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The NADPH-dependent consumption of O(2) by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O(2) molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O(2) uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O(2) and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.

Although much has been learned about cytochrome P450 as an oxygenating catalyst since its discovery as a carbon monoxide-binding pigment in liver microsomes over 40 years ago, two major problems remain unsolved. These are the role of the heme sulfur ligand and the identity of the “activated oxygen” species generated during the reduction of molecular oxygen. To address these questions, mutagenesis of pertinent amino acid residues in NH2-terminal-truncated microsomal P450s 2B4 and 2E1, has been carried out. Clones for these cytochromes with the active-site cysteine replaced by histidine, serine, or tyrosine were expressed in Escherichia coli. Despite instability, some of the resulting hemoproteins were purified. The histidine-437 mutant of P450 2E1 is reduced by NADPH in the presence of the reductase and yields H2O2 in the reconstituted system. However, no activity was detected in the oxidation of several substrates, thus indicating a functional role for the sulfur ligand. In other experiments, the active-site threonine residue that facilitates proton transfer was replaced with alanine. Changes in the rates of catalysis of aldehyde deformylation, olefin epoxidation, ipso-substitution, and other reactions by P450 2B4:T302A and 2E1:T303A mutants provided evidence for peroxo-iron, hydroperoxo-iron, and oxenoid-iron as discrete functional oxygenating species. The availability of multiple oxidants is believed to contribute to the unmatched versatility of the P450 cytochromes.

The function of the unique axial thiolate ligand of cytochrome P450 has been investigated by mutagenesis of the active-site cysteine with other amino acids in NH(2)-truncated P450s 2B4 and 2E1. The expressed Ser-436 variant of P450 2B4 was highly purified but incurred considerable heme loss. The pyridine hemochrome spectrum of C436S is characteristic of protoporphyrin IX, and the absolute spectra display Soret maxima at 405 nm (ferric), 422 nm (ferrous), and 413 nm (ferrous CO). 2B4:C436S catalyzes the NADPH- and time-dependent formation of H(2)O(2) in the reconstituted enzyme system, with maximal rates at approximately equimolar amounts of P450 reductase and C436S hemeprotein. The 2-electron oxidase activity with saturating reductase is directly proportional to the concentration of 2B4:C436S, and the turnover is 60-70% of that of the wild-type enzyme. In contrast, the C436S variant is devoid of oxygenase activity with typical substrates such as d-benzphetamine, 1-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H(2)O(2)-supported peroxidation of guaiacol and pyrogallol is comparable with 2B4 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2B4 nor 2B4:C436S catalyzes H(2)O(2) decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2B4 mainly into a 2-electron oxidase.

Evidence for multiple functional active oxidants in cytochrome P450-catalyzed reactions was previously obtained in this laboratory with mutants in which proton delivery was perturbed by replacement of the highly conserved threonine residue in the active site by alanine, thus apparently interfering with the conversion of the peroxo-iron to the hydroperoxo-iron and the latter to the oxenoid-iron species. These enzymes have now been employed to examine the reaction in which cytochrome P450 in liver microsomes is known to effect ipso -substitution, the elimination of p -substituents in phenols to yield hydroquinone. As shown with purified NH 2 -truncated cytochromes in a reconstituted enzyme system, the reaction exhibits an absolute requirement for cytochrome P450 and NADPH–cytochrome P450 reductase. Under optimal conditions truncated cytochrome P450 2E1 is active with 10 of the p -substituted phenols examined. Of particular interest, the corresponding cytochrome with threonine-303 replaced by alanine is from 1.5- to 50-fold higher in activity with the p- chloro, -bromo, -nitro, -cyano, -hydroxymethyl, -formyl, and -acetyl derivatives, and the reaction with the p -benzoyl, -methyl, and - t -butyl compounds is catalyzed by the mutant enzyme only. The results implicate the hydroperoxo-iron species as an electrophilic active oxidant in cytochrome P450-catalyzed aromatic ipso -substitution.

