The cytochromes P450 and b5 and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b₅ (cyt b₅). Numerous structural studies of cyt P450s, cyt b₅, and their reductases have given considerable insight into fundamental structure-function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be α-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b₅, to show what has already been possible and what seems to be viable in the very near future.     

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.     

The alpha-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.     

Modulatory effect of Urtica dioica L. (Urticaceae) leaf extract on biotransformation enzyme systems, antioxidant enzymes, lactate dehydrogenase and lipid peroxidation in mice.

The effects of two doses (50 and 100 mg/kg body wt given orally for 14 days) of an ethanol-water (80%-20%) extract of Urtica dioica L. and butylated hydroxyanisole (BHA) were investigated, for phase I and phase II enzymes, antioxidant enzymes, lactate dehydrogenase, lipid peroxidation and sulfhydryl groups in the liver of Swiss albino mice (8-9 weeks old). A modulatory effect of two doses and BHA was also observed for the activities of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase in the kidney, lung and forestomach, as compared with the control group. The activities of cytochrome b5 (cyt b5), NADH-cytochrome b5 reductase (cyt b5 R), glutathione S-transferase (GST), DT-diaphorase (DTD), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) showed a significant increase in the liver at both dose levels of extract. Both extract-treated showed significantly lower activity of cytochrome P450 (cyt P450), lactate dehydrogenase (LDH), NADPH-cytochrome P450 reductase (cyt P450 R), total sulfhydryl groups (T-SH), nonprotein sulfhydryl groups (NP-SH) and protein-bound sulfhydryl groups (PB-SH). BHA-treated Swiss albino mice showed a notable increase in levels of cyt b5, DTD, T-SH, PB-SH, GPx, GR, and SOD in the liver while, LDH, cyt P450, cyt P450 R, Cyt b5 R, GST, NP-SH, and CAT levels were reduced significantly as compared to control values. The extract was effective in inducing GST, DTD, SOD and CAT activity in the forestomach and SOD and CAT activity in the lung at both dose levels. BHA-treated Swiss albino mice induced DTD, GST and all antioxidative parameters in the kidney, lung and forestomach.     

Resonance Raman studies of cytochrome P450 2B4 in its interactions with substrates and redox partners

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.     

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

Human Cytochrome P450 17A1 Conformational Selection: MODULATION BY LIGAND AND CYTOCHROME b5*

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b₅ (b₅). Notably, titration of b₅ to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b_5, providing structural evidence consistent with the reported ability of b₅ to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.     

Human colon tumor cell line LS174T drug metabolizing system

The effects of culture variables on the specific content and activity of various enzymes of the drug mmetabolizing system were assessed in colon tumor cell line LS174T. The NADH reduced cytochrome b₅ (cyt b₅)⁴ spectrum of these cells was similar to rat liver cyt b₅. When released from the membrane by trypsin and concentrated, the cyt b₅ was found to cross react with rabbit antibody to rat liver cyt b₅ and human liver cyt b₅. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 µol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b₅ and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone showed a consistent, but not always significant increase in the NADPH and NADH cyt c reduction and benzanthracene an increase in the NADH cyt c reducing activity and cyt b₅ content. Griseofulvin lowered the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5 mM) caused a significant decrease in the specific activity of all enzymes, as judged by a student's t test, with a p<0.001.Abbreviations cyt b₅ cytochrome b₅ - cyt c cytochrome c - cyt P450 cytochrome P450 - PB Phenobarbital - HC Hydrocortisone - ALA -Aminolevulinic acid - GRIS Griseofulvin - PENT Pentagastrin - PASS Cell Passage - DMH Dimethylhydrazine - BA Benzanth Acene     

Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.     

Chemopreventive and therapeutic modulation of green tea polyphenols on drug metabolizing enzymes in 4-Nitroquinoline 1-oxide induced oral cancer

Oral cancer is one of the most common cancers in the world. Drugs can modulate the expression of drug metabolizing enzymes and are useful in chemoprevention as well as therapy in cancer. 4-Nitroquinoline 1-oxide (4-NQO) is used to induce oral cancer in the present study. In the present investigation, the effect of green tea polyphenols (GTP) on the activities of cytochrome b5, cytochrome P450, cytochrome b5 reductase (cyt b5 R), cytochrome P450 reductase (cyt P450 R), arryl hydrocarbon hydroxylase (AHH), DT-diaphorase (DTD)(Phase I enzymes) and glutathione-S-transferase (GST) and UDP-glucuronyl transferase (UDP-GT) (Phase II enzymes) were assessed in tongue and oral cavity. In induced rats, there was a decrease in the activity of Phase II enzymes and an increase in the activity of Phase I enzymes. On supplementation of GTP by both simultaneous and post treatment mode (200mg/kg) there was a significant increase in the activity of GST and UDP-GT and a significant decrease in the activity of Phase I enzymes. There was a significant decline in the number of tumors, tumor volume and oral squamous cell carcinoma in both simultaneous and post GTP treated animals relative to 4-NQO induced animals; on comparing simultaneous and post GTP treated animals the number of tumors, tumor volume and oral squamous cell carcinoma was significantly reduced in post treated animals. Thus inhibition of Phase I enzymes could be attributed to the protective efficacy of GTP which deactivates carcinogen and GTP induced the expression of Phase II enzymes that detoxifies the 4-NQO. It can be proposed that GTP plays role as a detoxifying agent by which its modulating role prevented/inhibited the formation of tumor.     

Solid-state NMR structures of integral membrane proteins

Abstract

Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.     

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

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.     

Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

Evaluation of the modulatory influence of food additive-garam masala on hepatic detoxication system.

Potential chemopreventive role of an Indian food additive-garam masala has been assessed through its impact on the hepatic levels of detoxication enzymes like glutathione S-transferase (GST), cytochrome b5 (cyt. b5) and cytochrome P-450 (cyt. P-450), and acid soluble sulfhydryl (-SH) content in 8-9 weeks old Swiss albino mice of either sex fed on the 0.5%, 1.0% and 1.5% (w/w) garam masala in the diet for 10 days. The data from this short term study revealed the significant but dose-independent alteration in the levels of detoxication system enzymes. The results suggest the possible chemopreventive potency of this widely used food additive by being a bifunctional inducer of detoxication system.