The active sites of cytochromes P450 IA1, IIB1, IIB2, and IIE1. Topological analysis by in situ rearrangement of phenyl-iron complexes

The reactions of cytochromes P450 IA1, IIB1, IIB2, and IIE1 with phenyldiazene yield complexes with absorption maxima at either 474 or 480 nm. Anaerobic extraction of the prosthetic group from the phenyldiazene-treated proteins followed by its exposure to oxygen and strong acid produces an equal mixture of the four possible N-phenylprotoporphyrin IX regioisomers. Exposure of the anaerobically extracted heme complexes to oxygen in the absence of strong acid results in their decomposition to heme and products other than N-phenylprotoporphyrin IX. These results show that the 474/480 nm absorption maxima are due to sigma phenyl-iron complexes. Treatment of the intact hepatic cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 474/480 nm band. Extraction of the prosthetic group then yields only the two N-phenylprotoporphyrin IX regioisomers with the N-phenyl group on pyrrole rings A and D. The same regioisomer pattern is obtained if the cytochrome P450IA1 phenyl-iron complex is simply warmed to 37 degrees C, but this thermal rearrangement occurs much less readily, if at all, with the complexes of the other isozymes. The regioisomers with the N-phenyl on pyrrole rings A and D are obtained in a 2:1 ratio with isozyme IA1, 1:1 with IIB2, 1:1.7 with IIB1, and 1:2 with IIE1. These results establish that the active sites of these cytochrome P450 isozymes have a common architecture despite their gross differences in substrate specificity. In this architecture, the region directly above pyrrole rings A and D is relatively open whereas that above pyrrole rings B and C is occluded by protein residues.     

Active site topologies of bacterial cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3). In situ rearrangement of their phenyl-iron complexes.

The reactions of cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3) with phenyldiazene result in the formation of phenyl-iron complexes with absorption maxima at 474-478 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 decreases the 474-478 nm absorbance and shifts the phenyl group from the iron to the porphyrin nitrogens. Acidification and extraction of the prosthetic group from each of the ferricyanide-treated enzymes yields a different mixture of the four possible N-phenylprotoporphyrin IX regioisomers. The ratios of the regioisomers with the phenyl ring on pyrrole rings B, A, C, and D (in order of elution from the high performance liquid chromatography column) are, respectively: cytochrome P450cam, 0:0:1:4; P450terp, 0:0:0:1; and P450BM-3, 2:10:2:1. The isomer ratio for recombinant cytochrome P450BM-3 without the cytochrome P450 reductase domain (2:9:2:1) shows that the reductase domain does not detectably perturb the active site topology of cytochrome P450BM-3. Potassium ions modulate the intensity of the spectrum of the phenyl-iron complex of cytochrome P450cam, but do not alter the N-phenyl isomer ratio. Computer graphics analysis of the crystal structure of the cytochrome P450cam phenyl-iron complex indicates that the active site of cytochrome P450cam is open above pyrrole ring D and, to a small extent, pyrrole ring C, in complete agreement with the observed N-phenylprotoporphyrin IX regioisomer pattern. The regioisomer ratios indicate that the active site of cytochrome P450terp is only open above pyrrole ring D, whereas that of cytochrome P450BM-3 is open to some extent above all the pyrrole rings but particularly above pyrrole ring A. The bacterial enzymes thus have topologies distinct from each other and from those of the mammalian enzymes so far investigated, which have active sites that are open to a comparable extent above pyrrole rings A and D.     

A constitutive form of guinea pig liver cytochrome P450 closely related to phenobarbital inducible P450b(e)

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 3/46, and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16 beta-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.     

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.     

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.     

Expression and characterization of a rabbit liver cytochrome P450 belonging to P450IIB subfamily with the aid of the baculovirus expression vector system

Using baculovirus a cDNA for cytochrome P450 (P450F1) belonging to rabbit P450IIB subfamily was expressed in Spodoptera frugiperda cells, where P450F1 was located on electron-dense structures (derived from the endoplasmic reticulum) present in both the cytoplasm and nucleus. Partially purified P450F1 exhibited absorption spectra similar to those of P450(1), the major phenobarbital-inducible form in rabbit liver. Like P450(1), P450F1 could oxidize aminopyrine, benzphetamine, 7-ethoxycoumarin, 1-nitropropane, and 2-nitropropane, though its activities toward benzphetamine and 7-ethoxycoumarin were about 50 and 5%, respectively, of those of P450(1). It is concluded that the members of P450IIB subfamily can act on a variety of xenobiotics, although substrate preferences are different among them.     

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.     

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.     

Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1).

Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.     

Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.     

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.     

Molecular basis for a functionally unique cytochrome P450IIB1 variant.

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.     

Nanosecond fluorometry of the single tryptophan in cytochrome P-450e (P450IIB2)

Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.     

The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences

Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP 19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys 119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles ofAsp309 and His480 in the aromatization of the steroid A ring.