Oxidation of the mechanistic probes trans,trans-2-methoxy-3-phenylmethylcyclopropane and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that different structural rearrangements occur for the two types of intermediates. The P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from rabbit), the expressed truncated isozymes P450 Δ2B4 and P450 Δ2E1 (ethanol-inducible, from rabbit), and mutants of the latter two in which an active site threonine was replaced with alanine, Δ2B4 T302A, and Δ2E1 T303A. Cationic rearrangement products were found from both probes. Oxidations of trans,trans-2-methoxy-3-phenylmethylcyclopropane gave small amounts of radical-derived rearrangement products indicating that hydroxylation occurs via insertion reactions with transition state lifetimes in the 80−200 fs range. A mechanistic description of cytochrome P450-catalyzed hydroxylations that is in accord with ...

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis - or trans -butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO 2 H) 3+ and oxenoid-iron (FeO) 3+ , can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

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.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides ...

The nasal mucosa of some mammalian species are susceptible to the toxicity of aflatoxin B1 (AFB1), a potent hepatocarcinogen, but little is known about the nasal enzymes involved in the metabolic activation of AFB1 or the metabolites produced. In the present study, the metabolism of AFB1 was studied with nasal microsomes from rats and rabbits and with several purified isozymes of rabbit P450 in a reconstituted enzyme system. The rates of AFB1-N7-guanine DNA adduct formation with rabbit and rat nasal microsomes are over 3- and 10-fold higher, respectively, than with liver microsomes from the same species. On the other hand, the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1) products known to be less toxic, are lower with nasal than with liver microsomes. Of particular interest, nasal microsomes produce high levels of six unidentified polar metabolites that are not formed by microsomes from liver or several other tissues. These same products are also generated by P450 NMa purified from rabbit nasal microsomes in a reconstituted system, but not by five other isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6) that are known to be present in nasal microsomes. AFB1-DNA adducts are formed by P450 NMa at a rate 3-fold higher than that by nasal microsomes. The DNA adducts are formed at much slower rates by P450s 2G1, 2B4, and 1A2, and adducts are not formed at measurable rates by P450s 2E1 and 3A6. Moreover, AFB1-DNA adduct formation is also catalyzed by cDNA-derived, heterologously expressed P450s 2A10 and 2A11, both of which are known to be present in the purified P450 NMa preparation. The K m and V max values of the two isozymes for DNA adduct formation are comparable to those for nasal microsomes. Furthermore, the formation of AFB1-DNA adducts by nasal microsomes is decreased by nicotine, a known inhibitor of P450 NMa. These data indicate that members of the P450 2A gene subfamily play an important role in the metabolic activation of AFB1 in rabbit and rat nasal mucosa and suggest a molecular basis for assessing the health risk associated with inhalation exposure to this procarcinogen in humans

The NADPH-dependent consumption of O(2) by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O(2) molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O(2) uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O(2) and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Several purified isoforms of microsomal cytochrome P450 were previously shown in this laboratory to catalyze the oxidative deformylation of a variety of alpha- or beta-branched aldehydes with the production of olefins and formic acid. In the present study, 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC, numbered according to the convention for steroids) was synthesized as a bicyclic analog of the aldehyde that is known to be the terminal intermediate in the enzymatic conversion of androgens to estrogens. ODEC undergoes aromatization in a reconstituted enzyme system containing liver microsomal cytochrome P450 2B4 and NADPH-cytochrome P450 reductase, along with NADPH and phosphatidylcholine, under aerobic conditions. The products, 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and formic acid, were identified by mass spectrometry. The corresponding 10-carbinol does not undergo oxidative aromatization with P450 2B4, and with ODEC as substrate, other microsomal P450 cytochromes are either weakly active (isoforms 2C3 and 3A6) or inactive (isoforms 2E1, 1A2, and 2G1). Cytochrome b5 stimulates the P450 2B4-catalyzed reaction with ODEC about 2.6-fold but has no effect with the other P450s. In two respects the conversion of the bicyclic model compound to HTN with P450 2B4 was shown to be similar to that of the steroid aromatase reaction. Deuterium in the formyl group of ODEC was retained in the formic acid that was produced and isolated as the 4-nitrobenzyl derivative, and with preparations of ODEC containing deuterium in the 1 alpha position or the 1 alpha and 2 alpha positions, it was shown that the desaturation reaction is specific for removal of the 1 beta-hydrogen, thus involving a stereospecific cis elimination of formate. Cytochrome b5 has no effect on the stereospecificity of the reaction.

