Effect Of Cytochrome B5 Research Articles

Steroidogenic cytochrome P450 17A1 structure and function

Cytochrome P450 17A1 (CYP17A1) is a critical steroidogenic enzyme, essential for producing glucocorticoids and sex hormones. This review discusses the complex activity of CYP17A1, looking at its role in both the classical and backdoor steroidogenic pathways and the complex chemistry it carries out to perform both a hydroxylation reaction and a carbon-carbon cleavage, or lyase reaction. Functional and structural investigations have informed our knowledge of these two reactions. This review focuses on a few specific aspects of this discussion: the identities of reaction intermediates, the coordination of hydroxylation and lyase reactions, the effects of cytochrome b5, and conformational selection. These discussions improve understanding of CYP17A1 in a physiological setting, where CYP17A1 is implicated in a variety of steroidogenic diseases. This information can be used to improve ways in which CYP17A1 can be effectively modulated to treat diseases such as prostate and breast cancer, Cushing's syndrome, and glioblastoma.

The effect of cytochrome b5 on a reaction cycle of cytochrome P450 1A1

The effect of cytochrome b5 on a reaction cycle of cytochrome P450 1A1

P.8.b.009 Inhaled loxapine for the acute treatment of agitation: clinical experience from eastern and northern Finland

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine–DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b5 on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine–DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6 > 2C19 > 1B1 > 1A2 > 2E1 and >2C9. Cytochrome b5 increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9 > 3A4 > 2D6 > 2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N2-oxide. CYP3A4 and 1A1 in the presence of cytochrome b5 are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine–DNA adduct 2. These ellipticine–DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC50 value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine–DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b5 on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine–DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b5 increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N2-oxide. CYP3A4 and 1A1 in the presence of cytochrome b5 are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine–DNA adduct 2. These ellipticine–DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC50 value of ellipticine in these cells was 0.7μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

Comparison of CYP3A4 and CYP3A5: The Effects of Cytochrome b5 and NADPH-cytochrome P450 Reductase on Testosterone Hydroxylation Activities

CYP3A4 and CYP3A5 require cytochrome b5 (b5) and NADPH-cytochrome P450 oxidoreductase (CPR) for optimum metabolism, but little is known about the specific requirements for b5 and CPR to produce optimal activities for these enzymes. The metabolism of testosterone (TT) by CYP3A4 and CYP3A5 was analyzed by various combinations of b5 and CPR using a fixed amount of recombinant P450 which had been purified from an Escherichia coli expression system. CYP3A4 and CYP3A5 required 4- and 8-fold more of CPR than of the P450s, respectively, for optimal activity. The requirement of b5 for optimal activity showed the same pattern for both CYP3A4 and CYP3A5, exhibiting a gradual stimulation of the activity reaching a maximum at 16 fold more b5 than P450. Although CYP3A4 exhibited higher activities than CYP3A5 in all combinations, both enzymes exhibited the same dependency profile for b5 and CPR. Therefore, the stronger activity of CYP3A4 compared to CYP3A5 appears to be intrinsic to the CYP3A4 protein itself and not to different requirements for b5 and CPR. Since the relative amounts of b5 and CPR are important in the maintenance of CYP3A4 and CYP3A5 activities, different levels of these proteins in vitro and in vivo may cause altered metabolism of their substrates or misinterpretation of enzyme properties.

Cytochrome b5 shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy

Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b5 alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine–DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b5 enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine–DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine–DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b5 might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b5 in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b5 and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.

Effects of cytochrome b5 on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5 with CYP3A4 but not CYP3A7

Effects of cytochrome b 5 ( b 5) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b 5 in approximately one third of the substrates employed in this study. Such enhancement by b 5 was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b 5 with any substrates employed. V max values for midazolam 1′-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b 5, which was also seen with CYP3A4, although the extent of the effects of b 5 on S 50 ( K m ) and Hill coefficient differed dependent on substrates used. In contrast, b 5 did not alter any of these kinetic parameters of CYP3A7. The effects of b 5 on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b 5 in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.

Comparison of substrate metabolism by cytochromes P450 2B1, 2B4, and 2B6: relationship of heme spin state, catalysis, and the effects of cytochrome b5

The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b 5 ( b 5) on these reactions were assessed. There did not appear to be any trends regarding the effects of b 5 when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b 5 were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b 5 effects. The rates of benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes.

