Coupled motions direct electrons along human microsomal P450 Chains

Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10⁻¹⁰ mol/cm² and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s⁻¹, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.     

Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 °C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug-drug interaction studies.     

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.     

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane‐bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2‐CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2‐CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid‐solubilized CYP2J2‐CPR system by increasing the K_m and decreasing the V_max, and effect that is size‐dependent. Crowding also affects the CYP2J2‐CPR‐ND system by decreasing both the K_m and V_max for Dextran‐based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2‐CPR membrane protein system.     

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

Active-site structure,binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.     

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.     

Mosquito NADPH‐cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3‐mediated deltamethrin metabolism

The NADPH‐cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane‐bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane‐bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR‐mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c–reducing activity indicated that the wild‐type and mutant flAnCYPORs followed a non‐classical two‐site Ping‐Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH‐regenerating system, increased CYP6AA3‐mediated deltamethrin degradation compared to the wild‐type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3‐mediated activity supported by wild‐type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3‐mediated metabolism. © 2010 Wiley Periodicals, Inc.     

Reactivities of organic phase biosensors: 6. Square-wave and differential pulse studies of genetically engineered cytochrome P450(cam) (CYP101) bioelectrodes in selected solvents

Cytochrome P450(cam) (CYP101) bioelectrodes suitable for application in organic phases were prepared from genetically engineered CYP101 and vesicular dispersions of didodecyldimethylammonium bromide. The amperometric biosensor system was characterised under anaerobic conditions by cyclic and square-wave voltammetric methods. Cyclic- and square-wave-voltammetry studies showed that the biosensors exhibited direct reversible electron transfer between the haem iron atom and the glassy carbon electrode surface. The formal redox potential estimated for the electrode in acetonitrile was -380 mV/Ag-AgCl. The formal potential shifted anodically as the organic phase biosensor responded irreversibly to substrate (camphor) under anaerobic and aerobic conditions in acetonitrile. Differential pulse analysis of the reactivities of the CYP101 enzyme electrode confirmed the square-wave voltammetry result, which showed that the binding of substrate decreased the redox potential necessary for initiating the monooxygenation reaction of cytochrome P450(cam).     

RNA‐Interference Approach to Study Functions of NADPH : Cytochrome P450 Oxidoreductase in Human Hepatocytes

Human NADPH : cytochrome P450 oxidoreductase (POR) is encoded by a single gene on chromosome 7q11.2. This flavoprotein donates electrons derived from NADPH to a variety of acceptor proteins, including squalene monooxygenase, heme oxygenase, cytochrome b₅, and many microsomal cytochromes P450 (CYPs), which are involved in oxidative drug metabolism, steroidogenesis, and other functions. Numerous aspects related to cellular POR expression have not been systematically investigated. Interestingly, POR expression is lower compared to CYPs and may thus be limiting for monooxygenase activities, but conversely, POR knock‐out in mice resulted in compensatory upregulation of CYPs. POR may also influence intracellular cholesterol and lipid homeostasis. To systematically investigate such effects, we developed specific POR gene silencing in cell lines and primary human hepatocytes by RNA interference using small interfering RNAs (siRNAs). In HepG2 cells, POR mRNA could be reduced by 95% over 4 days accompanied by reduced protein content and activity. In primary human hepatocytes, POR mRNA knock‐down was less effective and more variable. Analysis of CYPs indicated induction of CYP3A4 but not CYP1A2 or CYP2D6. These results demonstrate that POR can be efficiently and almost completely silenced in HepG2 cells and, at least partially, in primary human hepatocytes. This will allow systematic studies of various consequences of POR variability in human cells.     

Recombinant Escherichia coli cells immobilized in Ca-alginate beads for metabolite production

Milligram amounts of metabolites of drug candidates are required to identify toxic products. Human drug metabolites are currently produced selectively in a time- and cost-efficient manner in bioreactor systems containing recombinant Escherichia coli co-expressing a human cytochrome P450 isoenzyme/NADPH cytochrome P450 reductase (hCYP/HR) complex. For further optimization, immobilization of the catalytic system in Ca-alginate microbeads was considered. This new concept was designed for CYP3A4 with testosterone as substrate. Immobilized E. coli cells had a high maximal and homogeneously distributed biomass. Viability was stable over at least 1 week of culture and even longer during storage. Gene expression was ideally initiated 6 h after immobilization. Although immobilized E. coli cells expressed a highly functional enzyme system after 2 days, they did not metabolize testosterone, probably due to cell permeability problems resulting from immobilization. Therefore, immobilized cell membranes displaying testosterone bioconversion activity, even after long-term storage, will be used in bioreactors with high organic solvent content.     

Strategies for the development of highly selective cytochrome P450 inhibitors: Several CYP targets in current research

Cytochromes P450 (CYPs) play an important role in the metabolism of endogenic and xenobiotic substances, especially drugs. In addition, many CYPs may serve as targets for disease treatment. However, due to the presence of a common heme, the hydrophobicity of the CYP binding cavity, and the high homology within the binding pocket, most CYP inhibitors lack selectivity, which often leads to drug-drug interactions. Therefore, it is meaningful to develop highly selective CYP inhibitors. In this review, we summarize some of the strategies that have been used to develop highly selective CYP inhibitors, such as the weakening of the heme-binding group interaction, reduction of molecular lipophilicity and introduction of small structural changes within compounds.     

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

What is the Functional Role of N-terminal Transmembrane Helices in the Metabolism Mediated by Liver Microsomal Cytochrome P450 and its Reductase?

We sought to clarify on the hitherto unresolved role of N-terminal transmembrane segments (TMS) of cytochrome P450 (CYP) and its’ reductase (CPR) in protein interaction/catalysis. TMS analyses show little evolutionary conservation in CYPs. The conserved CPR’s TMS poses limited scope for predictable/consistent hetero-recognition with the wide bevy of CYPs’ TMS, as evident from preliminary analyses and TMhit server predictions for inter-helical binding. Further, experimentations with four different CPR preparations (preps) and two liver microsomal CYPs (2C9 and 2E1) shows that the hydroxylated product formation rate is not quantitatively correlated to the extent of integrity of the CPR N-terms. Incorporation of cytochrome b ₅ in some reactions afforded similar rates while employing either fully intact or partially intact CPR. A survey of literature shows that liver microsomal CYPs function quite well even without the TMS or with significantly altered TMS. These observations negate the hypothesis that N-term TMS of CPR or CYP is obligatory for CYP–CPR interaction and catalysis. Also, in CYP2E1-mediated hydroxylation of para-nitrophenol, the extent of intactness or truncation did not significantly affect the CPR preps’ catalytic role at very low or high substrate concentrations. To interpret these results, we draw support from recently published research on reduced nicotinamide adenide dinucleotide phosphate oxidase (Takac et al., J Biol Chem, 286:13304–13313, 2011) and from our pertinent earlier works. We infer that CPR’ free TMS segment could alter the diffusible reactive oxygen species’ dynamics in the microenvironment, thereby altering the reaction outcome. Based on the evidence, we conclude that TMS merely facilitates “interaction/catalysis” by anchoring the CYP and CPR in the lipid interface.     

Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism

The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.