Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.     

Fatty acid-induced alteration of the porphyrin macrocycle of cytochrome P450 BM3.

Surface-enhanced resonance Raman scattering (SERRS) of substrate-free and substrate-bound forms of the P450 domain of cytochrome P450 BM3 are reported and assigned. Substrate-free P450 yields mixed spin heme species in which the pentacoordinate high-spin arrangement is dominant. The addition of laurate or palmitate leads to an increase in high spin content and to an allosteric activation of heme mode v29, which is sensitive to peripheral heme/protein interactions. Differences between laurate and palmitate binding are observed in the relative intensities of a number of bands and the splitting of the heme vinyl modes. Laurate binding to P450 results in different protein environments being experienced by each vinyl mode, whereas palmitate binding produces a smaller difference. The results demonstrate the ability of SERRS to probe substrate/prosthetic group interactions within an active site, at low protein concentrations.     

Mechanism of monoclonal antibody inhibition/stimulation of reactions catalyzed by cytochrome P450IIB1

We describe two monoclonal antibodies (MAbs) against rat cytochrome P450IIB1 and investigate the mechanisms by which they influence P450IIB1-mediated catalysis. MAb ce9 partially inhibits the activities toward p-nitroanisole, 7-ethoxycoumarin, and benzphetamine as well as NADPH oxidation. These findings can be explained by the observation that ce9 cross-links P450 to form large aggregates resulting in the inhibition of the functional interaction with NADPH cytochrome P450 reductase. Binding of ce9 to P450IIB1 does not affect the spin state of the P450 heme, as revealed by comparing the magnetic circular dichroism (MCD) spectra of free and antibody-bound P450IIB1. On the other hand, the second antibody tested, MAb 14E10, induces a remarkable low to high spin transition upon binding to P450IIB1, as shown by MCD difference spectroscopy. This MAb stimulates activities toward p-nitroanisole and 7-ethoxycoumarin without affecting the rate of NADPH oxidation. This observation indicates that MAb 14E10 may increase the efficiency of electron utilization by P450IIB1. Benzphetamine metabolism remains unchanged in the presence of MAb 14E10.     

Interaction of cytochrome P-450 with hydrocarbons

Hydrocarbons of different structures interact with microsomal and solubilized cytochrome P-450 from liver of phenobarbital-pretreated rats forming a high spin enzyme-substrate type complex. The affinity of cytochrome P-450 for hydrocarbons increases with increasing lipophilicity independently of the chemical structure. The binding capacity of microsomal P-450 for aliphatic hydrocarbons is generally higher than for aromates. Mutual influence in binding of two different hydrocarbons by microsomal P-450 is stronger among aromatic than among aliphatic hydrocarbons; in both cases it appears to be effected rather by specific interaction of both substances with the binding site than by a nonspecific influence on the microsomal membrane. Only one fraction of low spin form of solubilized cytochrome P-450 from rat liver interacts with hydrocarbons. The binding capacity for aromatic and aliphatic substances corresponds to that found in microsomes. The affinity for the most lipiphilic substrate, perhydrophenanthrene, is equal in microsomal and solubilized preparation; with decreasing lipophilicity the affinity of solubilized P-450 decreases faster than in microsomes. The LM2 fraction of cytochrome P-450 from phenobarbital-pretreated rabbits interacts only with aliphatic hydrocarbons with wide variation of the binding capacity. The affinity is generally one order of magnitude lower than in microsomes. Active fractions of solubilized P-450 from both species are rapidly converted to P-420 by dithionite. The extent of this conversion is strongly reduced by saturation with substrate.     

Selective potent restriction of P450b- but not P450e-dependent 7,12-dimethylbenz[a]anthracene metabolism by the microsomal environment

The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, differ by only 13 amino acids and yet purified P450b is considerably more active than P450e for all known substrates. A unique regioselectivity difference between cytochromes P450b and P450e for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and a genetic deficiency in P450e expression in the Marshall (M520/N) rat strain have been exploited to determine the microsomal contributions of the respective forms toward the metabolism of DMBA. The total contribution to metabolism by each isozyme has been assessed based on the sensitivity to rabbit anti-P450b/e IgG and comparison with microsomal P450b and P450e content as measured by Western blots. Liver microsomes from untreated M520/N rats do not express detectable levels of P450e but express P450b at a level that is 2-fold higher than that of P450e in liver microsomes from untreated F344 rats (50 pmol/mg). However, only 4% of the constitutive DMBA metabolizing activity of liver microsomes from the M520/N rat strain could be inhibited by anti-P450b/e IgG. A 30-fold induction of hepatic P450b by phenobarbital (PB) was also completely ineffective in increasing P450b-dependent DMBA metabolism. PB treatment had no appreciable effect on either the levels of expression of P450b protein or P450b-dependent DMBA metabolism, in M520/N lung and adrenal microsomes. In contrast, PB treatment of F344 rats considerably increased P450b/e-dependent metabolism by liver, lung, and adrenal microsomes. The regioselectivity of the anti-P450b/e-sensitive metabolism (predominantly 12-methyl hydroxylation), however, indicated a much greater contribution from P450e than P450b in every tissue examined despite a several fold higher expression of P450b than of P450e. P450b was expressed constitutively in lung microsomes from both strains but again failed to exhibit appreciable DMBA metabolizing activity. Based on these activities and microsomal P450b contents, P450b consistently exhibited turnover numbers (0.02-0.15 nmol/nmol P450b/min) that were at least 10-fold lower than those of pure P450b. In contrast, the calculated turnover numbers for microsomal P450e were consistently comparable to those of pure P450e (approximately 1 nmol/nmol P450e/min).     

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: evidence for structural similarity between trout P450 LMC5 and human P450IIIA4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.     

Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).     

Changes in the amount of cytochrome P450s in rat hepatic microsomes with starvation

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2 alpha- and 16 alpha-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6 beta-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB-4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P450 PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7 alpha-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid omega- and (omega-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.     

Rapid conformational dynamics of cytochrome P450 2E1 in a natural biological membrane environment

Among the members of the cytochrome P450 superfamily, P450 2E1 is most often associated with the production of reactive oxygen species and subsequent cellular toxicity. We sought to identify a structural basis for this distinguishing feature of P450 2E1 by examining its carbon monoxide binding kinetics as a probe of conformation/dynamics. We employed liver microsomes from wild-type and P450 2E1 knockout mice in order to characterize this P450 in a natural membrane environment. The CO binding kinetics of the P450s of wild-type microsomes had a rapid component that was absent in the knockout microsomes. Data analysis using the maximum entropy method (MEM) correspondingly identified two distinct kinetic components in the wild-type microsomes and only one component in the knockout microsomes. The rapid kinetic component in wild-type microsomes was attributed to endogenous P450 2E1, while the slower component was derived from the remaining P450s. In addition, rapid binding kinetics and a single component were also observed for human P450 2E1 in a baculovirus expression system, in the absence of other P450s. Binding kinetics of both mouse and human P450 2E1 were slowed in the presence of ethanol, a modulator of this P450. The unusually rapid CO binding kinetics of P450 2E1 indicate that it is more dynamically mobile than other P450s and thus able to more readily interconvert among alternate conformations. This suggests that conformational switching during the catalytic cycle may promote substrate release from a short-lived binding site, allowing activated oxygen to attack other targets with toxic consequences.     

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin

Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.     

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 > 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.     

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His(6)-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.     

The effect of glycerol on cytochrome P450scc (CYP11A1) spin state,activity, and hydration

We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity.