Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films

Abstract

NADPH-cytochrome P450 reductase (CPR) serves as electron donor for cytochrome P450 catalyzed monooxygenase reactions utilizing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as electron transfer cofactors. Here, stable films of human and rabbit CPRs with didodecyldimethylammonium bromide (DDAB), dimyristoylphosphatidyl choline (DMPC), and poly(diallyldimethylammonium) (PDDA) were made on pyrolytic graphite (PG) electrodes for comparative structural and electrochemical studies. CD and UV-VIS absorbance spectra suggested that near native CPR conformation is retained in PDDA films, and some conformational changes occur in DMPC or DDAB films. Cyclic voltammetry of these films gave quasireversible pairs of peaks at average formal potential À 0.246 AE 0.008 V vs. NHE. In human CPR-DDAB (H-CPR-DDAB), a second pair of peaks at þ 0.317 V vs. NHE was found that depended strongly on identity of buffer and salt. Excepting H-CPR in DDAB, films showed similar voltammetry, formal potentials, and k s values. While CPR-PDDA films had near native CPR structures, electrochemical parameters did not differ significantly from CPR-DMPC films. The relative independence of film voltammetry from the influence of film materials for CPRs is in contrast with heme iron proteins that, while retaining near native structures, have formal potentials that depend significantly on identity of the film material.

Electrocatalysis by subcellular liver fractions bound to carbon nanostructures for stereoselective green drug metabolite synthesis

“…On the other hand, no detectable H 2 O 2 amount was formed in the absence of HLM on PGE/MWNT electrodes under similar electrolysis conditions, suggesting the catalytic role of microsomal heme proteins in forming colorimetrically measurable peroxide levels from oxygen reduction (Figure 2). 13 We suggest the possible role of H 2 O 2 towards microsomal lipid peroxidation that could cause a detrimental effect on the organization of membrane-bound microsomal enzyme films and associated electrocatalytic bioactivity. 21,22 Thus, the observed decrease in product yield with number of reuses of the PGE/MWNT/HLM electrodes was likely due to the extensive lipid peroxidation by repeated H 2 O 2 stress caused by the electrolysis process.…”

“…Electrochemical Michaelis-Menten fitting of the testosterone catalytic currents offered apparent Michaelis-Menten affinity constants ( K M app ) of 290 ± 33 μM for PGE/MWNT/HLM and 480 ± 51 μM PGE/HLM electrodes towards testerone substrate (Figure 3C a and b). 13a The lower K M app of PGE/MWNT/HLM indicates the stronger affinity of HLM adsorbed to MWNT for testosterone.…”

“…They additionally reported layer-by-layer films of genetically engineered specific CYP with CPR or rat liver microsomes or HLM assembled with polyions for direct electrochemistry and chemical toxicity evaluations. 13 Gilardi et al engineered CYP-fused CPR proteins to enhance catalytic activity by controlling the lifetime of the active ferryloxy oxidant form of CYP. 14 Mie et al designed thiolated gold electrodes with hydrophobic units to immobilize supersomes and demonstrated electrocatalytic properties.…”

“…The cyclic voltammograms of the HLM adsorbed onto PGE/MWNT electrodes displayed a redox pair at a formal potential of −0.46 V vs. Ag/AgCl (Figure 1), which is in agreement with the formal potential of only microsomal CPR film adsorbed on similar electrodes (Figure S2) and with results of other studies. 13,18 No redox peaks at this potential region were observed for control electrodes of PGE adsorbed with a phospholipid (PL) layer or PGE/MWNT in the absence of HLM (Figure S3a,b). Thus, the cyclic voltammetry results confirmed the existence of direct electronic communication between HLM and MWNT-modified electrodes.…”

“…13,15 By obtaining the calibration plot of standard 6β-hydroxytestosterone (Figure S7), we were able to determine that 2.2 ± 0.3 and 0.45 ± 0.06 nmol of metabolite were formed by the PGE/MWNT/HLM and PGE/HLM electrodes, respectively, per unit PGE geometric area (in cm 2 ). This suggests a 5-fold enhancement in the amount of metabolite produced by HLM immobilized on MWNT-modified electrodes.…”