Cytochrome P450 as an oxene transferase.

In the presence of iodosobenzene, liver microsomes catalyze the O-dealkylation of 7-ethoxycoumarin. The reaction proceeds in the absence of NADPH and O₂ and is dependent on cytochrome P450. The results indicate that cytochrome P450 acts as an oxene transferase probably involving [FeO]³⁺ as the transient intermediate of active oxygen.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Multiple mechanisms of cytochrome P450-catalyzed substrate hydroxylations

We have examined the 5-$\text{exo}$-hydroxylation of camphor by cytochrome P450 in [¹⁸O] water/buffer solution. In the $\text{NADH}\text{O}{}_2\text{-dependent}$ reaction of the reconstituted multienzyme system, no ¹⁸O-label is observed in the product alcohol. Similarly, in the m-chloroperbenzoic acid or cumene hydroperoxide supported reactions with ferric P450, solvent oxygen is not incorporated into hydroxycamphor. When the analagous reaction is carried out using iodosobenzene as the exogenous oxidant, however, the alcoholic oxygen of the product is derived entirely from the solvent. These results cannot be explained by equilibration of the iodosobenzene oxygen with solvent water before reacting with P450, and suggest a unique mechanism for iodosobenzene-supported P450 oxygenations. We propose two distinct mechanistic activities for cytochrome P450: a hydroxylase, and an oxene transferase, with the former encompassing the classic oxygenase as well as “peroxygenase” reactions.     

Source of oxygen in cytochrome P-450 catalyzed carbinolamine formation

N-Methylcarbazole was incubated in H₂O¹⁸ and under an ¹⁸O atmosphere. N-Hydroxymethylcarbazole, 1-OH- and 3-OH-N-methylcarbazole were isolated by HPLC and analyzed for ¹⁸O content In incubations containing ¹⁸O, all three metabolites showed >95% ¹⁸O incorporation. In incubations containing H₂O¹⁸, the N-hydroxymethyl metabolite showed ¹⁶O incorporation equal to control incubations in 100% H₂O. These data demonstrate that the sole source of oxygen in cytochrome P-450 catalyzed, NADPH supported N-hydroxymethylcarbazole formation is atmospheric oxygen.     

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b₅ from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol (α,α-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H₂¹⁸O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.     

Uncoupling of oxygen activation from hydroxylation in the steroid C-21 hydroxylase of bovine adrenocortical microsomes

Androst-4-ene-3,17-dione, although not hydroxylated by the C-21 hydroxylase, induces Type I spectral change in cytochrome P-450 and stimulates NADPH oxidation and oxygen consumption. The androstenedione-dependent oxygen activation results in reduction of oxygen to water without producing a free peroxide intermediate as evidenced by the ratio of NADPH oxidized to O₂ consumed being equal to 2 in the presence of this steroid.     

Decomposition of unsaturated phospholipid by iron-ADP-adriamycin co-ordination complex

The system, which contains NADPH, purified cytochrome P-450 reductase, and adriamycin, produces H₂O₂ and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in appreciable amounts with oxygen consumption and NADPH oxidation under aerobic conditions. Such an adriamycin-induced NADPH oxidation system, however, does not cause the decomposition of unsaturated fatty acids in microsomal phospholipid micelles, suggesting no direct participation of the active oxygen species and semiquinone radicals of adriamycin in lipid peroxidation. Adriamycin produces a co-ordination complex with Fe³⁺ and ADP, which, but no Fe³⁺-ADP complex, could be reduced by NADPH-cytochrome P-450 reductase at the expence of NADPH. The decomposition of unsaturated fatty acids in phospholipid micelles is achieved by the Fe³⁺-ADP-adriamycin complex and strikingly enhanced by enzymatically reduced iron-ADP-adriamycin complex.     

Environment-dependent mechanism of dehalogenation by Rhodococcus chlorophenolicus PCP-1

Cells and cell-free preparations of a soil-bioremediating organism, Rhodococcus chlorophenolicus PCP-1, dehalogenated polychlorophenols both under aerobic and anaerobic conditions. Under aerobic conditions molecular O₂ served as the source of oxygen for the dechlorinating para-hydroxylation reaction. Chlorophenols were dehalogenated and para-hydroxylated also under anaerobic conditions by a cyt P-450 enzyme. Water was used anaerobically as an oxygen source but the reaction required the presence of sulphite ions or iodosobenzene. When the dehalogenating enzyme was given a choice between molecular O₂ and water in the presence of sulphite ions or iodosobenzene, the oxygen was preferably derived from water.Dedicated to the honour of Prof. Dr. Norberto Palleroni for the occasion of his 70th birthday Correspondence to: J. S. Uotila     

The final step of aldosterone biosynthesis is catalyzed by an NADPH-dependent and molecular oxygen-requiring enzyme

Using sonicated mitochondria fraction prepared from bovine adrenal glomerulosa cells, aldosterone biosynthesis from 18-hydroxycorticosterone was examined as its final step, as production of [³H]-aldosterone from [³H]-corticosterone was strongly reduced by addition of non-radioactive 18-hydroxycorticosterone during the incubation. Significant conversion of 18-hydroxycorticosterone to aldosterone by the mitochondria sonicate was observed in the presence of NADPH, but not NADP⁺. This reaction was almost completely inhibited in the atmosphere of 100% carbon monoxide in the presence of either NADP⁺, or NAD⁺, and significantly reduced in the mixture of carbon monoxide and oxygen (90:10) in the presence of NADPH. Several drugs, such as SU compounds, spironolactone, amphenone B and SKF 525A which affect cytochrome P-450 blocked production of aldosterone from 18-hydroxycorticosterone. From these results, we conclude that a mixed function oxidase involving a cytochrome P-450 is engaged in the final course of aldosterone biosynthesis.     

