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Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of which trans-4-hydroxy-2-nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two pathways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanism-based inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanism-based inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme.

Nasal microsomal P450 form a (NMa), a major cytochrome P450 isozyme in rabbit olfactory and respiratory nasal mucosa with high activity toward a variety of odorants and environmental toxicants, was previously purified to electrophoretic homogeneity from rabbit nasal microsomes. In the present study, a cDNA library constructed from poly(A)+ RNA from rabbit respiratory nasal mucosa was screened with antibodies to P450 NMa, and five immunopositive clones were isolated and characterized. Sequence analysis indicated that the clones encode two highly similar P450s that contain 494 amino acid residues, with the first 20 corresponding to P450 NMa, and differ from each other in only 8 residues scattered throughout the polypeptide chains. On the basis of structural homology the two proteins are designated as CYP2A10 and CYP2A11 and are the first members of the P450 2A subfamily to be identified in nasal tissue. Genomic blot analysis indicated that 2A10 and 2A11 are apparently not allelic variants. Both genes are expressed in liver and lung as well as in nasal tissues, as judged by RNA blot analysis, but the relative levels of the two mRNAs differ. Both enzymes were partially purified after expression of the cDNAs in Escherichia coli and shown to catalyze the oxygenation of a variety of substrates, including ethanol and procarcinogens such as N-nitrosodiethylamine and phenacetin. P450 2A10 is generally more active than P450 2A11 and strikingly so in the conversion of testosterone to androstenedione.

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.

We provide here a list of 154 P450 genes and seven putative pseudogenes that have been characterized as of October 20, 1990. These genes have been described in a total of 23 eukaryotes (including nine mammalian and one plant species) and six prokaryotes. Of 27 gene families so far described, 10 exist in all mammals. These 10 families comprise 18 subfamilies, of which 16 and 14 have been mapped in the human and mouse genomes, respectively; to date, each subfamily appears to represent a cluster of tightly linked genes. We propose here a modest revision of the initially proposed (Nebert et al., DNA 6, 1-11, 1987) and updated (Nebert et al., DNA 8, 1-13, 1989) nomenclature system based on evolution of the superfamily. For the gene we recommend that the italicized root symbol CYP for human (Cyp for mouse), representing cytochrome P450, be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. We suggest that the human nomenclature system be used for other species. This system is consistent with our earlier proposed nomenclature for P450 of all eukaryotes and prokaryotes, except that we are discouraging the future use of cumbersome Roman numerals.

The rotation of cytochrome P-450 LM2 (CYPIIB4) incorporated into large microsomal-like lipid vesicles was investigated by saturation transfer EPR using 15N- and 2H-substituted spin labels. In combination with rotational diffusion, the distribution and size of protein particles in the bilayer were studied by freeze-fracture electron microscopy. The data from both methods suggest an oligomeric and membrane-spanning aggregate for the topology of microsomal cytochrome P-450.

We report here the unexpected finding that recombinant or hepatic microsomal NADPH-cytochrome P450 reductase catalyzes the oxidative deformylation of a model xenobiotic aldehyde, 2-phenylpropionaldehyde, to the n-1 alcohol, 1-phenylethanol, in the absence of cytochrome P450. The flavoprotein and NADPH are absolute requirements, and the reaction displays a dependence on time and on NADPH and reductase concentration. Not surprisingly, the hydrophobic tail of the flavoprotein is not required for catalytic competence. The reductase domain of neuronal nitric oxide synthase is about 30% more active than P450 reductase, and neither flavoprotein catalyzes conversion of the aldehyde to the carboxylic acid, by far the predominant metabolite with P450s in a reconstituted system. Reductase-catalyzed deformylation is unaffected by metal ion chelators and oxygen radical scavengers, but is strongly inhibited by catalase, and the catalase-mediated inhibition is prevented by azide. These results, together with observed parallel increases in 1-phenylethanol and H(2)O(2) formation as a function of NADPH concentration, are evidence that free H(2)O(2) is rate-limiting in aldehyde deformylation by the flavoprotein reductases. This contrasts sharply with the P450-catalyzed reaction, which is brought about by iron-bound peroxide that is inaccessible to catalase.

