Chapter 6. Cytochromes P450: Tailoring a Class of Enzymes for Biosensing

Dodhia, Vikash R.; Gilardi, Gianfranco

doi:10.1039/9781847559777-00153

Cited by 8 publications

(21 citation statements)

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“…Recent studies suggest that biocatalysis using cyt P450 fusion proteins can be optimized by controlling the initial electron transfer rate. 22 …”

Section: Summary and Future Perspectivesmentioning

confidence: 99%

“…79 Also, recent reviews have emphasized protein engineering to optimize catalytic properties of cyt P450s for synthetic applications. 22,80 While obtaining sufficient materials for these application can be time consuming, one approach is to use genetically engineered bacteria to isolate bicistronic membrane-bound materials featuring lipid, a single cyt P450, and CPR. These systems can be used directly to fabricate LbL films.…”

Section: Summary and Future Perspectivesmentioning

confidence: 99%

“…19 Since that time, there has been considerable research involving cyt P450 thin film voltammetry. Much of this work is summarized in reviews, including articles by Scheller et al covering direct electrochemistry and biosensors, 20 Fleming et al 21 addressing large-amplitude Fourier transform ac voltammetry, and Gilardi et al 22 discussing electrochemistry of cyt P450s, microsomes, and fusion proteins with emphasis on genetic engineering.…”

Section: Introductionmentioning

confidence: 99%

“…22 On the other hand, electrochemical biocatalysis utilizing direct electron transfer to cyt P450s in films in aerobic solution features enzyme-catalyzed reduction of oxygen to hydrogen peroxide, which in turn reacts with cyt P450Fe III to form the active ferryloxy species that oxidizes substrates. 19-26 Neither approach fully mimics the natural catalytic pathway, which is desirable to retain all catalytic properties and features of the enzyme pathways.…”

Section: Introductionmentioning

confidence: 99%

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Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

2011

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Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This feature article discusses the direct electrochemistry of cyt P450s in thin films, and the utilization of such films for electrochemically-driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway, and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems.

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“…Recent studies suggest that biocatalysis using cyt P450 fusion proteins can be optimized by controlling the initial electron transfer rate. 22 …”

Section: Summary and Future Perspectivesmentioning

confidence: 99%

Section: Summary and Future Perspectivesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

2011

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“…Thin-film voltammetry of a number of cyt P450s has been studied [5–9]. Three general pathways to activate cyt P450s on electrodes have been elaborated: (a) direct electron transfer to pure cyt P450Fe III in films in aerobic solutions results in catalytic reduction of oxygen to hydrogen peroxide (H 2 O 2 ), which transfers oxygen to the heme iron [5]; (b) fusion proteins with an electron acceptor protein linked to a cyt P450 feature electron flow from electrode, to electron acceptor protein, to the cyt P450 [7]; and (c) microsomal CPR/cyt P450 films where electrons flow from electrode to CPR to the cyt P450 as in the natural catalytic pathway [8]. All these approaches substitute the electrode for NADPH as an electron donor.…”

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confidence: 99%

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

et al. 2012

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The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of −0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H2O2, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.

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