P-450 reductase

Asymmetric oxidation of this sulphide was also catalyzed by two isocytochromes P 450 purified from phenobarbital induced rat liver309. Both P 450 isocytochromes, termed PB-1 and PB-4, when reconstituted with purified rat liver NADPH-cytochrome P 450 reductase and cytochrome b5 afforded ethyl p-tolyl sulphoxide with S-configuration at the sulphur atom. In the case of PB-1 optical purity of this sulphoxide was 58% whereas with PB-4 it was 78%. 

DMN oxidative demethylation has been shown to be a liver mi-crosome cytochrome P-450 monooxygenase (10) Lotlikar et al. (11) found that a reconstituted enzyme system, consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidyl choline was effective in catalyzing the demethylation of DMN. The most commonly accepted mechanism for the oxidative demethylation of DMN and, by extension, of other dialkyInltrosamlnes is shown in Scheme 1. 

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

Baskin, L.S., and Yang, C.S. (1980a) Cross-linking studies of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase. Biochemistry 19, 2260-2264. 

D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, and S.H. Snyder, Cloned and expressed nitric-oxide synthase structurally resembles cytochrome-P-450 reductase. Nature 351, 714-718 (1991). 

Microsomal NADPH-Cytochrome P-450 Reductase and NADH cytochrome b5 Reductase... 

In addition to nitric oxide, superoxide, and peroxynitrite, NO synthases are able to generate secondary free radicals because similar to cytochrome P-450 reductase, the reductase domain can transfer an electron from the heme to a xenobiotic. Thus it has been found [158,159] that neuronal NO synthase NOS I catalyzed the formation of CH3CH(OH) radical from ethanol. It was suggested that the perferryl complex of NOS I is responsible for the formation of such secondary radicals. Miller [160] also demonstrated that 1,3-dinitrobenzene mediated the formation of superoxide by nNOS. It was proposed that the enhancement of superoxide production in the presence of 1,3-dinitrobenzene converted nNOS into peroxynitrite-produced synthase and may be a mechanism of neurotoxicity of certain nitro compounds. 

MICROSOMAL NADPH-CYTOCHROME P-450 REDUCTASE AND NADH CYTOCHROME b5 REDUCTASE... 

The primary function of flavoprotein NADPH-cytochrome P-450 reductase is the hydro-xylation of various substrates, which occurs during electron transfer from NADPH to cytochrome P-450 [1] ... 

While cytochrome P-450 catalyzes the interaction with substrates, a final step of microsomal enzymatic system, flavoprotein NADPH-cytochrome P-450 reductase catalyzes the electron transfer from NADPH to cytochrome P-450. As is seen from Reaction (1), this enzyme contains one molecule of each of FMN and FAD. It has been suggested [4] that these flavins play different roles in catalysis FAD reacts with NADPH while FMN mediates electron... 

It has been believed that P-450 reduction by NADPH cytochrome P-450 reductase is a biphasic process, but it was recently shown [7] that some P-450 cytochromes are reduced with single-exponential kinetics and that the presence of substrate is not an obligatory condition for the reduction of all P-450 forms. Thus, the kinetics of reduction of various ferric P-450 cytochromes possibly depends on many factors such as substrate, rate-limiting step, etc. 

Although it is still unclear whether the formation of oxidized and hydroxylated products, which is the main pathway of catalytic activities of cytochrome-R-450 reductase, is mediated by free radicals, mitochondrial enzymes are certainly able to produce oxygen radicals as the side products of their reactions. It has been proposed in earlier studies [14,15] that superoxide and hydroxyl radicals (the last in the presence of iron complexes) are formed as a result of the oxidation of reduced NADPH cytochrome-P-450 reductase ... 

Superoxide generation was detected via the NADPH-dependent SOD-inhibitable epinephrine oxidation and spin trapping [15,16], Grover and Piette [17] proposed that superoxide is produced equally by both FAD and FMN of cytochrome P-450 reductase. However, from comparison of the reduction potentials of FAD (-328 mV) and FMN (190 mV) one might expect FAD to be the most efficient superoxide producer. Recently, the importance of the microsomal cytochrome h558 reductase-catalyzed superoxide production has been shown in bovine cardiac myocytes [18]. 