Enzymes that effect with ease one of the most difficult chemical reactions, hydroxylation of an unfunctionalized alkyl group, are of particular interest because highly reactive intermediates must be produced. A typical example, the hydroxylation of fatty acids in the omega position, is now known to occur widely in nature. The catalysts, which can be called "omega-oxygenases," also insert molecular oxygen into a variety of other substrates at positions removed from activating functional groups, as in steroids, eicosanoids, and numerous drugs and other xenobiotics. Progress in the characterization of bacterial nonheme-iron enzymes, and plant, bacterial, and mammalian P450 cytochromes that catalyze fatty acid omega-oxidation, and evidence for multiple functional oxidants are summarized.

The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.

Cytochrome P450 NMa, which was first identified in this laboratory in rabbit nasal microsomes, is now known to represent two distinct gene products, P450s 2A10 and 2A11. In the present study, chimeric and site-directed mutants of 2A11 were constructed to determine which of the eight different amino acid residues are responsible for the much greater activity of 2A10 toward coumarin and testosterone. Mutation of Arg62 and Asp63 of 2A11 to the corresponding residues in 2A10, or mutation of Thr120 to Ser, as found in 2A10, did not change the activities. However, mutation of Arg62, Asp63, Gln104, Ala117, and Thr120 of 2A11 to the corresponding residues in 2A10 resulted in a protein that is as active as 2A10 in coumarin hydroxylation and approximately half as active as 2A10 in androstenedione formation. Mutation of Arg372 in 2A11 to His, as found in 2A10, resulted in a significant increase in the rate of hydroxylation of testosterone, but not of coumarin. Our findings indicate that the identify of the amino acid at position 104 and/or 117 is important for activity with testosterone and for regioselectivity at the 17 position, as well as for optimal activity with coumarin. In contrast, the identity of the residue at position 372 is important for optimal activity with testosterone but not the regioselectivity at the 17 position and does not influence the activity with coumarin.

We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992. These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes. Of 36 gene families so far described, 12 families exist in all mammals examined to date. These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively. To date, each subfamily appears to represent a cluster of tightly linked genes. This revision supersedes the previous updates [Nebert et al., DNA 6, 1-11, 1987; Nebert et al., DNA 8, 1-13, 1989; Nebert et al., DNA Cell Biol. 10, 1-14 (1991)] in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. "P" ("p" in mouse) after the gene number denotes a pseudogene. If a gene is the sole member of a family, the subfamily letter and gene number need not be included. We suggest that the human nomenclature system be used for all species other than mouse. The mRNA and enzyme in all species (including mouse) should include all capital letters, without italics or hyphens. This nomenclature system is identical to that proposed in our 1991 update. Also included in this update is a listing of available data base accession numbers for P450 DNA and protein sequences. We also discuss the likelihood that this ancient gene superfamily has existed for more than 3.5 billion years, and that the rate of P450 gene evolution appears to be quite nonlinear. Finally, we describe P450 genes that have been detected by expressed sequence tags (ESTs), as well as the relationship between the P450 and the nitric oxide synthase gene superfamilies, as a likely example of convergent evolution.

A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.

The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, is described. Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein. Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters. With the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed in increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein. The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein. The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein. Microsomal assays for ethylene formation from N-nitrosodiethylamine and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Furthermore, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes. A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.

Rabbit nasal olfactory and respiratory microsomes demonstrate high activity toward [3H]-(S)-nicotine, with specific activities of 22.2 and 6.5 nmol/min/mg protein, respectively. The major metabolite produced is (S)-nicotine delta 1'; 5'-iminium ion, with lesser amounts of nornicotine and the N'-oxide. Reconstitution of the rabbit nasal microsomal system with cytochromes P-450 NMa and NMb indicated that only P-450 NMa has significant activity toward nicotine, and the metabolite profile and turnover are similar to that observed with nasal microsomes. The low Km (35 microMs) and high Vmax (28 min-1) suggest that a significant portion of inhaled nicotine is metabolized by nasal tissues in the rabbit.