The mechanisms of oxidation of primary and secondary benzylic alcohols to the corresponding carbonyl compounds by purified rabbit liver cytochrome P450 forms 2B4 and 2E1 in a reconstituted enzyme system has been examined by linear free energy relationships, intramolecular and steady-state deuterium isotope effects, and the incorporation of an O2-derived oxygen atom or solvent-derived deuterium. The kcat and Km values were found to be relatively insensitive to the presence of electronic perturbations at the para position. The Hammett reaction constants for the oxidation of benzyl alcohols by P450s 2B4 and 2E1 are -0.46 and -0.37, respectively, and with 1-phenylethyl alcohols the corresponding reaction constants are -1.41 and -1.19, respectively. With [1-2H1]benzyl alcohol, P450s 2B4 and 2E1 show similar intramolecular deuterium isotope effects of 2.6 and 2.8, respectively, whereas with [1-2H2]benzyl alcohol under steady-state conditions, the deuterium isotope effects on the catalytic constants are 2.8 and 1.3, respectively. No significant isotope effect on the catalytic constant was noted for either form of P450 with 1-phenylethyl alcohol. In D2O, acetophenone formed by either form of P450 from 1-phenylethyl alcohol does not contain a deuterium atom at the methyl group, whereas under an atmosphere of 18O2 approximately 30% of the labeled oxygen is incorporated into the carbonyl group with either form of the cytochrome. The results are consistent with a mechanism that involves stepwise oxidation of the alcohol to a carbon radical alpha to the alcohol function, followed by oxygen rebound to yield the gem-diol, dehydration of which gives the carbonyl product.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The cytochrome P450 gene superfamily encodes many isoforms that are unusual in the variety of chemical reactions catalyzed and the number of substrates attacked. The latter include physiologically important substances such as steroids, eicosanoids, fatty acids, lipid hydroperoxides, retinoids, and other lipid metabolites, and xenobiotics such as drugs, alcohols, procarcinogens, antioxidants, organic solvents, anesthetics, dyes, pesticides, odorants, and flavorants. Accordingly, it is not surprising that these catalysts have come under intensive study in recent years in fields as diverse as biochemistry and molecular biology, endocrinology, pharmacology, toxicology, anesthesiology, nutrition, pathology, and oncology. In this review, recent advances in our knowledge of the catalytic properties, reaction mechanisms, and regulation of expression and activity of the P450 enzymes are briefly summarized. In addition, the prospects for research in this field are considered, and advances are predicted in four broad areas: improved basic knowledge of enzyme catalysis and regulation; synthesis of fine chemicals, including drug design and screening; removal of undesirable environmental chemicals; and biomedical applications related to steroid, drug, carcinogen, and alcohol metabolism.

As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts.

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.

Olfactory-specific cytochrome P450NMb was previously purified to electrophoretic homogeneity from microsomes of rabbit nasal mucosa in this laboratory. In the present study, a cDNA library made from poly(A)+ RNA from rabbit nasal mucosa was screened with antibodies to this P450, and eight immunopositive clones were isolated and characterized. The sequence determined from two overlapping clones contained an open reading frame of 1446 nucleotides, with the predicted first 39 amino acids corresponding to residues 12 to 50 of purified NMb, except for position 46, where Leu was encoded instead of the Glu residue that was found earlier by Edman degradation analysis. The complete polypeptide, including residues 1 to 11, contains 494 amino acid residues and has a molecular weight of 56,640. Sequence comparisons indicated that NMb is more than 50% identical to members of the rabbit P450 gene II family, including IIB4, IIC3, IIC5, IIE1, and IIE2, and 83% identical to rat P450olf1 (IIG1). Hybridization of NMb to electrophoretically fractionated rabbit nasal poly(A)+ RNA revealed 3.6- and 2.1-kb species, but with a probe derived from the 3'-nontranslated portion of the cDNA only the 3.6-kb band was observed, suggesting the use of alternate polyadenylation sites or splicing. In agreement with the known tissue-specific distribution of NMb protein, NMb transcripts were found in olfactory mucosa, but not in liver, lung, intestine, or kidney. Genomic hybridization analysis indicated that there may be only one copy of the NMb gene present in the rabbit genome.

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.

As shown previously in this laboratory, purified rabbit liver microsomal cytochrome P-450 form 2 (P-450 IIB4) catalyzes the reductive cleavage of hydroperoxides to yield hydrocarbons and either aldehydes or ketones. We have proposed that lipid hydroperoxides are the physiological substrates for the cleavage reaction and have shown that with 13-hydroperoxy-9,11-octadecadienoic acid the formation of pentane is roughly equimolar with respect to the NADPH consumed. In the present study, the other product was isolated and identified as 13-oxo-9,11-tridecadienoic acid. Of particular interest, the alcohol-inducible form of liver microsomal cytochrome P-450 form 3a (P-450 IIE1) is the most active of the isozymes examined in the reductive beta-scission of the 13-hydroperoxide derived from linoleic acid and the 15-hydroperoxide derived from arachidonic acid as well as the model compounds cumyl hydroperoxide (alpha, alpha-dimethylbenzyl hydroperoxide) and t-butyl hydroperoxide. In general, the forms of P-450 with lower activity, as judged by the rate of NADPH oxidation in the reconstituted system, give less of the cleavage products (hydrocarbon and oxo compound) and catalyze direct reduction of the hydroperoxides to the corresponding hydroxy compounds. The occurrence of the reductive cleavage reaction in liver microsomal membranes was demonstrated, and microsomes from animals treated with ethanol or acetone (P-450 IIE1 inducers) or phenobarbital (a P-450 IIB4 inducer) were more active than those from untreated animals. We suggest that the alcohol-inducible P-450, in addition to its known deleterious effects in chemical toxicity and chemical carcinogenesis, may enhance the reductive cleavage of lipid hydroperoxides with a resultant loss in membrane integrity.

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.

▪ Abstract The author describes studies that led to the resolution and reconstitution of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence for multiple isoforms of the oxygenases with distinct chemical and physical properties and different but somewhat overlapping substrate specificities. Present knowledge of the individual steps in the P450 and reductase reaction cycles is summarized, including evidence for the generation of multiple functional oxidants that may contribute to the exceptional diversity of the reactions catalyzed.

Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.

Cytochrome P450 enzymes catalyze a number of oxidations in nature including the difficult hydroxylations of unactivated positions in an alkyl group. The consensus view of the hydroxylation reaction 10 years ago was that a high valent iron-oxo species abstracts a hydrogen atom from the alkyl group to give a radical that subsequently displaces the hydroxy group from iron in a homolytic substitution reaction (hydrogen abstraction-oxygen rebound). More recent mechanistic studies, as summarized in this review, indicated that the cytochrome P450-catalyzed "hydroxylation reaction" is complex, involving multiple mechanisms and multiple oxidants. In addition to the iron-oxo species, another electrophilic oxidant apparently exists, either the hydroperoxo-iron intermediate that precedes iron-oxo or iron-complexed hydrogen peroxide formed by protonation of the hydroperoxo-iron species on the proximal oxygen. The other electrophilic oxidant appears to react by insertion of OH(+) into a C-H bond to give a protonated alcohol. Computational work has suggested that iron-oxo can react through multiple spin states, a low-spin ensemble that reacts by insertion of oxygen, and a high-spin ensemble that reacts by hydrogen atom abstraction to give a radical.

Norcarane was employed as a mechanistic probe in oxidations catalyzed by hepatic cytochome P450 enzymes and by the soluble methane monooxygenase (sMMO) enzyme from Methylococcuscapsulatus (Bath). In all cases, the major oxidation products (>75%) were endo- and exo-2-norcaranol. Small amounts of 3-norcaranols, 2-norcaranone, and 3-norcaranone also formed. In addition, the rearrangement products (2-cyclohexenyl)methanol and 3-cycloheptenol were detected in the reactions, the former possibly arising from a radical intermediate and the latter ascribed to a cationic intermediate. The formation of the cation-derived rearrangement product is consistent with one or more reaction pathways and is in accord with the results of previous probe studies with the same enzymes. The appearance of the putative radical-derived rearrangement product is in conflict with other mechanistic probe results with the same enzymes. The unique implication of a discrete radical intermediate in hydroxylations of norcarane may be the consequence of a minor reaction pathway for the enzymes that is not manifest in reactions with other probes. Alternatively, it might reflect a previously unappreciated reactivity of norcaranyl cationic intermediates, which can convert to (2-cyclohexenyl)methanol. We conclude that generalizations regarding the intermediacy of radicals in P450 and sMMO enzyme-catalyzed hydroxylations based on the norcarane results should be considered hypothetical until the origin of the unanticipated results can be determined.

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the gamma-meso-alkyl heme derivative.

Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5' flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA-protein interactions revealed that Sp1 and NFkappaB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1alpha (IL-1alpha) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1alpha-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1alpha treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1alpha in HepG2 cells.

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.

Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.

Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring. The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer. Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics. Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion. To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface. The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices. The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane.

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.

The inactivation of cytochrome P450 2B4 by aldehydes in a reconstituted enzyme system requires molecular oxygen and NADPH and is not prevented by the addition of catalase, superoxide dismutase, epoxide hydrolase, glutathione, or ascorbic acid. A strong correlation between loss of enzymatic activity and bleaching of the heme chromophore was established, and the inactivation was shown to be irreversible upon dialysis. In general, saturated aldehydes are more inhibitory than those with alpha,beta-unsaturation, as indicated by the k(inact) values, and primary aldehydes are more potent inactivators than the structurally related secondary and tertiary aldehydes. Consistent with recent studies on catalytic specificity of the T302A mutant of this cytochrome [Vaz, A. D. N., Pernecky, S. J., Raner, G. M., & Coon, M. J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4644-4648], the rate of aldehyde deformylation, as determined by formation of the alcohol with one less carbon atom, is greatly stimulated over that of the wild-type enzyme. Of particular interest, the rate of oxidation of aldehydes to carboxylic acids is decreased with the mutant, whereas the rate of inactivation via heme destruction is enhanced. Furthermore, comparative deuterium isotope effects and the relative rates of inactivation and product formation suggest that the mechanism of aldehyde inactivation of P450 2B4 involves the deformylation reaction and is unrelated to carboxylic acid formation. Finally, in the reaction of P450 2B4 with 3-phenylpropionaldehyde, the formation of a heme adduct with a molecular weight corresponding to that of native heme plus 104 mass units confirms the loss of the carbonyl group from the aldehyde prior to reaction with the chromophore. We conclude that inactivation of P450 by aldehydes occurs via homolytic cleavage of a peroxyhemiacetal intermediate to give an alkyl radical that reacts with the heme.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), 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 is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.

Publisher Summary This chapter discusses the N-terminal modifications that alter P450 membrane targeting and function. The hydrophobic N-terminal region that is present in mammalian P450 cytochromes serves as the signal peptide for cotranslational insertion of the protein into the endoplasmic reticulum, while the following hydrophobic proline-rich segment promotes stabilization of the enzyme. The role of the N-terminal region in promoting extensive aggregation of P450 enzymes is demonstrated by disaggregation of shortened P450s to monomers by the detergent cholate at concentrations that do not alter the multimeric aggregation state of the full-length enzymes. The retention of catalytic activity and the monomerization of P450 cytochromes at low detergent concentrations, achieved through N-terminal modification, are used to improve the suitability of these enzymes as candidates for crystallization attempts. The chapter describes the methods for the heterologous expression, determination of subcellular localization, and purification of shortened P450 cytochromes as well as examination of their catalytic properties.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

A series of esters and several amides were shown to undergo oxidative cleavage with the formation of carbonyl products in the presence of purified isoforms of liver microsomal cytochrome P450 (P450) in a reconstituted enzyme system. The reaction also requires NADPH and NADPH-cytochrome P450 reductase and is stimulated by phosphatidylcholine. Kinetic constants were determined in experiments in which the predicted aldehyde product was identified and quantitated by gas chromatography. A relationship was seen with P450 2E1 between the structures of the esters and the Vmax values, with the rates decreasing in the series of methyl formate to methyl valerate, and similarly in the series of methyl, ethyl, propyl, butyl, and amyl acetates. Furthermore, a clear correlation exists between the Km values of the ethyl esters examined and the log of the octanol/water partition coefficients of these substrates. With P450 2E1, the Km decreases significantly between one and four carbon atoms in the chain length of the acyl component of the ester but is unaffected by a further increase in length. However, no correlation was found between the Km value and the chain length of the alcohol moiety of the esters. Similarly, with P450 2B4 a large decrease in Km occurs between one and five carbons in the acyl component of the ethyl esters but is unaffected by a further increase in chain length. The observed correlation is presumed to arise from hydrophobic interactions between the access channel to the active site of P450 and the acyl side chain of the esters. P450 1A2 is also active in ester cleavage, and the three cytochromes examined with esters are active in the conversion of N-alkyl amides to aldehydes, as are P450s 2C3, 1A1, and 3A6. Studies on 2-butyl acetate oxidation by P450 2B4 in the presence of 18O2 showed 88% 18O incorporation into the product, 2-butanone. This is consistent with a mechanism that involves hydroxylation at the alpha-carbon of the alcohol component of the ester to yield an unstable geminal hydroxy ester, as proposed earlier by F. P. Guengerich et al. (1988, J. Biol. Chem. 263, 8176-8183) for several dihydropyridine carboxylic esters. Our results further indicate that such an intermediate decomposes by a nonhydrolytic mechanism and also rule out the possibility of transient ester hydrolysis with subsequent oxidation of the alcohol formed. In addition, they establish that oxidative cleavage is a widespread reaction among P450 cytochromes and commonly used esters and amides.

This laboratory previously expressed cDNAs encoding rabbit liver cytochrome P450 2E1 (the ethanol-inducible isoform) and the corresponding protein lacking amino acids 3-29, a proposed membrane anchor, in Escherichia coli. Unexpectedly, the shortened protein, like the full-length form, was found to be predominantly located in the bacterial inner membrane rather than the cytosol and to have full catalytic activity. Additional proteins with alterations in the NH2-terminal region of P450 2E1 or P450 2B4 (the phenobarbital-inducible isoform) were similarly expressed, and it was concluded that such modifications can change the cytochrome to an increased cytosolic localization and that the first two hydrophobic segments are not uniquely involved in attachment to the bacterial membrane (Pernecky et al., 1993, Proc. Natl. Acad. Sci. USA 90, 2651-2655). In the present study, three chimeric cytochromes were produced to determine the effect on subcellular localization: 2E1:2B4, in which the first 17 residues of 2E1 (delta 3-29) replaced the corresponding 17 residues in 2B4 (delta 2-27), and BM-3:2B4 and BM-3:2E1, in which the first 19 residues of P450BM-3 replaced the first 17 in 2B4 (delta 2-27) and 2E1 (delta 3-29), respectively. Of the total cytochrome expressed, the localization in the E. coli cytosol was about 60, 70, and 80% for the respective chimeras, with 80% being the highest for any P450 we have examined. A plot of the extent of membrane binding versus hydropathy of the NH2-terminal region showed that the terminal sequence strongly influences the subcellular distribution and that a group of 2E1 proteins and a group of 2B4 proteins each have other regions that characteristically determine the extent of membrane attachment. The role of the NH2-terminal region in the high level of aggregation of purified full-length P450 is indicated by the finding that the multimeric state of 2E1 or 2B4 is unaffected by sodium cholate at concentrations that convert 2E1 (delta 3-29) or 2B4 (delta 2-27) to the monomeric state. In contrast to our earlier experience with P450 2E1, purified P450 2B4 (delta 2-27) has on the average only about half the activity of full-length 2B4 with substrates that undergo oxidative dealkylation or oxygenation at a hydroxyl group.