Expression of Modified Cytochrome P450 2C10 (2C9) in Escherichia coli, Purification, and Reconstitution of Catalytic Activity

The human cytochrome P450 (P450) 2C gene family is complex and heterologous expression methods are needed to facilitate the isolation of individual P450 proteins and the elucidation of their catalytic specificities. We prepared a series of constructs of P450 2C10 in the plasmid vector pCW, with modification of the 5′ end of the coding sequence of the cDNA. Some were not expressed at all in Escherichia coli; two were expressed at levels of 5-20 nmol membrane-bound P450 (liter culture)−1-one (2C1028) with original codons 2-7 altered by substitution of the 5′-terminal sequence described by Barnes et al. (Barnes, H. J., Arlotto, M. P., and Waterman, M. R., Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and one (2C1029) with original codon 2 modified, codons 3-20 deleted, and alteration of the immediate downstream codons. In both cases the P450 2C10 proteins were found essentially only in the bacterial membranes. These proteins could be purified to a high degree by solubilization and a single DEAE chromatography step. Typical P450 Fe2+ · CO absorption spectra were observed in the bacterial membranes and the purified preparations. The P450 2C1029 protein was found to have its N-terminal Met removed and the expected residues 2(Ala)-24 were identified by amino acid sequence analysis. However, the other P450 (2C1028) was apparently blocked at the N-terminus. Three native P450 2C9/10 preparations isolated from human liver showed the expected sequences (beginning with Met) for at least the first 17 residues. The blocked N-terminus in the P450 2C1028 protein may be the result of the MALLLAVF sequence, which was also used in the expression of P450 3A4 and resulted in a blocked protein. Catalytic activities of P450 2C1028 and P450 2C1029 for tolbutamide hydroxylation were similar to those measured with purified liver P450 2C9/10 in the presence of cytochrome b5, although the effect of cytochrome b5 did not always show the same pattern as with the isolated liver enzyme. The recombinant P450 2C10 enzymes did not catalyze (S)-mephenytoin 4′-hydroxylation.

The role of cytochrome b5 in adrenal microsomal steroidogenesis

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17α- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-45017α,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.

Effects of Cytochrome b5 on Aniline Hydroxylation Catalyzed by a Reconstituted System Containing Acetone or 2,2'-Bipyridine

Cytochrome b5 did not exert any effect on NADPH-dependent aniline hydroxylation in the absence of acetone or 2,2'-bipyridine, whereas cytochrome b5 exhibited a stimulatory effect on the reaction in the presence of acetone or 2,2'-bipyridine. In addition, cytochrome b5 did not have any significant effect on the cumene hydroperoxide-dependent reaction in the presence of acetone or 2,2'-bipyridine.

Studies on cytochrome P-450 (P-450 17α,lyase)from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5

We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b 5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450 17α,lyase). P-450 17α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b 5 to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b 5. With the addition of cytochrome b 5, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b 5 plays some important role in controlling the dual activities of P-450 17α,lyase.

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5.

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein.

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.

Lipid-protein interactions as determinants of activation or inhibition by cytochrome b5 of cytochrome P-450-mediated oxidations.

Activation or inhibition by cytochrome b5 of benzphetamine N-demethylation was studied in micelle-reconstituted systems containing cytochrome P-450 LM2, NADPH-cytochrome P-450 reductase, and dilauroyl-phosphatidylcholine. The effects of cytochrome b5 were critically dependent on both protein:protein and lipid:protein ratios. A 200% stimulation of N-demethylation by cytochrome b5 was obtained at cytochrome P-450 reductase:cytochrome P-450 ratios similar to those in microsomes, compared to only a 20% stimulation at a ratio of 1:1. At lipid:protein ratios less than 50:1, the addition of cytochrome b5 caused significant inhibition of benzphetamine N-demethylation. Such an inhibition could be partially reversed by increasing phospholipid content of micelles and was not seen in vesicle-reconstituted systems at cytochrome b5:cytochrome P-450 ratios of 1:1 or lower. At high cytochrome P-450 reductase:cytochrome P-450 ratios, addition of cytochrome b5 did not alter the efficiency (80%) with which NADPH was utilized: however, at ratios similar to those in microsomes, an increase in efficiency from 42% to 80% was observed. The function of cytochrome b5 was interpreted in terms of a model in which inhibition of cytochrome P-450-mediated reactions results from changes in phospholipid-protein interactions and activation occurs via facilitation of electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane.