The formation and autoxidative decomposition of the oxygenated forms of two isozymes of rabbit liver microsomal cytochrome P-450 were studied: 5,6-benzoflavone- or isosafrole-inducible P-450LM4, isolated in the high spin state, and P-450LM3b, isolated in the low spin state. When an anaerobic solution of photochemically reduced isozyme 4 was mixed with aerobic buffer in a stopped flow spectrophotometer, the dioxygen complex with absorption maxima at 555 and 418 nm was rapidly formed. The monophasic reaction had a pseudo-first order rate constant of about 58 s-1. Autoxidation of the complex, which was complete in about 20 s, exhibited biphasic first order kinetics at 580 nm with rate constants of about 0.92 and 0.22 s-1. The results obtained with isozyme 3b were similar, except that the decomposition of the ferrous oxy intermediate appeared to be triphasic. Superoxide could not be detected as a product of the autoxidative decomposition of the oxy form of P-450 isozyme 4. Hydrogen peroxide was produced in about 70% yield when oxygen was in excess, whereas in a titration in which small increments of oxygen were added to an excess of the ferrous cytochrome the reduction apparently led to the formation of water. The biphasic kinetics of some of the reactions involving purified mammalian cytochrome P-450 has in some instances been attributed to the formation of aggregates by these hydrophobic proteins. This was ruled out as an explanation of the kinetics observed for the autoxidative decay of ferrous dioxygen P-450LM4, since two phases were also observed with a preparation converted from the usual aggregated state to the monomeric state by exposure to a zwitterionic detergent.

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

Cytochrome P-450 is known to catalyze the following oxygen transfer reaction: RH + PhIO----ROH + PhI where RH represents a variety of hydroxylatable substrates and PhIO a variety of iodosobenzene derivatives that serve as oxygen donors, and neither molecular oxygen nor an external electron donor is required. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interaction with a variety of substituted iodosobenzenes and iodobenzene diacetates have been determined by stopped flow spectrophotometry. The reaction of phenobarbital-induced rabbit liver microsomal cytochrome P-450 form 2 with iodosobenzenes or iodobenzene diacetates leads to the reversible formation of three spectral intermediates, termed E, F, and G. Complex E is characterized by a type I difference spectrum, representing the iodosobenzene-dependent partial shift of the low spin hexacoordinate form of the ferric enzyme to the high spin pentacoordinate form, F represents a transient intermediate whose spectrum cannot be determined for kinetic reasons, and G represents a blue-shifted intermediate with an absorption maximum at about 393 nm in the absolute spectrum. The striking and principal feature of these observations is that the spectrum of Complex G does not vary with structural differences in the iodosobenzene derivatives, in contrast to the transient species observed in previous studies in this laboratory in the reaction between cytochrome P-450 form 2 and aromatic peroxy compounds. Complex G exhibits the spectral properties one might anticipate for an iron-oxo intermediate containing only one oxygen atom derived from the starting iodosobenzene.

The ethanol-induced rabbit liver microsomal cytochrome P-450, P-450LM3a, has been shown previously to efficiently catalyze the demethylation of N-nitrosodimethylamine (NDMA) with a Km of 2.9 mM. Since the predominant Km in hepatic microsomes from ethanol-treated rabbits is 0.07 mM, the role of P-450LM3a in the activation of this carcinogen has been uncertain. In the present study, antibodies to P-450LM3a were shown to almost completely inhibit NDMA demethylation by the purified P-450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the high-Km NDMA demethylase activity in the microsomes. These results indicate that P-450LM3a is the major P-450 responsible for the low-Km NDMA demethylase activity. In addition, evidence is provided for the existence of a cytochrome immunochemically similar to P-450LM3a in liver microsomes from rats, mice, and guinea pigs that effectively catalyzes the demethylation of NDMA.

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.

