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.     

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.     

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.     

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.     

Electrochemical investigations on the oxygen activation by cytochrome P-450.

The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.     

P450 reductase and cytochrome b5 interactions with cytochrome P450: effects on house fly CYP6A1 catalysis

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.     

Cytochrome P450 (CYP) enzymes and the development of CYP biosensors

Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.     

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.     

Ethanol-Induced Oxygen Radical Formation and Lipid Peroxidation in Rat Brain: Effect of Chronic Alcohol Consumption

Abstract: The effect of chronic and in vitro ethanol exposure on brain oxygen radical formation and lipid peroxidation was analyzed. Ethanol induces a dose-dependent increase in lipid peroxidation in brain homogenates. The peroxidative effects of alcohol seem to be related to both cytochrome P450 and the ethanol-inducible form of cytochrome P450 (CYP2E1), because preincubation with metyrapone (an inhibitor of cytochrome P450) or with an antibody against CYP2E1 abolished the ethanol-increased lipid peroxidation. Using the formation of dichlorofluorescein, we also demonstrated that both in vitro and chronic alcohol exposure significantly enhanced the formation of oxygen radical species in synaptosomes. Chronic alcohol treatment also leads to an induction of cytochrome P450 (230%), NADPH cytochrome c reductase (180%), NADPH oxidation (184%), and CYP2E1 in brain microsomes. In addition, this treatment produced a decrease in the GSH/GSSG ratio in brain and significantly enhanced the levels of superoxide dismutase and catalase activities. This mechanism could be involved in the toxic effects of ethanol on brain and membrane alterations occurring after chronic ethanol intake.     

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.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Reductive metabolism of nitroprusside in rat hepatocytes and human erythrocytes.

The metabolism of nitroprusside by hepatocytes or subcellular fractions involves a one-electron reduction of nitroprusside to the corresponding metal-nitroxyl radical. Thiol compounds also reduced nitroprusside to the metal-nitroxyl radical apparently via a thiol adduct. The nitroprusside reduction by microsomes was shown to be due to cytochrome P450 reductase as an antibody to cytochrome P450 reductase inhibits the microsomal reduction of nitroprusside, and the inhibitors of cytochrome P450 such as carbon monoxide or metyrapone had no effect. The reduction of nitroprusside by mitochondria in the presence of NADH or NADPH also produced the metal-nitroxyl radical. In hepatocytes, both mitochondria and the cytochrome P450 reductase are involved in the reduction of nitroprusside. The reductive metabolism of nitroprusside was found to produce toxic by-products, namely, free cyanide anion and hydrogen peroxide. We have also detected thiyl radicals formed in the thiol compound reduction of NP. We propose that cyanide and hydrogen peroxide are important toxic species formed in the metabolism of nitroprusside. The rate of reductive metabolism of nitroprusside by rat hepatocytes was much higher than with human erythrocytes. Therefore the major site of nitroprusside metabolism in vivo may be liver and not blood as originally proposed.     

Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.     

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.     

An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

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   

Cytochrome P450 3A4-mediated oxidative conversion of a cyano to an amide group in the metabolism of pinacidil

The conversion of nitriles to amides is generally considered to be a hydrolytic process that does not involve redox chemistry. We demonstrate here that cytochrome P450 (CYP) is responsible for the conversion of the cyano group of pinacidil to the corresponding amide. The reaction in human liver microsomes was NADPH-dependent and was nearly completely inhibited by an anti-CYP3A4 antibody. Incubations of pinacidil with recombinant CYP enzymes confirm that CYP3A4 is the principal catalyst of this reaction. The kinetics of pinacidil amide formation by CYP3A4 yielded an apparent K(m) of 452 +/- 33 microM and k(cat) of 0.108 min(-1) (k(cat)/K(m) = 0.238 mM(-1).min(-1)). Incubation of pinacidil with CYP3A4 in the presence of (18)O(2) or H(2)(18)O showed that the amide carbonyl oxygen derived exclusively from molecular oxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubations were carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explained by a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe(3+)-O-O(-)) on the carbon atom of the -C triple bond N triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed by cleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe(3+)-O-O(-) intermediate to a -C=N pi-bond in a P450 system resembles the analogous reaction catalyzed by the nitric oxide synthases.     

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.     

TNF-alpha -induced endothelial cell adhesion molecule expression is cytochrome P-450 monooxygenase dependent

It is strongly suspected thatcytokine-induced gene expression in inflammation is oxidant mediated;however, the intracellular sources of signaling oxidants remaincontroversial. In inflammatory bowel disease (IBD) proinflammatorycytokines, such as TNF-, trigger gene expression of endothelialadhesion molecules including mucosal addressin cell adhesion molecule-1(MAdCAM-1). MAdCAM-1 plays an essential role in gut inflammation bygoverning the infiltration of leukocytes into the intestine. Severalgroups suggest that endothelial-derived reduced NADP (NADPH) oxidaseproduces signaling oxidants that control the expression of adhesionmolecules (E-selectin, ICAM-1, VCAM-1). In addition to NADPH oxidase,cytochrome P-450 (CYP450) monooxygenases have also beenshown to trigger cytokine responses. We found that in high endothelialvenular cells (SVEC4-10), multiple inhibitors of CYP450 monooxygenases(SKF-525a, ketoconazole, troleandomycin, itraconazole) attenuatedTNF- induction of MAdCAM-1, whereas NADPH oxidase inhibition (PR-39)did not. Conversely, E-selectin, ICAM-1, and VCAM-1 inductionrequires both NADPH oxidase and CYP450-derived oxidants. We show herethat MAdCAM-1 induction may depend exclusively on CYP450-derivedoxidants, suggesting that CYP450 blockers might represent a possiblenovel therapeutic treatment for human IBD.