Stimulatory effect of cytochrome b5 induced by p-nitroanisole and diisopropyl 1,3-dithiol-2-ylidenemalonate on rat liver microsomal drug hydroxylations.

The relationship between the changes in the amount of the components of the liver microsomal electron transport systems and drug hydroxylase activities on administration of p-nitroanisole was investigated. The content of cytochrome P-450 in the p-nitroanisole-treated rats was not significantly different from that in the controls during the treatment. The cytochrome b5 content increased 2.2-fold over that of the controls after 14 days of treatment. Demethylation activities per mg microsomal protein of p-nitroanisole, aminopyrine, and benzphetamine were enhanced 2.7-, 2.4-, and 2.8-fold, respectively, but aniline hydroxylase activity was not enhanced. Similar results were obtained with microsomes from diisopropyl 1,3-dithiol-2-ylidenemalonate (NKK-105)-treated rats. The time-course study of changes in the amount of the components of drug hydroxylase systems suggested that the increase in cytochrome b5 content enhanced the demethylation activities. In the reconstituted system containing cytochrome P-450 partially purified from p-nitroanisole- or NKK-105-treated rats, cytochrome b5 was required for the maximal activities of the demethylation reactions, but did not participate in aniline hydroxylation. These findings showed good correlation between the change of cytochrome b5 content and the demethylation activities and suggested that the increase in cytochrome b5 content might enhance the demethylation activity by stimulating supply of a second electron to cytochrome P-450.

Effect of cytochrome b5 on fatty acid omega- and (omega-1)-hydroxylation catalyzed by partially purified cytochrome P-450 from rabbit kidney cortex microsomes.

Cytochrome P-450 was solubilized from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B and heparin-Sepharose CL-6B columns. Fatty acid omega- and (omega-1)-hydroxylation activity was reconstituted from the partially purified cytochrome P-450 and NADPH-cytochrome c reductase, with phosphatidylethanolamine or phosphatidylcholine. The activity was further stimulated by addition of detergent-solubilized cytochrome b5 from rabbit liver microsomes. Trypsin-solubilized or boiled detergent-solubilized cytochrome b5 had no effect. Among fatty acids tested, caprate, laurate, myristate, and palmitate were the most effective substrates. When caprate and laurate were used as the substrates, the products were the corresponding omega- and (omega-1)-hydroxy fatty acids. The ratio of these products was altered by addition of cytochrome b5. On the other hand, when myristate and palmitate were the substrates, small amounts of unknown polar fatty acids were also formed besides omega- and (omega-1)-hydroxy fatty acids, and the ratio of these products was not affected by addition of cytochrome b5. Benzo(a)pyrene hydroxylation activity was also reconstituted from the same cytochrome P-450 preparation, NADPH-cytochrome c reductase, and phosphatidylserine. However, cytochrome b5 showed only a slight stimulation. The possibility that different cytochrome P-450 species are involved in fatty acid and benzo(a)pyrene hydroxylations is discussed.

Effect of cytochrome b5 on the size, density, and permeability of phosphatidylcholine vesicles

Cytochrome b5, isolated from rabbit liver by a procedure using detergent, was incubated with phosphatidylcholine bilayer vesicles at 37 degrees for 30 min. A comparison of a number of physical properties was made between the cytochrome b5-phosphatidylcholine complex (at a molar ratio of 1:1000) and the phosphatidylcholine vesicles. The binding of the protein to the vesicle caused no aggregation and no detectable change in Stokes radius of the vesicle as monitored by gel filtration. Only small increases in s20 (from 2.67 up to 3.82 X 10(-13) s) and density (from 1.025 up to 1.042 g ml(-1)) were observed upon binding of the cytochrome b5 to phosphatidylcholine vesicles. At molar ratios of 5:1000, and above, two types of complexes could be detected by sucrose density gradient centrifugation: one had a molar ratio of approximately 1.066 g ml(-1)) the other, a more constant ratio of 20:1000 (density greater than 1.107 g ml(-1)). Cytochrome b5 was also incubated with phosphatidylcholine vesicles prepared with ferricyanide trapped inside. The leakage of the ferricyanide from inside the vesicles was increased when cytochrome b5 was present, but the vesicles, although leaking, were not completely depleted of their ferricyande, and so must still be intact. It is suggested that at molar ratios of cytochrome b5 to phosphatidylcholine below 5:1000, the binding of the protein causes minimal change in vesicle structure.