Earlier findings in several laboratories have suggested that the various forms of cytochrome P-450 within a given species or from different species are not closely related. However, our present studies on the primary structure of the phenobarbital-inducible cytochrome from rabbit liver microsomes (P-450LM2) show that this protein is about 80% identical to the corresponding rat protein (P-450b), the sequence of which was recently deduced by others. For example, of the first 70 residues of the NH2-terminal sequences, 52 are identical, with most differences structurally conservative and attributable to point mutations. No evidence was found for heterogeneity in the region of the rabbit cytochrome corresponding to the variable region proposed for the rat cytochrome. A comparison of the known primary structure of P-450LM2 with that of P-450b and that of the bacterial camphor-hydroxylating cytochrome (P-450cam) as recently reported by others reveals only one highly conserved cysteine-containing peptide present in approximately the same position in each cytochrome. This peptide in the rabbit enzyme, -Arg-Ile-Gln-Glu-Glu-Ala-Arg-Cys147-Leu-Val-Glu-Glu-Leu-Arg-, and the corresponding peptides containing Cys152 in P-450b and Cys134 in P-450cam may serve an essential function such as providing the axial thiolate ligand to the heme iron atom.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.

The exon-intron organization of two rabbit genes that hybridize with cytochrome P-450 3a (P-450ALC) cDNA has been determined by restriction mapping and sequence analysis. Gene 1 encodes cytochrome P-450 3a as judged by the complete identity of its coding nucleotide sequence with P-450 3a cDNA. Gene 2 encodes a previously uncharacterized cytochrome P-450 that is 97% identical in primary structure to P-450 3a, with 16 amino acid differences scattered throughout the protein. Genes 1 and 2, which are 10 and 9 kilobases in length, respectively, are comprised of 9 exons with exon-intron junctions occurring at identical positions along the mRNA sequences. Each gene contains two transcription start sites approximately 27 and 33 nucleotides upstream from the translation initiation codon, as determined by primer extension and S1 nuclease protection experiments. The predicted lengths of gene 1 and 2 transcripts from the first transcription start site to the poly(A) attachment site are 1999 and 1660 nucleotides, respectively. This difference in size is primarily the result of a 338-base pair deletion in the 3' nontranslated portion of the gene 2 transcript relative to that of gene 1. The two genes show considerable similarity in their 5' flanking regions, including a "TATAA" transcriptional promoter element at position -28. However, a 32-base pair element that is repeated in gene 1 is present only as a single inexact copy in gene 2. By use of synthetic oligonucleotides as hybridization probes, gene 2 transcripts were shown to be present in poly(A)+ RNA from untreated rabbit liver at approximately 50% of P-450 3a mRNA levels. In kidney, however, no gene 2 mRNA was detected although 3a mRNA was present at approximately 10% of the level in liver.

Two genes that hybridize to the cDNA for alcohol-inducible cytochrome P-450 form 3a (P-450ALC) have been isolated from a rabbit genomic library and characterized by restriction mapping, hybridization, and partial sequence analysis. The genes show extensive sequence similarity as judged by hybridization at high stringency to the coding region of P-450 3a cDNA. However, only gene 1 hybridizes under these conditions to the 3′ nontranslated segment of P-450 3a cDNA. The hybridizing fragments derived from both cloned genes were found to be present in the genome of all rabbits examined by Southern blot analysis, indicating that the genes represent separate loci and are not polymorphic alleles. Partial sequence analysis indicated that gene 1 encodes P-450 3a. Gene 2, if transcribed, would encode a protein with greater than 96% sequence identity with P-450 3a in the NH2-terminal region.

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200–300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octylglucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.

