Demethylation of tertiary amines by a reconstituted cytochrome P-450 enzyme system: kinetics of oxygen consumption and hydrogen peroxide formation

Initial reaction rates of oxygen consumption and hydrogen peroxide formation in a cytochrome P-450 catalyzed reaction are practically independent of the nature of tertiary amines that were used as substrates. From the kinetic studies and the substrate conversion results that the amount of water formed in a side reaction is determined by the substrate specificity. Both hydrogen peroxide and water formation lower the efficiency of the monooxygenatic activity of cytochrome P-450.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin

The kinetic characteristics and mechanism of flavonoid inhibition of cytochrome P-450-mediated reactions were examined in rat liver microsomes, using the naturally occurring flavonoid, quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin inhibited the O-deethylation of ethoxyresorufin in beta-naphthoflavone-induced microsomes by 15-80% at concentrations of 10-250 nM. The pattern of inhibition was dependent on quercetin concentration. Quercetin also inhibited p-nitroanisole demethylation and benzo(a)-pyrene hydroxylation, but did not change the proportions of the individual benzo(a)pyrene metabolites in comparison to controls. Specific steps in the P-450 reaction pathway were tested for sensitivity to quercetin inhibition. The Km values of the P-450 substrates tested were increased in the presence of quercetin; competition for and/or alteration of the substrate binding site contributes to the mechanism of inhibition. In experiments under anaerobic, carbon monoxide-saturated conditions, quercetin did not inhibit cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase. The cumene hydroperoxide-supported O-deethylation of ethoxyresorufin was inhibited by quercetin (15-60% inhibition at concentrations of 50-300 nM), suggesting that quercetin may interfere with the formation or breakdown of the oxygenated heme complex. Stoichiometry experiments established that quercetin is a potent uncoupler of P-450 reactions, elevating the rates of H2O2 formation almost twofold. Structure/activity studies indicated that certain other naturally occurring flavonoids were at least as potent inhibitors of ethoxyresorufin deethylation as quercetin. These findings are of interest in light of the significant dietary exposure of the human population to the flavonoids.     

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.     

Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.     

Substrate specificity of the carbon monoxide-dependent cytochrome P-450 kinetics

The substrate-dependent kinetics of the carbon monoxide-inhibited cytochrome P-450 activity and its light reversibility is reinvestigated in microsomal preparations. In order to find out whether the substrate specificity is mediated by an isoenzyme-specific binding of carbon monoxide with different dissociation constants an experimental design has been chosen where it could be established that essentially the same isoenzyme component was involved in two different monooxygenase reactions, i.e., the O-dealkylation of 7-ethoxycoumarin and the 7-hydroxylation of coumarin. The dissociation constant kD(CO) of the ferrous cytochrome P-450 carbon monoxide complex is 6-fold higher in the presence of 7-ethoxycoumarin than in the presence of coumarin. But the light-induced relative changes of the Warburg partition coefficient for the 7-ethoxycoumarin deethylation and for coumarin 7-hydroxylation do not differ remarkably from each other. These relative changes are shown to represent the ratio of the photoinduced rate constant to the spontaneous rate constant of the dissociation for the ferrous cytochrome P-450 carbon monoxide complex. The differences in the dissociation constants are assigned to substrate specific effects on the carbon monoxide binding, indicating a substrate-specific change of the free binding enthalpy for carbon monoxide.     

The Aqueous Two-Phase System Polyethylene Glycol/Dextran as a Reaction Medium for the Microsomal Monooxygenase System

An aqueous two-phase system of dextran and polyethylene glycol was investigated as a reaction medium for pig liver microsomes in order to find out if the partition of the microsomes, of the substrate 7-ethoxycoumarin and of the product 7-hydroxycoumarin favoured any biotechnological perspectives. Cytochrome-P-450 and NADPH-cytochrome P-450 reductase concentrations and the monooxygenase 7-ethoxycoumarin deethylation activity were measured under a variety of the system parameters. Microsomes totally partition into the bottom phase whereas the concentration ratio of substrate to product is higher in the microsome free top phase. An unfavourable effect is the specific partial deactivation of the cytochrome P-450 by polyethylene glycol.     

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.     

Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam: kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate

The kinetics of NADH consumption, oxygen uptake, and hydrogen peroxide production have been studied for norcamphor metabolism by cytochrome P-450cam. The kinetic deuterium isotope effects on these processes, with specifically deuteriated norcamphor, are 0.77, 1.22, and 1.16, respectively. Steady-state UV-visible spectroscopy indicates that transfer of the second electron to the dioxy ferrous P-450 is the rate-limiting step, as it is when camphor is the substrate. The inverse deuterium isotope effect for NADH consumption is consistent with an isotope-dependent branching between monooxygenase and oxidase activity, where these reactivities differ in their NADH:oxygen stoichiometries. However, no isotope-dependent redistribution of steady-state intermediates was detected by isotopic difference UV-visible spectroscopy in the presence of norcamphor. The kinetic isotope effects and steady-state spectral results suggest that the high-valent iron-oxo hydroxylating intermediate [FeO]3+ is reduced by NADH and the physiological electron-transfer proteins to afford water.     

