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.     

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.     

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.     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

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.     

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128.

We have investigated mechanisms of mitochondrial targeting of the phenobarbital-inducible hepatic mitochondrial P450MT4, which cross-reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N-terminus. Inducers of cAMP and protein kinase A-mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N-terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1-DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.     

Ontogenic development of liver progesterone metabolism in female sheep. Contribution of cytochrome P4502B and P4503A subfamilies.

Age-related changes in progesterone hepatic metabolism were measured in Lacaune ewes in the foetal, neonatal (1 and 4 weeks), growing (7 months), pregnant (11 months) and adult (6 years) stages. 6 beta-Hydroxylation and 20 alpha-reduction were found to be the most efficient metabolic process in ovine microsomes. These activities were detected in 3-month-old foetuses and they increased rapidly during the first month of life, in a similar manner to the developmental expression of the cytochrome P4503A subfamily. 16 alpha- and 21-hydroxylation of progesterone were characterized by low, constant turn over in sheep liver microsomes during development. The hepatic ovine P4502B isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, hydroxylapatite and CM cellulose chromatographic separations. This hemoprotein had an apparent molecular weight of 51 kDa and was characterized by spectral data, NH2-terminal amino-acid sequence, immunological and catalytic properties. The relative contribution of this form and of the previously purified ovine P4503A subfamily was investigated in liver progesterone metabolism by immunoinhibition studies using polyclonal antibodies raised in rabbits and from the existence of induction and of significant correlations between microsomal activity and specific P450 content. In sheep liver microsomes, it would appear that cytochrome P4502B is involved in progesterone 21-hydroxylation whereas P4503A participates in the 6 beta- and 16 alpha-hydroxylation and possibly in the reductive conversion of progesterone in its 20 alpha-hydroxy derivative.     

Isolation of regioisomers of N-alkylprotoporphyrin IX from chick embryo liver after treatment with porphyrinogenic xenobiotics

Several porphyrinogenic xenobiotics cause mechanism-based inactivation of cytochrome P450 (P450) isozymes with concomitant formation of a mixture of four N-alkylprotoporphyrin IX (N-alkylPP) regioisomers, which have ferrochelatase inhibitory properties. To isolate the four regioisomers of N-methylprotoporphyrin IX (N-methylPP), 3,5-diethoxycarbonyl, 1-4-dihydro-2,4,6-trimethylpyridine (DDC) was administered to untreated, beta-naphthoflavone-, phenobarbital-, and glutethimide-pretreated 18-day-old chick embryos. Separation of the N-methylPP regioisomers by high pressure liquid chromatography (HPLC) revealed no marked difference in the regioisomer pattern among the different treatments. After administration of griseofulvin, allylisopropylacetamide (AIA), or 1-[4-(3-acetyl-2,4,6-triemethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime (ATMP) to untreated and glutethimide-pretreated 18-day-old chick embryos, an N-alkylPP was isolated after AIA administration only. This finding strengthened previous reports of the species specificity of N-alkylPP formation with griseofulvin and ATMP. A series of dihydropyridines, namely 4-ethylDDC, 4-hexylDDC, and 4-isobutylDDC were administered to untreated and glutethimide-pretreated 18-day-old chick embryos and hepatic N-alkylPPs were isolated and separated by HPLC into regioisomers. The regioisomer patterns obtained did not support a previous proposal of masked regions above both rings B and C in the heme moieties of the P450 isozymes responsible for N-alkylPP formation. However, the data support the hypothesis of a partially masked region above ring B alone. The regioisomer patterns were in agreement with results previously obtained in rats showing that the percentage of Nc and (or) ND regioisomers in the regioisomer mixture increases as the length and bulk of the 4-alkyl substituent of a DDC analogue increase. Differences in the regioselectivity of heme N-alkylation may be due to intrinsic chemical features of DDC analogues themselves or to differences in the P450 isozymes inactivated.     

Frequency of a Specific Cytochrome P4502D6B (CYP2D6B) Mutant Allele in Clinically Differentiated Groups of Patients with Parkinson Disease

The frequency of the cytochrome P4502D6B CYP2D6B (29B) mutant allele has been determined in three clinically distinct groups of patients with Parkinson disease. No differences in mutation frequency among the patients with the rigidity-akinetic and monosymptomatic tremor forms has been observed compared to the healthy control group, while in the group with akinetic-rigidity tremor symptoms the frequency of heterozygous wt/29B individuals was significantly increased. Therefore, individuals bearing the CYP2D6B mutation appear to be predisposed to the development of this particular form of Parkinson disease, and the presence of the 29B mutation in the genotype may serve as an additional diagnostic criteriaum for the clinical differentiation of patients with Parkinson disease.     

Dilinoleoylphosphatidylcholine decreases ethanol-induced cytochrome P4502E1.

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.     

Optical biosensor study of ternary complex formation in a cytochrome P4502B4 system

The optical biosensor method was used for the revelation of ternary complexes, formed by the full-length NADPH-cytochrome P450 reductase (d-Fp) and cytochromes P4502B4 (d-2B4) and b5 (d-b5) in the course of their interactions within the reconstituted d-2B4-containing system. Based on the lack of competition between d-b5 and d-Fp for the binding sites on immobilized 2B4 (3) as well as on the analysis of data obtained in the three proteins' dissociation reactions, the possibility of formation of ternary complexes through the interactions between membranous hydrophobic fragments of proteins was substantiated. All the complexes obtained were productive.     

Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: Effects of xenobiotics and sex steroids

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, β-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.     

Mechanism of cytotoxicity of aflatoxin B1: role of cytochrome P1-450

A mouse hepatoma cell line, Hepa-1, is highly sensitive to the toxic effects of Aflatoxin B1 (AFB1). Half maximal survival (LD50) of cells occurs at 0.068 ug AFB1/ml. Benzo(a)anthracene, which induces aryl hydrocarbon hydroxylase and cytochrome P1-450 in Hepa-1, causes a slight increase in the toxicity of AFB1 (LD50 = 0.034 ug/ml). An aryl hydrocarbon hydroxylase- and cytochrome P1-450-deficient mutant of Hepa-1 is, however, over 100 times more resistant to AFB1 than Hepa-1. Almost no decline in survival is observed at 5 ug AFB1/ml. Cytochrome P1-450 thus effects strongly on the cytotoxicity of AFB1 in these cells. The basal activity in Hepa-1 is enough to elicit an almost full toxic effect. AFB1, although a substrate for cytochrome P1-450, does not act as an inducer of aryl hydrocarbon hydroxylase.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex

Cytochrome P-450cam reacts with phenyldiazene (PhN = NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a sigma phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-A resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10 degrees in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that two water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T.L., & Howard, A.J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of N-phenylprotoporphyrin IX from the red cells and spleen of the phenylhydrazine-treated rat

Blood and spleens of phenylhydrazine-injected rats were treated with a solution of acidic methanol and zinc ion to isolate a green pigment. The pigment was resolved into two, I and II, by thin-layer chromatography. Pigment I was a mixture of two isomers of zinc complex of esterified N-phenylprotoporphyrin IX, in which vinyl-substituted pyrrole rings A and B were phenylated; and pigment II was a mixture of two isomers of the porphyrin complex with the N-phenyl group on propionic acid-substituted rings C and D. These pigments were also chemically prepared from the reaction of phenylhydrazine with oxyhemoglobin, independently characterized, and used to confirm the structures of the biological pigments. Determination revealed that the total amount of pigments found in the blood and spleen at 24 h after injection of phenylhydrazine corresponds to about 0.4% of the injected phenylhydrazine.     

Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.