Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Abstract

Microsomal cytochromes (cyt) 3 P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids. Cyts P450 require two electrons and two protons to carry out catalysis that leads to insertion of a single oxygen atom into the substrate. In the mammalian microsomal cyt P450 system, the two electrons are delivered to cyt P450 by NADPH-dependent cytochrome P450 reductase (CPR). Like cyt P450, CPR is membrane-bound and located in the membrane of the endoplasmic reticulum. CPR contains two flavin molecules, FMN and FAD. The diflavin moiety of CPR is essential for sequential electron transfer to cyt P450 as it permits CPR to accept two electrons from NADPH and transfer one electron at a time to cyt P450. The first electron from CPR reduces ferric cyt P450 to ferrous cyt P450, which rapidly binds oxygen to form oxyferrous cyt P450. The second electron is then delivered to oxyferrous cyt P450. This is followed by protonation of the reduced oxyferrous intermediate leading to heterolytic cleavage of the oxygen bond to form water and an oxyferryl intermediate, the putative, active, oxidizing species of cyt P450. An oxygen atom is inserted into the substrate, and the more hydrophilic product dissociates from the enzyme. Readers are referred to a recent review for further details about the cyt P450 reaction cycle (1).An alternative electron donor to cyt P450 is cyt b 5 , another microsomal hemoprotein also located in the endoplasmic reticulum membrane. Because of its relatively high mid-point redox potential (ϩ25 mV versus NHE), cyt b 5 can deliver only the second electron to oxyferrous cyt P450 but not the first electron to ferric cyt P450. It has been recognized for 3 decades that cyt b 5 may either increase, decrease, or not alter the activity of selected cyts P450 (2, 3). Cyt b 5 has been reported to affect the catalytic activity of more than 20 cyt P450 isoforms, including the majority of the human drug-metabolizing cyt P450 isoforms like cyt P450 3A4, 2B6, 2C9, 2C19, and 2E1 (1, 4 -8). The effect of cyt b 5 has also been shown to depend on the cyt P450 isozyme and substrate (2, 9). In the case of cyt P450 2B4 and 2E1, the electron donating properties of cyt b 5 are required for its stimulatory activity (6, 7, 9 -11), although some studies suggest that apo-cyt b 5 can stimulate the activity of cyt P450 3A4 via an allosteric effect (12). At present, the ability of apo-cyt b 5 to stimulate cyt P450 3A4 is controversial (13). Experiments performed in the reconstituted system with purified proteins have demonstrated that ferrous cyt b 5 can rapidly reduce oxyferrous cyt P450 2B4 (14,15).It is known that cyt P450 2B4 forms a 1:1 complex with CPR and with cyt b 5 in a purified, reconstituted system (16,17). A site-directed mutagenesis study of the interactions of cyt P450 2B4 with CPR and cyt b 5 has identified residues, primarily in the * This work was supported by National Institutes of Health Grant ...

Effects of theCYP2B6*6Allele on Catalytic Properties and Inhibition of CYP2B6 In Vitro: Implication for the Mechanism of Reduced Efavirenz Metabolism and Other CYP2B6 Substrates In Vivo

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Effects of a Commonly Occurring Genetic Polymorphism of Human CYP3A4 (I118V) on the Metabolism of Anandamide

“…The rates of reduction of the wild-type and the mutant P450 were determined at 25°C using a Hi-Tech SF61DX2 stopped-flow spectropho-tometer (Hi-Tech Scientific, Salisbury, Wiltshire, UK) as described previously (Zhang et al, 2008). The CYP3A4 (3 M) was reconstituted with 3 M cytochrome P450 reductase (CPR) and 10 g/ml lipid in 50 mM HEPES buffer, pH 7.4, in a final volume of 2 ml on ice for 60 min.…”

Evidence That Cytochromeb5and Cytochromeb5Reductase Can Act as Sole Electron Donors to the Hepatic Cytochrome P450 System

“…It seems that where inhibitory effects are observed they are due to competition between Cyb5 and POR, such as for a binding site on the proximal surface of CYP2B4 whereby formation of a Cyb5-P450 complex prevents ferric P450 from accepting an electron from POR and initiating the catalytic cycle. Where stimulatory effects are observed, they are due to an increase in the rapidity and efficiency of catalysis in the presence of Cyb5 compared with POR; where no effect is observed, this represents a balance between these two opposing effects (Zhang et al, 2008).…”