%. Based on these data, we can assume that intake of vitamin B2 (riboflavin) improves catalytic efficiency of CYP2C9. This finding is essential for the modulation of catalytic activity of CYP2C9. The proposed electroanalytic approach is a sensitive and robust method for drug metabolism assay.

This study aimed to investigate whether the water-soluble pharmaceutical form of phosphatidylcholine nanoparticles (wPC) stimulated the catalytic activity of CYP enzymes 2C9 and 2D6. We have shown that electroenzymatic CYP2C9 catalysis to nonsteroidal anti-inflammatory drug naproxen as a substrate was enhanced from 100% to 155% in the presence of wPC in media. Electroenzymatic CYP2D6 activity in the presence of the adrenoceptor-blocking agent bisoprolol as a substrate was elevated significantly from 100% to 144% when wPC was added to potassium phosphate buffer solution. These results indicate the ability of wPC in the form of the phospholipid ultra-small nanoparticles to work as a membrane additive and crowding agent to accelerate the electroenzymatic reactions of cytochrome P450.