If the mechanism of superoxide production in microsomes by NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, and cytochrome P-450 is well documented, it cannot be said about microsomal hydroxyl radical production. There are numerous studies, which suggest the formation of hydroxyl radicals in various mitochondrial preparations and by isolated microsomal enzymes. It has been shown that the addition of iron complexes to microsomes stimulated the formation of hydroxyl radicals supposedly via the Fenton... 

Then, A-hydroxyphentermine supposedly reacts with superoxide generated by NADPH-cytochrome P-450 reductase and forms the final product 2-methyl-2-nitro-l-phenylpropane ... 

Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

It has earlier been suggested to make cytochrome c a more specific reagent for superoxide detection by its acetylation or succinoylation [9-11], It was proposed that acetylation and succinoylation must cause a greater decrease in the reaction of cytochrome c with NADPH cytochrome P-450 reductase than with superoxide due to a decrease in the electrostatic charge of native cytochrome c [12]. However, the rate constant for the most selective succinoylated cytochrome c became about 10% of native cytochrome [13], making this assay even less sensitive. 

Fig. 10.8 Selected cDNAs isolated in recent years that encode enzymes involved in the biosynthesis of various classes of isoquinoline alkaloids. 6-OMT, norcoclaurine 6-0-methyltransferase 23 CYP80A1, berbamunine synthase 19 CYP80B1, (S)-A-methylcoclaurine 3 -hydroxylase 20 CPR, cytochrome P-450 reductase 29 4 -OMT, (5)-3 -hydroxy-A-methylcoclaurine 4 -0-methyltransferase 30 BBE, berberine bridge enzyme 12 SalAT, salutaridinol 7-O-acetyltransferase 28 COR, codeinone reductase.25...

While the cytochrome P-450 monooxygenase reaction described in Eq. (1) often involves hydroxylation of carbon, many other reactions are catalyzed by these enzyme systems. These reactions include oxidation of nitrogen and sulfur, epoxidation, dehalogenation, oxidative deamination and desulfuration, oxidative N-, O-, and S-dealkylation, and peroxidative reactions (56). Under anaerobic conditions, the enzyme system will also catalyze reduction of azo, nitro, N-oxide, and epoxide functional groups, and these reductive reactions have been recently reviewed (56, 57). Furthermore, the NADPH-cytochrome P-450 reductase is capable of catalyzing reduction of quinones, quinonimines, nitro-aromatics, azoaromatics, bipyridyliums, and tetrazoliums (58). 

Vermilion, J.L. and Coon, M.J. (1978) Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. Journal of Biological Chemistry, 253 (8), 2694-2704. 

Bhattacharyya, A.K., Lipka, J.J., Waskell, L. and Tollin, G. (1991) Laser flash photolysis studies of the reduction kinetics of NADPH cytochrome P-450 reductase. Biochemistry, 30 (3), 759-765. 

Figure 6.27 Passage of reducing power from NADPEH to form water. One atom of oxygen is incorporated into the toxic compound ( oxidation). NADPEI-cytochrome P-450 reductase is a flavin-containing protein...

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

Komiyama, T., Kikuchi, T., and Sugiura, Y., 1986, Interactions of anticancer quinone dmgs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C, with NADPH-cytochrome P-450 reductase, xanthine oxidase and oxygen, J. Pharmacobiodyru 9 651-664. 

Qrunones can accept one or two electrons to form the semiquinone anion (Q ") and the hydroquinone dianion (Q ). Single-electron reduction of a quinone is catalyzed by flavoenzymes with relatively low substrate selectivity (Kappus, 1986), for instance NADPH cytochrome P-450 reductase (E.C. 1.6.2.3), NADPH cytochrome b5 reductase (E.C. 1.6.2.2), and NADPH ubiquinone oxidoreductase (E.C. 1.6.5.3). The rate of reduction depends on several interrelated chemical properties of a quinone, including the single-electron reduction potential, as well as the number, position, and chemical characteristics of the substituent(s). The flavoenzyme DT-diphorase (NAD(P)H quinone acceptor oxidoreductase E.C. 1.6.99.2) catalyzes the two-electron reduction of a quinone to a hydroquinone. 

Anandatheerthavarada HK, Boyd MR, Ravindranath V. 1992. Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and... 

Ravindranath V, Anandatheerthavarada HK, Shankar SK. 1990. NADPH cytochrome P-450 reductase in rat, mouse and human brain. Biochem Pharmacol 39 1013-1018. 

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