Oxycytochrome P-450: its breakdown to superoxide for the formation of hydrogen peroxide.

Four different experimental studies are described which were designed to evaluate the role of oxycytochrome P-450 in the formation of superoxide anions and hydrogen peroxide. The use of lipophilic copper chelates with superoxide dismutase like activity revealed that the primary site of interaction of these agents is related to the inhibition of the flavoprotein. NADPH-cytochrome P-450 reductase. Measurements of the proton assisted nucleophilic displacement of superoxide from oxycytochrome P-450 by high concentrations of sodium azide indicated an increase in the rate of hydrogen peroxide formation concomitant with the inhibition of the N-demethylation of ethylmorphine. Studies on the effect of NADH on the rate of hydrogen peroxide formation during NADPH oxidation by liver microsomes failed to reveal a stimulatory or synergistic effect in a manner analogous to results obtained during the cytochrome P-450 dependent oxidation of substrates such as ethylmorphine. These results suggest that hydrogen peroxide formation may not require the reduction of oxycytochrome P-450 to peroxycytochrome P-450. Measurements of the reduction of succinylated cytochrome c using purified cytochrome P-450 and the flavoprotein, NADPH-cytochrome P-450 reductase, directly demonstrate the formation of superoxide anions. It is concluded that oxycytochrome P-450 may decompose to generate hydrogen peroxide.     

Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups

The role of cytochrome b5 heme propionate groups in the functional interactions between cytochromes P-450 RLM5 and b5 has been investigated by comparing the capacity of RLM5 to interact with both native b5 and a b5 derivative in which the native heme was replaced with ferric protoporphyrin IX dimethyl ester (DME-b5). Both forms of b5 interacted with RLM5 causing an increase in the RLM5 spin state from 28 to 68% high-spin RLM5 at saturation, as judged using uv-visible spectrophotometry. However, DME-b5 exhibited a 7-fold weaker affinity for RLM5. The apparent dissociation constant (Kd) for the interaction between RLM5 and b5 was also shown to be a strong function of ionic strength, in a manner consistent with the involvement of electrostatic attraction in complex formation. Reconstitution of b5 into an RLM5-dependent monooxygenase system stimulated the p-nitroanisole demethylase rate about 25-fold and 7-ethoxycoumarin deethylase about 6-fold. DME-b5, however, produced only 30% of the stimulation of RLM5-dependent turnover of p-nitroanisole observed at equivalent concentrations of native b5 without a change in Km. With 7-ethoxycoumarin, turnover was 50% diminished. The diminished capacity of DME-b5 to stimulate RLM5-dependent substrate turnover was shown not to be due to impairment of electron flow between NADPH-cytochrome P-450 reductase and DME-b5, since the Km of reductase for DME-b5 is 2.5-fold lower, and the Vmax is actually increased, but rather to an impairment of some aspect of functional interaction between the DME-b5 and RLM5. The data show that complex formation between cytochrome P-450 and b5 involves electrostatic attraction mediated in part by cytochrome b5 heme propionate groups.     

Effects of hydrogen peroxide on cytochrome P-450 inactivation

Inactivation rate of purified oligomeric cytochrome P-450 LM2 has been investigated in glucose oxidase system and under the action of exogenous hydrogen peroxide (400 microM). It has been found that hydrogen peroxide has a distinct inactivating effect on cytochrome P-450. The enzyme inactivation is accompanied by the loss of heme and the decrease in SH-group content in the protein molecule. Benzphetamine, a substrate specific for this enzyme isoform, exerts a protective effect by decreasing the rate of cytochrome P-450 inactivation and SH-group oxidation. Similar results have been obtained during the investigation of cytochrome P-450 inactivation in the monomerized system. It has been found that the inactivation process is accompanied by the formation of the enzyme aggregates. The changes in the aggregate state are due to the formation of intermolecular covalent bonds.     