The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and phospholipid. The two enzymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of Renex-690, a nonionic detergent. These steps were followed by treatment with Amberlite XAD-2 and calcium phosphate gel. Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome b5 and contained no significant amount of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, nonheme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis of the partially purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of two major polypeptide bands, one with a molecular weight of 47,000 to 49,000 and the other 52,000 to 53,000. The component of lower molecular weight corresponded to the major polypeptide induced by phenobarbital treatment, as shown by electrophoresis of the proteins in normal and induced microsomes. The partially purified preparation accepted 2 electrons from dithionite per molecule of cytochrome P-450 under anaerobic conditions, indicating the presence of another electron acceptor in addition to the iron atom of the hemeprotein. The partially purified NADPH-cytochrome P-450 reductase preparations, which catalyzed the reduction of 8.6 to 10.0 µmoles of cytochrome c per min per mg of protein at 30°, were free of cytochrome P-450 and contained only a trace of NADH-cytochrome c reductase. FMN and FAD were found to be present in equimolar amounts, and labile sulfide was absent. The reconstituted enzyme system was active in the hydroxylation of fatty acids (laurate), hydrocarbons (hexane, cyclohexane, and octane), drugs (benzphetamine, hexobarbital, and ethylmorphine), and aniline. The presence of both phosphatidylcholine and deoxycholate was necessary for maximal activity.

Two forms of highly purified liver microsomal cytochrome P-450, P-450LM2, and P-450LM4, have been titrated with standardized solutions of sodium dithionite under anaerobic conditions. Only 1 electron was consumed per hemin molecule, and reoxidation of the reduced heme is accompanied by the transfer of 1 electron to oxidizing agents such as cytochrome c, cytochrome b5, or potassium ferricyanide. The present results are in disagreement with earlier dithionite titrations and reoxidation experiments which indicate that liver microsomal cytochrome P-450 is a 2-electron acceptor, but are in accord with previous potentimetric titrations and product yield data which indicate that this hemeprotein is a 1-electron acceptor. The cause for the discrepancy between the present and previous results is not fully understood.

somal cytochrome P-450 (P-450~~) with aromatic hydroperoxides and peracids two transient spectral intermediates are produced reversibly: Complex C, which is directly involved in the hydroxylation pathway, and Complex D, which is formed in a nonproductive side reaction. In the present study the broad substrate specificity of P-450~~ was exploited to examine the effect of structural changes on the reaction Toluene + cumene hydroperoxide -j benzyl alcohol + cumyl alcohol Determination of Complexes C and D by stopped flow spectrophotometry and measurement of the initial rate of benzyl alcohol formation in the presence of 3-acetylpyridine adenine dinucleotide and alcohol dehydrogenase indicated that a number of m- and p-substituted toluenes and cumene hydroperoxides could be used in place of the parent compounds. The rate-limiting decay of Complex C was found to be dependent on the structure of both the peroxy compound and the hydroxylatable substrate. These results are compatible with a homolytic mechanism of oxygen-oxygen bond cleavage but not with the heterolytic formation of a common iron-oxo intermediate from the various peroxides.

SUMMARY: Isosafrole serves as an excellent inducing agent in rabbits for Wq, the same isozyme as that which is induced by 3-methylcholanthrene and 5,6-benzoflavone. Since the isosafrole adduct formed with isozyme 4 is unstable, the uncomplexed cytochrome may be purified in very good yield (over 30%, based on total P-450) from liver microsomes of animals given this agent. Isosafrole forms adducts with both isozyme 2 (the phenobarbital-inducible form of P-450) and isozyme 4 in the reconstituted system containing NADPHcytochrome P-450 reductase, phosphatidylcholine, and NADPH under aerobic conditions, but with the latter cytochrome the reaction has a lower Km and maximal rate. The addition of synergists to insecticides has a sparing action, so that lower doses of the insecticide then have the same effect (11. Many synergists were found to exhibit substrate binding spectra upon addition to hepatic microsomes (21, and it was proposed that the methylenedioxyphenyl synergists may act by affecting the integrity of the electron transfer chains involved in mixed function oxidation reactions (3). Philpot and Hodgson (4) reported that an ethyl isocyanide-like difference spectrum with absorption maxima at 455 and 428 nm was produced by the addition of piperonyl butoxide and a reduced pyridine nucleotide to mouse liver microsomes, and Franklin (5) demonstrated that various methylenedioxyphenyl compounds (piperonyl butoxide, methylenedioxybenzene, safrole, and isosafrole) are metabolized by rat liver microsomes in the presence of oxygen and NADPH (but not NADH) to give products which exhibit an isocyanide-like absorption spectrum with the reduced cytochrome P-450. Safrole and isosafrole cause the induction of a unique hepatic microsomal hemoprotein in the rat (6) which is present as a stable complex (7); this form of cytochrome P-450 has been purified to electrophoretic homogeneity as the isosafrole metabolite complex (8,9). The dissociation of the complex by added substrates to generate free cytochrome P-450 has been described (10,111, and a linear relationship has been observed between the amount of complex decomposed and the ability of the

