Protein Film Electrochemistry of Microsomes Genetically Enriched in Human Cytochrome P450 Monooxygenases

Abstract

This communication demonstrates direct electron delivery from electrodes to cyt P450 reductases in stable films ( approximately 100 nm thick) of genetically enriched CYP1A2 and CYP3A4 microsomes made by layer-by-layer assembly with polyions. Reversible voltammetry of films containing genetically enriched cyt P450 monooxygenase microsomes was shown to involve cyt P450 reductase by comparison with the pure rabbit reductase and by lack of characteristic reactions of iron heme enzymes, such as reaction of the FeII form with CO and catalytic electrochemical reduction of oxygen and hydrogen peroxide. The microsome films were activated electrochemically to catalyze styrene epoxidation, consistent with the pathway utilized in the human liver, although further work is required to establish this definitively.

Thin film voltammetry of metabolic enzymes in rat liver microsomes

“…This is considerably negative of the potential expected for CYP enzymes in similar films (ca. −0.3 V vs SCE) [8,12] but very similar to the peak potential observed for rabbit cyt P450 reductase in polyion films (−0.49 V at pH 7.4), and films of genetically enriched Supersomes containing human monooxygenase systems [5]. PDDA/ microsome peaks were not shifted by addition of CO, and they show very little catalytic reduction of oxygen or hydrogen peroxide.…”

“…We recently reported direct electrochemistry of so called Supersomes, or microsomes genetically enriched in CYP1A2 or CYP3A4 [5]. The voltammetric peaks found were attributed to oxidoreductases such as CYP NADPH-reductase, not CYPs.…”

“…These oxidoreductases include NADPH-cytochrome P450 reductase, the natural electron donor for all microsomal cytochrome CYP-catalyzed oxidations [1,3]. Electrochemical reduction of these oxidoreductases in the supersome films was able to convert styrene to styrene oxide in low yield, presumably via electron transfer to oxidoreductases, and then to the CYPs [5].…”

Protein film voltammetry: electrochemical enzymatic spectroscopy. A review on recent progress

“…The minimization of the adsorptive surface denaturation of proteins and keeping the electrode surface as clean as possible, are essential factors which facilitate the direct electron exchange between an electrode and the redox site of the proteins. Several metal electrodes coated with metal oxides, carbon electrodes coated with organic monolayers, and pyrolytic graphite edge plane electrodes are currently known as the most suitable surfaces fulfilling these requirements [5,[11][12][13][14][15][16][17][18][19]. In the last few years, basal plane pyrolytic graphite electrodes, glassy carbon electrodes, and even unmodified metallic electrodes have been extensively used in PFV [5].…”

“…These electrodes contain a big fraction of the negative carboxylate groups in their three-dimensional structure that can interact electrostatically (or by hydrogen bonds) with positively charged amino acids of many proteins [12][13][14][15][16][17][18][19]. This contributes to better sticking of many proteins to the electrode surface.…”