Conformational change of cytochrome P-450 indicated by the measurement of fluorescence-energy transfer

The conformation of cytochrome P-450 was studied in relation to the interaction with its substrate by the measurement of fluorescence energy transfer. Cytochrome P-450 I-c and cytochrome P-450 II-d, both obtained from the hepatic microsome of polychlorinated biphenyl-treated rats, were used as P-450 type and P-448 type, respectively, and benzo[a]pyrene and 7-ethoxycoumarin were used as substrate. The distance between the donor and acceptor, which was described by the F?rster equation, was calculated on the basis of the fluorescence-energy transfer between the substrate as a donor and the heme of the enzyme as an acceptor. The distance was apparently changed by incorporation of the enzyme into phospholipid or by reduction of the heme, indicating that the treatments changed the conformation of the enzyme. Differences in the conformational change were observed between the two enzymes, and the conformational change of cytochrome P-450 II-d using benzo[a]pyrene as a substrate was different from that using 7-ethoxycoumarin. The results suggest that the substrate-binding sites of the two enzymes differ in position toward the heme, and that there are at least two different substrate sites in cytochrome P-450 II-d, one for benzo[a]pyrene and another for 7-ethoxycoumarin.     

On the mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 by chloramphenicol

The mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (P-450 PB-B2) by chloramphenicol has been investigated. Preparations of the enzyme from animals treated in vivo with chloramphenicol (CAP PB-B2) have been isolated, and their catalytic, spectral, and physical properties have been compared with those of the native PB-B2. The CAP PB-B2 exhibited: 1) a 60-70% loss in the rate of NADPH-supported monooxygenase activity with the substrates benzphetamine, 7-ethoxycoumarin, and p-nitroanisole; 2) a 60% decrease in the extent of enzymatic P-450 reduction catalyzed by NADPH-cytochrome P-450 reductase under both aerobic and anaerobic conditions; 3) a 60% decrease in the steady-state level of the ferrous dioxygen complex in the presence of substrates; 4) a 60% decrease in the magnitude of the type I spectral change induced by benzphetamine; and 5) a shift in the wavelength maximum for the chemically reduced ferrous-carbonyl complex from 450 to 451.5 nm. On the other hand, the ability of the CAP PB-B2 to catalyze the iodosobenzene-supported metabolism of 7-ethoxycoumarin and p-nitroanisole was unaltered. The results are consistent with a scheme whereby the binding of metabolites of chloramphenicol to amino acid residues in the PB-B2 close to the heme moiety blocks electron transport from NADPH-cytochrome P-450 reductase, thereby leading to a loss of monooxygenase activity.     

Roles of residues 129 and 209 in the alteration by cytochrome b5 of hydroxylase activities in mouse 2A P450S.

Cytochrome b5 stimulates the coumarin 7-hydroxylation activity of P450coh. A mutation of Arg-129 in P450coh, however, abolishes the stimulation. Moreover, this mutant P450coh binds loosely to cytochrome b5-conjugated Sepharose 4B, whereas wild-type P450coh binds tightly. Consistent with this, the mutation increases the Ka value for b5 binding approximately 6-fold. The identity of residue 209 also alters the stimulation of the activity of P450coh depending on the type of the substrates used and products formed. Coumarin 7-hydroxylation activity is greatly stimulated by cytochrome b5 only when Phe is at position 209, while cytochrome b5 stimulates testosterone hydroxylation activity of P450coh in which Phe, Asn, Ser or Lys substitutes residue 209. P450coh changes its rate of hydrogen peroxide formation depending on the identity of residue 209 and substrate used. Cytochrome b5 decreases the hydrogen peroxide formation of some P450coh whose activities are stimulated by the cytochrome; however, the decrease does not always result in stimulating the activity. The results indicate, therefore, that residues 129 and 209 play different roles in stimulating P450coh activity by cytochrome b5; Arg-129 is a key residue in the cytochrome b5-binding domain and is essential for the stimulation. Residue 209, however, alters the efficiency of electron transport for substrate oxidation as a residue which resides near the sixth ligand of heme and in the substrate-binding site.     

Ligand binding of organic sulfides to microsomal cytochrome P-450.

Octyl methyl-, butyl methyl- and pentamethylene sulfide react with about 50% of oxidized cytochrome P-450 in liver microsomes from phenobarbital-pretreated rats by formation of optical difference spectra with maxima at 435 and 552 nm and concomitant shifts in the electron paramagnetic resonance spectrum. Reduction by NADPH or sodium dithionite yielded a Soret absorption band at 449 nm and alpha and beta bands at 573 and 545 nm, respectively. The ligand metyrapone and the substrate n-octane competitively inhibited the formation of these difference spectra and pentamethylene sulfide was a strong competitive inhibitor of the 0-deakylation of 7-ethoxycoumarin. These results indicate a direct ligand binding of the sulfides to cytochrome P-450 with concomitant blocking of the hydrophobic substrate binding site. Some sulfides did not interact as ligands but as substrates, in variation, however, with the source of microsomes.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.