The administration of polycyclic aromatic compounds such as beta-naphthoflavone or 3-methylcholanthrene is known to cause the induction of many liver microsomal monoxygenase activities and the appearance of a distinct cytochrome called P-448 in genetically responsive, but not in nonresponsive, inbred mouse strains. However, the administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin induces these activities and cytochrome P-448 formation to the same extent in both responsive and nonresponsive inbred strains. In contrast, phenobarbital or pregnenolone-16 alpha-carbonitrile induces in both responsive and nonresponsive strains a different profile of enzyme activities and the appearance of cytochrome P-450 (rather than cytochrome P-448). In the present studies, electrophoresis of liver microsomal proteins from inbred C57BL/6N and DBA/2N and recombinant inbred AKXL-38 and AKXL-38A mouse strains revealed the presence of four polypeptides whose relative staining intensity could be correlated with the induction state of the microsomes as determined by enzymatic and spectral methods. Of these four bands, Band 4 (55,000 daltons) was increased whenever spectral measurements revealed an increase in the cytochrome P-448 content due to administration of beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Administration of pregnenolone-16alpha-carbonitrile caused an increase in Band 3 (54,000 daltons), whereas administration of phenobarbital caused an increase primarily in Band 2 (51,000 daltons) but also smaller increases in Band 1 (49,000 daltons) and Band 4. The changes observed for phenobarbital and pregnenolone-16alpha-carbonitrile were the same for both responsive and nonresponsive strains. The same electrophoretic technique was used to measure the incorporation of radioactive leucine into microsomal proteins. Microsomes were prepared from liver combined from responsive mice (C57BL/6N) treated with beta-naphthoflavone and L-[14C]leucine and nonresponsive mice (DBA/2N) treated with beta-naphthoflavone and L-[3H-4,5]leucine. A significant increase in the 14C/3H ratio was observed for Band 4, and decreases were seen for Bands 1 and 2. In similar experiments with other mice and phenobarbital as the inducing agent with L-[14C]leucine and the vehicle alone with L-[3H-4,5]leucine, the 14C/3H ratio was markedly increased for Band 2, and smaller increases were observed for Bands 1 and 4. These results and other data presented indicate that the increased formation of cytochrome P-448 and P-450 by beta-naphthoflavone and phenobarbital, respectively, is primarily the result of an increased rate of de novo protein synthesis rather than a decreased degradation rate or a conversion of pre-existing polypeptides.

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+—-2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to catalyze electron transfer to cytochrome P-450, whereas the trypsin-solubilized reductase does not. In the present study, treatment of the highly purified detergent-solubilized rabbit liver enzyme (m.w. 77,700) with trypsin was shown to yield a small peptide (m.w. 6,100) as well as the large peptide (m.w. 71,200) which retains the flavin prosthetic groups. The small peptide, which is hydrophobic in nature as shown by its amino acid composition and solubility properties, is apparently the moiety in the native reductase involved in binding to cytochrome P-450 and to the microsomal membrane. The C-terminal amino acid sequences of the native reductase and large fragment are identical [-Trp-(Leu, Val)-Asp-Ser-COOH], thereby indicating that the hydrophobic peptide is located in the N-terminal region of the enzyme.

Cytochrome P-450 isozyme 2 from rabbit liver microsomes fluoresces upon excitation at 295 nm due to the single tryptophyl residue (Trp121) in the protein. The fluorescence spectrum, which is not altered by the presence of phospholipid or substrates, has a maximum at 335 nm, which suggests that the environment of the residue is hydrophobic. The fluorescence intensity decreases linearly with increase of specific content of the cytochrome preparations, and the holoenzyme was estimated to exhibit, at most, 6% as much fluorescence as the apoenzyme. This indicates that the fluorescence of the tryptophan is quenched by energy transfer to the heme. The distance between the tryptophyl residue and the heme was estimated to be less than 40 A. From enhancement of the fluorescence by methanol and ethanol, 30 to 50% of the Trp residue was found to be accessible to these solvents. On the other hand, the accessibility to iodide and cesium ions, as estimated by quenching effects, is less than 14%. From such evidence, the tryptophyl residue is believed to be partly buried. Since Trp121 is conserved at or near the same position in all mammalian P-450's so far sequenced, the results obtained may be applicable to these related cytochromes as well.

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.

NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and b5 and NADPH-cytochrome c reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.

The reaction of purified rabbit liver microsomal P-450 isozyme 2 with 4,4′-dithiobis(2-nitrobenzoate) (DTNB) exhibits first order kinetics and results in the modification of a single thiol, but causes no net loss of the native ferrous-carbonyl spectrum. Inclusion of both phospholipid and a tight-binding nitrogenous ligand, 1-benzylimidazole, in the reaction medium produces a burst-phase of DTNB modification, but the stoichiometry remains one thiol modified per polypeptide chain. The site of isozyme 2 rapidly labeled by DTNB and by monobromobimane, a fluorescent reagent for thiol groups, was shown to be Cys 152 . Results obtained strongly suggest that Cys 152 does not provide the proximal thiolate ligand to the heme iron atom. Since Cys 152 represents one of the two highly conserved cysteine-containing regions in the P-450 cytochromes, it appears likely that the other region, containing Cys 436 in this rabbit cytochrome (corresponding to Cys 355 in bacterial P-450 cam, Cys 436 in rat P-450 b or e, Cys 461 in rat P-450 c, Cys 456 in rat P-450 d or mouse isozyme 3, and Cys 458 in mouse isozyme 1) is the source of the thiolate ligand to the heme.

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMred → O 2 Complex I→Complex II→P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome b 5 is added. The latter protein does not appear to function as an electron carrier in this process.

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.

Cytochrome P-450 isozyme 3a, isolated from hepatic microsomes of rabbits treated chronically with ethanol, was found to have a unique substrate specificity when compared with isozymes 2, 3b, 3c, and 4. Form 3a has unusually high activity in the p-hydroxylation of aniline and in the oxidation of alcohols to aldehydes. These properties are reflected in the increased activities of these substrates in microsomes from ethanol-treated rabbits as compared to microsomes from untreated animals or those administered phenobarbital or 5,6-benzoflavone. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH cytochrome P-450 reductase and is stimulated by the presence of phospholipid, was shown not to be due to contaminating catalase or an NAD- or NADP-dependent alcohol dehydrogenase. Isozyme 3a catalyzes the oxidation of methanol, 1-propanol, and 1-butanol as well as ethanol; the relationships between the apparent Km values for these alcohols and their octanol-water partition coefficients is in accord with the known hydrophobic nature of the P-450 binding site. Whereas typical substrates of isozyme 2 are known to be metabolized with the stoichiometry predicted of a monooxygenase reaction, with isozyme 3a the sum of acetaldehyde formed from ethanol and of hydrogen peroxide generated is inadequate to account for the NADPH and oxygen consumed. Free hydroxyl radicals appear to mediate the slow oxidation of ethanol in the presence of the reductase alone but not the faster rate catalyzed by P-450 isozyme 3a. The results obtained, however, do not rule out the involvement of hydroxyl radical equivalent generated and bound at the active site of the cytochrome.

The cytochrome P-450 of Pseudomonas putida (P-450 cam ) and that of phenobarbital-induced liver microsomes (P-450 LM ) differ markedly in substrate specificity, solubility, and the requirement of the former for an iron-sulfur protein and the latter for a phospholipid for hydroxylation activity. Despite these differences, highly purified P-450 cam and P-450 LM show immunological cross reaction by competitive binding and inhibition of catalytic activity and are of similar subunit molecular weight and amino acid composition. Upon treatment with cyanogen bromide they yield small heme-containing peptides of highly similar amino acid composition.