Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Mechanisms of hydroxylation by cytochrome P-450: Exchange of iron-oxygen intermediates with water

Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from ¹⁸O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H₂¹⁸O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from ¹⁸O₂ into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O₂ plus NADPH.     

Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.     

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome $\text{c}$ per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase.

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

Ethanol and drug metabolism in mouse liver microsomes subsequent to lipid peroxidation-induced destruction of cytochrome P-450

Preincubation of mouse liver microsomes with NADPH resulted in malondialdehyde formation, destruction of cytochrome P-450, and decreased rates of aniline hydroxylation and N-demethylation of aminopyrine and ethylmorphine. These phenomena were more pronounced in phosphate than in Tris buffer. No reduction in rates of NADPH-linked oxidation of ethanol or in the activities of NADPH oxidase and NADPH-cytochrome c reductase was observed. While addition of EDTA to preincubation mixtures prevented lipid peroxidation, loss of cytochrome P-450, and inactivation of the drug-metabolizing capacity of microsomes, it did not alter ethanol oxidation rates and the activities of NADPH oxidase and NADPH-cytochrome c reductase. These findings argue against the involvement of cytochrome P-450 in the microsomal ethanol-oxidizing system.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

7 Alpha-hydroxylation of cholesterol by reconstituted systems from rat liver microsomes

A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-¹⁴C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome $\text{c}$ as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min.     

Substrate reduction and oxygen activation during microsomal metabolism of quinones

2-Dimethylamino-3-chloro-1,4-naphthaquinone (DCNQ) was used to study oxygen and substrate activation in microsomal system. DCNQ was shown to be bound to microsomal cytochrome P-450 as a type I substrate; its N-demethylation was catalyzed by cytochrome P-450. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are capable of DCNQ reduction to semi- and hydroquinones. The OH-radical formed in the presence of DCNQ, NADPH and reductase was detected, using a spin trap (5,5-dimethylpyrroline-N-oxide). The OH-radical formation was shown to be stimulated by the Fe-EDTA complex. Using the OH-radical scavengers (mannitol, N-butanol, alpha-naphthol) and the catalase inhibitor sodium azide, it was shown that the OH-radical participates in microsomal oxidation of DCNQ and aminopyrine. It was assumed that in the course of microsomal oxidation the reduced DCNQ is responsible for: i) stimulation of molecular oxygen reduction to H2O2; ii) reduction of Fe ions (Fe3+----Fe2+) which cause the decomposition of H2O2 in the Fenton reaction resulting in the formation of a strong oxidizing agent--a hydroxyl radical.     

Characterization of 6 alpha-hydroxylation of taurochenodeoxycholic acid in pig liver

The properties of the species-specific 6 alpha-hydroxylation of taurochenodeoxycholic acid were studied in subcellular fractions from pig liver. The hydroxylation was observed in microsomes but not in mitochondria. A partially purified cytochrome P-450 fraction in the presence of NADPH-cytochrome P-450 reductase, NADPH, and phospholipid catalyzed 6 alpha-hydroxylation of taurochenodeoxycholic acid at a 160-fold higher rate than the microsomes. This cytochrome P-450 fraction did not catalyze 6 alpha-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol or testosterone, nor did it catalyze 7 alpha-hydroxylation of cholesterol.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

A highly purified preparation of cytochrome P-450 from microsomes of anaerobically grown yeast.

Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b₅, NADH-cytochrome b₅ reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic cytochrome P-450.