Intramolecular kinetic isotope effects (KIEs) were determined for cytochrome P450-catalyzed hydroxylation reactions of methyl-dideuterated trans-2-phenylcyclopropylmethane-d2 (1-d2), which gives two products from oxidation of the methyl group, trans-2-phenylcyclopropylmethanol (2) and 1-phenyl-3-buten-1ol (3). In oxidations of each enantiomer of 1-d2 with three P450 enzymes (CYP2B1, CYPDelta2E1, and CYPDelta2E1 T303A), the apparent intramolecular KIEs were different for products 2 and 3 in all cases and different for each enzyme-substrate combination. In oxidations of each enantiomer of undeuterated 1-d0 and trideuteriomethyl 1-d3 by CYP2B1 and CYPDelta2E1, the ratio of products 2/3 decreased for 1-d3 in comparison to 1-d0 in all cases. The results require multiple pathways for P450-catalyzed hydroxylation and are consistent with the "two-oxidants" model, where hydroxylation is effected by both the hydroperoxy-iron species and the iron-oxo species. The results are not consistent with predictions of the "two-states" model for P450-catalyzed hydroxylations, where oxidations occur from a low-spin state and a high-spin state of iron-oxo.

Cytochrome P450 2G1 (2G1), which is uniquely expressed in the olfactory mucosa in mammals, may have important physiological functions. In the present study, we have examined the catalytic activity of rabbit 2G1 toward a number of steroid sex hormones, including androstenedione, estradiol, progesterone, testosterone, and 5 alpha-dihydrotestosterone; the purified cytochrome is active toward all of these compounds in a reconstituted enzyme system with turnover numbers of 1.84, 0.34, 1.46, 1.04, and 0.84, respectively, at a substrate concentration of 5 microM. In the presence of cytochrome b5, the turnover numbers are 1.58, 0.66, 1.66, 2.74, and 1.34, respectively. Estradiol is converted to the 2-hydroxy compound (major product) and 4-hydroxy compound (minor product) by 2G1, and progesterone is converted to the 16 alpha-hydroxy derivative as well as the corresponding keto compound as a secondary product. The same products are formed in olfactory microsomal suspensions as major metabolites of progesterone, and the reactions are inhibited strongly by anti-2G1 IgG. In a reconstituted system, 2G1 has an apparent Km of 2.0 microM and a Vmax of 1.8 nmol/min/nmol P450 for the formation of the 16 alpha-hydroxyprogesterone. Of particular interest, 2G1-catalyzed progesterone metabolism is effectively inhibited by the boar pheromones, 5 alpha-androst-16-en-3-one and 5 alpha-androst-16-en-3 alpha-ol, and to a lesser extent by a variety of odorant compounds as well as by known P450 inhibitors, including ketoconazole and alpha-naphthoflavone. The broad substrate specificity and relatively high catalytic efficiency of 2G1 in sex steroid metabolism suggest a role for this unique P450 isozyme in the maintenance of steroid hormone homeostasis in the olfactory mucosa.