NADPH-Cytochrome P450 Reductase CPR

Cytochrome P450 reductase is the physiological reductant of a wide range of microsomal cytochrome P450 monooxygenase isozymes that 

During catalytic turnover, NADPH reduces FAD and the FAD subsequently reduces FMN by two electrons with the latter eyeling between the hydroquinone and semiquinone states while carrying out the 1-electron reduction of the P450 heme iron (Masters et al., 1966 Backes and Reker-Backes, 1988). The midpoint redox potentials of the FAD are El(,x/sq n290 mV and fi365 mV and of the FMN are El(,x/sq nl lOmV and 

E sq/red fl270mV (lyanagi et al., 1974). Electron transfer rates of FAD to FMN within CPR are difficult to measures and somewhat controversial and diseussion of them is beyond the scope of this chapter. 

Both the microsomal CPR and P450 are integral membrane proteins. CPR has two functional domains, an N-terminal 6kDa hydrophobic domain that serves to anchor the enzyme to the endoplasmic reticulum, and a 72kDa hydrophilic C-terminal catalytic domain (Kasper, 1971). In the absence of the N-terminal membrane anchor, which can be cleaved by proteolysis or omitted from the expression system in E. coll, CPR is incapable of reducing P450 but is able to reduce cytochrome c and several artificial electron acceptors. 

The electron transfer rates in P450BM3, measured by laser flash photolysis using semicarbazide-aetivated 5-deazflavin semiquinone, show that no reduction of the native BMP heme oeeurs even though FMN could be reduced rapidly to the semiquinone (Hazard et al., 1997). In the presence of earbon monoxide, which can displace water from the sixth coordination site of iron and convert the low-spin ferric iron to high spin, the intramolecular electron transfer rate is 18sec . In the presence of both CO and the substrate myristic acid, an intramolecular electron transfer rate of up to 250sec can be obtained. 

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Cinnamate 4-hydroxylase (C4H EC 1.14.13.11, also defined as CYP73A [36]) catalyzes the p-hydroxylation of trani-cinnamate to form trani -p-coumarate. The enzyme has a requirement for molecular oxygen and NADPH as well as association with the electron donor NADPH-cytochrome P450 reductase (CPR EC 1.6.2.4) for activity. C4H was the first characterized plant P450 [37, 38]. 

The crystal structure of NADPH-cytochrome P450 reductase (CPR), the common electron-transfer protein of Class 11 eukaryotic P450 systems, was reported in 1997. This was followed by the structures of adrenodoxin reductase (AdR) and adrenodoxin (Adx), the two electron-transfer proteins of the Class 1 mitochondrial P450 system. The crystal structure of a cross-linked AdR-Adx complex has also been reported. " Putidaredoxin reductase (PdR) and putidaredoxin (Pd) of the P450cam system have also been structurally characterized. ... 

Figure 7 The crystal structure of NADPH-cytochrome P450 reductase (CPR). The FAD and FMN domains are linked by a hinge domain...

The realization of functional human cytochrome P450 activity in bacteria requires delivering a supply of electrons via co-expression of the human NADPH cytochrome P450 reductase (CPR) with each cytochrome P450. In some cases modifications are required in either the coding sequence itself or in the codon usage to enhance translation in bacteria. Additionally, deletions and substitutions in the 5 -end of some of the genes have been made to allow more efficient expression in bacteria. Ultimately, the coupled enzymes must be capable of insertion into the membrane. 

The discovery of c5 ochromes P450 in mammalian tissues rich with these proteins, such as liver and the adrenal gland, resulted in intense scrutiny of their roles in xenobiotic metabolism and endogenous functions. Following their discovery came the realization that mammalian proteins required electron donor systems for activity, either NADPH-cytochrome P450 reductase (CPR) or adrenodoxin and adrenodoxin reductase in the endoplasmic reticulum or the mitochondria respectively Protein biochemistry and molecular biology revealed the multiplicity of CYP forms and mammalian genomes exhibited CYP diversity. 

LOPES CARDOSO, M.I., MEUER, A.H., RUEB, S., MACHADO, J.A., MEMELINK, J., HOGE, J.H.C., A promoter region that controls basal and elicitor-inducible expression levels of the NADPH cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds nuclear factor GT-1. Mol. Gen. Genet., 1997, 256, 674-681. 

Abbreviations used in text CYP, cytochrome P450 HMPA, hexamethylphosphoramide NDEA, N-nitrosodiethylamine NNK, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone CPR, NADPH-cytochrome P450 reductase RT-PCR, reverse transcriptase-polymerase chain reaction TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin AFB1, aflatoxin Bl BaP benzo(a)pyrene G.I., gastrointestinal. 

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