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome $\text{c}$ reductase and 70% in an atmosphere of CO:O₂ (9:1) when compared to an atmosphere of N₂:O₂ (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.     

Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction

The cytochrome P-450 (P-450_sccII) and its reductase, NADPH-cytochrome reductase [EC 1.6.2.4], associated with conversion of progesterone to 4-androstene-3,17-dione, were extensively purified from pig testis microsomes. Higher lyase activity (turnover number of 15 mol of the product formed/min/mol of P-450) could be restored by mixing the P-450_sccII, its reductase, pig liver cytochrome b₅ and cytochrome b₅-reductase [EC 1.6.2.2], and phospholipid in the presence of NADPH, NADH, and O₂. Omission of either cytochrome b₅ or NADH resulted in a significant loss of the lyase activity indicating actual participation of cytochrome b₅ in this P-450-mediated steroidogenic system in the testis.     

The oxidative metabolism of arachidonic acid by purified cytochromes P-450

Arachidonic acid is catalytically oxidized using either of two types of purified cytochrome P-450 reconstituted with the purified flavo-protein, NADPH-cytochrome P-450 reductase. The reaction is dependent on the presence of cytochrome P-450, NADPH, and oxygen. The patterns of products formed are unique for the type of cytochrome P-450 used. This suggests an enzyme-directed specificity of the site of attack on the unsaturated fatty acid by the hemeprotein. Additional experiments show a possible role for cytochrome b₅ since the addition of purified cytochrome b₅ enhances the rate of metabolism of arachidonic acid 2 to 3 fold.     

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.     

A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria

The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: Assay and characterization of the enzyme system

Microsomal preparations of $\text{Aspergillus ochraceus}$ TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and O₂ and exhibits a pH optimum of 8.0–8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistant with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.     

Further characterization of the 2-deoxyecdysone C-2 hydroxylase from Locusta migratoria

Additional data are provided on the enzyme 2-deoxyecdysone C-2 hydroxylase which has been shown in a previous study (Kappler et al., 1986) to be a mitochondrial hydroxylase with some classical characteristics of a cytochrome P-450 monooxygenase but which appeared to be insensitive to CO. Using ¹⁸O₂, we have now demonstrated that molecular oxygen is directly incorporated into ecdysone during the process of C-2 hydroxylation. Neither cumene hydroperoxide nor linoleyl hydroperoxide could support C-2 hydroxylation. When the reaction was sustained by α-ketoglutarate, addition of cofactors like Fe²⁺, ascorbate and catalase caused only a slight increase of the enzymatic activity whereas the α-ketoglutarate-dependent hydroxylation was largely decreased in the presence of malonate; these data eliminate the possible existence of a dioxygenase mechanism for C-2 hydroxylation.The paper also provides inhibition kinetics which indicate that 2-deoxy-20-hydroxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone are competitive inhibitors of the C-2 hydroxylase whereas the 3-epi isomer of 2-deoxyecdysone is a non-competitive inhibitor.     

Interactions of steroids with human placental cytochrome P-450 in the presence of carbon monoxide.

Spectral analyses of the carbon monoxide (CO) complex of human placental microsomal cytochrome P-450 revealed absorption maxima at 426 and 450 nm when NADPH (2×10⁻⁴M) was utilized as a reducing agent. Additional NADPH or NADH did not produce any further increases in the absorption maximum at 450 nm. A period of 10–15 minutes was required for the complete reduction. Various steroids were added to both sample and reference cuvettes to examine their interactions with the CO-cytochrome P-450 complex. The resulting spectral changes indicated that low concentrations of steroids (≃10⁻⁷M) such as androstenedione, 19-hydroxyandrostenedione, 19-oxoandrostenedione and testosterone completely eliminated the absorbance maxima at 450 nm while 19-norandrostenedione, 19-nortestosterone, pregnenolone and benzo[a]-pyrene did not eliminate this peak. Since ample time was allowed to reduce the cytochrome P-450 with NADPH, the observed interaction of steroids with cytochrome P-450 in the presence of CO does not represent an effect on reductase activity, but on the formation of the CO-cytochrome P-450 complex.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450_LM2, cytochrome b₅ enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or $\text{p}$-nitroanisole about 5-fold. Cytochrome b₅ did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b₅-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H₂O₂ or $\text{O}{}_2{}^{\mathrm{?}}$ in the system, thus strongly indicating cytochrome b₅ being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.