Improving the Efficiency of Electrocatalysis of Cytochrome P450 3A4 by Modifying the Electrode with Membrane Protein Streptolysin O for Studying the Metabolic Transformations of Drugs

Abstract

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 (ks) 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.

Purification, Characterization, and Ferrous Oxidation Kinetics of Iron Oxidase from Acidithiobacillus ferridurans

“…Furthermore, the molecular simulation and bioinformatics analysis result is rightly in line with the kinetics experimental result of the fast rate constant. Redox proteins can be used in electrochemical detection and electrocatalysis [48,49]. It was reported that genetic engineering and overexpression were successfully performed on some proteins of bioleaching microorganisms to improve their mineral bioleaching efficiency [50,51].…”

Comparative Analysis of Bioelectrocatalytic Cytochrome P450 3A4 Systems

“…При послойном нанесении на электрод ДДАБ, а затем стрептолизина O в мембранной пленке, образованной ДДАБ, наблюдалось появление полостей, визуализируемых на изображениях, полученных с помощью атомносиловой микроскопии (АСМ). Данный подход позволил зарегистрировать увеличение каталитического тока CYP 3А4 в присутствии субстрата эритромицина в 2 раза (с 0.6 мкА до 1 мкА) и повысить эффективность электрокатализа реакции N-деметилирования эритромицина в 2.97 раза [47].…”

Alternative Electron Sources for Cytochrome P450s Catalytic Cycle: Biosensing and Biosynthetic Application

“…Interaction with substrates also stabilizes the enzyme and can lead to changes in both electrochemical thermodynamics and parameters [47,73]. Enzymatic reactions in confined environments mimic the membranous surroundings and partially crowded cell media [42,[74][75][76][77][78][79]. The 2D → 3D transition by means of the incorporation of enzymes into 3D nanopores on a plane electrode makes it possible to study the electrochemical and catalytic activity of enzymes [76][77][78][79].…”

“…Enzymatic reactions in confined environments mimic the membranous surroundings and partially crowded cell media [42,[74][75][76][77][78][79]. The 2D → 3D transition by means of the incorporation of enzymes into 3D nanopores on a plane electrode makes it possible to study the electrochemical and catalytic activity of enzymes [76][77][78][79]. The enzyme immobilization in a volume-confined environment in a proper pore-forming nanomaterial could stabilize its conformation and reduce denaturation [76].…”

“…The pore-forming protein streptolysin O (SLO) was proposed to mimic the cellular environment for creating a more developed surface with protein cavities for effective immobilization of CYP3A4 enzyme based on hybrid biomembranes in the lipid-like bilayer of the electrode modifier didodecyldimethylammonium bromide (DDAB) [78]. The novelty of the approach proposed in this work is the enzyme incorporation in the three-dimensional composite DDAB/SLO, leading both to an improvement in the electron transfer properties and to the efficiency of CYP3A4 electrocatalysis.…”