Comparative aromatic hydroxylation and N-demethylation of MPTP neurotoxin and its analogs, N-methylated beta-carboline and isoquinoline alkaloids, by human cytochrome P450 2D6

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin is a chemical inducer of Parkinson's disease (PD) whereas N-methylated beta-carbolines and isoquinolines are naturally occurring analogues of MPTP involved in PD. This research has studied the oxidation of MPTP by human CYP2D6 (CYP2D6*1 and CYP2D6*10 allelic variants) as well as by a mixture of cytochrome P450s-resembling HLM, and the products generated compared with those afforded by human monoamine oxidase (MAO-B). MPTP was efficiently oxidized by CYP2D6 to two main products: MPTP-OH (p-hydroxylation) and PTP (N-demethylation), with turnover numbers of 10.09 min-1 and Km of 79.36+/-3 microM (formation of MPTP-OH) and 18.95 min-1 and Km 69.6+/-2.2 microM (PTP). Small amounts of dehydrogenated toxins MPDP+ and MPP+ were also detected. CYP2D6 competed with MAO-B for the oxidation of MPTP. MPTP oxidation by MAO-B to MPDP+ and MPP+ toxins (bioactivation) was up to 3-fold higher than CYP2D6 detoxification to PTP and MPTP-OH. Several N-methylated beta-carbolines and isoquinolines were screened for N-demethylation (detoxification) that was not significantly catalyzed by CYP2D6 or the P450s mixture. In contrast, various beta-carbolines were efficiently hydroxylated to hydroxy-beta-carbolines by CYP2D6. Thus, N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline (a close MPTP analog) was highly hydroxylated to 6-hydroxy-N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline and a corresponding 7-hydroxy-derivative. Thus, CYP2D6 could participate in the bioactivation and/or detoxification of these neuroactive compounds by an active hydroxylation pathway. The CYP2D6*1 enzymatic variant exhibited much higher metabolism of both MPTP and N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline than the CYP2D6*10 variant, highlighting the importance of CYP2D6 polymorphism in the oxidation of these toxins. Altogether, these results suggest that CYP2D6 can play an important role in the metabolic outcome of both MPTP and beta-carbolines.

ABCB1 and cytochrome P450 genotypes and phenotypes: influence on methadone plasma levels and response to treatment

BACKGROUND AND OBJECTIVE: The in vivo implication of various cytochrome P450 (CYP) isoforms and of P-glycoprotein on methadone kinetics is unclear. We aimed to thoroughly examine the genetic factors influencing methadone kinetics and response to treatment. METHODS: Genotyping for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, ABCB1, and UGT2B7 polymorphisms was performed in 245 patients undergoing methadone maintenance treatment. To assess CYP3A activity, the patients were phenotyped with midazolam. RESULTS: The patients with lower CYP3A activity presented higher steady-state trough (R,S)-methadone plasma levels (4.3, 3.0, and 2.3 ng/mL x mg for low, medium, and high activity, respectively; P = .0002). As previously reported, CYP2B6*6/*6 carriers had significantly higher trough (S)-methadone plasma levels (P = .0001) and a trend toward higher (R)-methadone plasma levels (P = .07). CYP2D6 ultrarapid metabolizers presented lower trough (R,S)-methadone plasma levels compared with the extensive or intermediate metabolizers (2.4 and 3.3 ng/mL x mg, respectively; P = .04), whereas CYP2D6 poor metabolizer status showed no influence. ABCB1 3435TT carriers presented lower trough (R,S)-methadone plasma levels (2.7 and 3.4 ng/mL . mg for 3435TT and 3435CC carriers, respectively; P = .01). The CYP1A2, CYP2C9, CYP2C19, CYP3A5, and UGT2B7 genotypes did not influence methadone plasma levels. Only CYP2B6 displayed a stereoselectivity in its activity. CONCLUSION: In vivo, CYP3A4 and CYP2B6 are the major CYP isoforms involved in methadone metabolism, with CYP2D6 contributing to a minor extent. ABCB1 genetic polymorphisms also contribute slightly to the interindividual variability of methadone kinetics. The genetic polymorphisms of these 4 proteins had no influence on the response to treatment and only a small influence on the dose requirement of methadone.

Expression and activity of the cytochrome P450 2E1 in patients with nonalcoholic steatosis and steatohepatitis

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver (NAFL) have a different prognosis and should be dealt with differently. The pathogenesis of NASH implicates the overexpression of cytochrome P450 2E1 (CYP2E1). We investigated whether the noninvasive determination of CYP2E1 activity could replace a liver biopsy in order to differentiate NASH from NAFL. METHOD: Forty patients referred for suspicion of NASH underwent liver biopsy. In these patients, CYP2E1 activity was determined noninvasively by the 6-hydroxychlorzoxazone/chlorzoxazone (CHZ) ratio (CHZ test). Expression of CYP2E1 on liver slides was assessed by immunohistochemistry, and immunostaining for smooth muscle actin was used to assess the activation of hepatic stellate cells (HSC). RESULTS: Thirty patients with NASH were compared with 10 subjects with NAFL. No statistically significant difference could be identified for the clinical and biochemical parameters between the two groups. In the histology, steatosis was more important in NASH than in NAFL (P<0.0001). There was no difference either in the activity (CHZ test) or in the expression of CYP2E1 (immunohistochemistry) between patients with NASH and patients with NAFL. The degree of HSC activation was also comparable between the two groups. A positive and significant correlation was found between the activity of CYP2E1 and body mass index (P<0.001) as well as with the degree of steatosis (P=0.008). CONCLUSION: For patients suspected to have NASH, noninvasive tests including the determination of the CYP2E1 activity are unable to distinguish them from patients with steatosis.

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues

OBJECTIVE: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. DESIGN: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. PATIENTS: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. RESULTS: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. CONCLUSIONS: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.

Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content

Echinacea preparations are one of the best selling herbal medicinal products with a well established therapeutic use in the prophylaxis of upper respiratory tract infections. Their consumption is increasing, but information about their ability to inhibit cytochrome P450 enzymes (CYP) is fragmentary. The picture is further complicated by a lack of phytochemical characterization of previously tested preparations. Due to its well characterized immunomodulatory activity, the standardized Swiss registered Echinacea purpurea (L.) Moench Echinaforce extract was selected for detailed study. With the single baculovirus-expressed CYP isoforms 1A2, 2C19, 2D9 and 3A4, inhibitory actions were measured by monitoring fluorescent metabolites derived from enzyme substrates (supersome assay). The Echinaforce extract induced mild inhibition of all these isoforms, with CYP 3A4 being the most, and CYP 2D6 the least sensitive enzyme. To assess whether CYP inhibition might be a general feature of Echinacea preparations, an additional nine commercially available preparations were screened using CYP 3A4. All tested preparations were able to inhibit CYP 3A4, but inhibitory potencies (expressed as median inhibitory concentration, IC50) varied by a factor of 150. The alkylamides are thought to be responsible for the immunomodulatory activity of Echinacea, and so the concentration of 2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and total alkylamide content were determined in all preparations, and the latter was found to be associated with their CYP 3A4 inhibitory potency. The chemically pure alkylamides dodeca-2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and dodeca-2E,4E-dieonoic acid isobutylamide (2) showed inhibitory activity on CYP 2C19, 2D6 and 3A4. However, unlike the Echinaforce extract, the alkylamides did not induce CYP 1A2 inhibition. Thus, other, as yet unidentified constituents also contribute to the overall weak inhibitory effects seen with Echinacea preparations in-vitro.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 μM and 6.06 μM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^