Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane of phosphatidylcholine vesicles.

Cytochrome P-450 and NADPH-cytochrome P-450 REDUctase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphoaditylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80--90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the sysTEM IN A SIMILAR WAY TO THE FAST-PHASE REDUCTION OF CYTOCHROME P-450, though the latter is not the rate-limiting step of the overall reaction.     

NADPH-cytochrome P-450 reductase of yeast microsomes.

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b₅ of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b₅ that observed in the yeast microsomes.     

Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.     

Possible association of NADPH-cytochrome P-450 reductase and cytochrome P-450 in reconstituted phospholipid vesicles

A fluorescent probe, N-(1-anilinonaphth-4-yl)-maleimide (ANM), was specifically labeled to SH group(s) in the hydrophilic moiety of NADPH-cytochrome P-450 reductase at a ratio of 1 +/- 0.1 ANM/mol of protein. The ANM-labeled reductase and P-450 were reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles in which all of the enzymes were functionally active. The reconstitution of the mixed-function oxidase system was found to be strongly dependent on both the lipid to protein molar ratio and phospholipid composition. The interactions of ANM-labeled reductase with P-450 in proteoliposomes were investigated by perturbation of the fluorescence of ANM. Upon incorporation of P-450 into the phospholipids vesicles (ANM-reductase/P-450/lipids identical to 1:1.4:800), a significant decrease of total fluorescence intensity and slight increase of emission anisotropy of ANM were observed. In the average fluorescence lifetime of ANM bound with reductase, an appreciable change was shown between the absence and presence of P-450 in the vesicles. These data provide clear evidence that significant molecular interactions occur between the two proteins in a membranous reconstituted system.     

Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase. Modification of carboxyl groups on the reductase with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, or taurine, respectively. The apparent Km for cytochrome P-450c or cytochrome P-450b was increased 1.3- to 5.2-fold in a reconstituted monooxygenase assay with 7-ethoxycoumarin or benzphetamine as substrate. There were varied effects on the Vmax. There was no significant change in the conformation of the reductase upon chemical modification with either acetic anhydride or EDC. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37°C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Rotation and interactions of genetically expressed cytochrome P-450IA1 and NADPH-cytochrome P-450 reductase in yeast microsomes

Rat liver cytochrome P-450IA1 and/or yeast NADPH-cytochrome P-450 reductase was expressed genetically in yeast microsomes. The ratio of P-450IA1 to the reductase was about 17:1 and 1:2 without and with coexpression of the reductase, respectively. Rotational diffusion of P-450IA1 was examined by observing the flash-induced absorption anisotropy, r(t), of the heme.CO complex. In only P-450IA1-expressed microsomes, 28% of P-450IA1 was rotating with a rotational relaxation time (phi) of about 1200 microseconds. The mobile population was increased to 43% by the presence of the coexpressed reductase, while phi was not changed significantly. Increased concentration of KCl from 0 to 1000 mM caused considerable mobilization of P-450IA1. The results demonstrate a proper incorporation of P-450IA1 molecules into yeast microsomal membranes. The significant mobilization of P-450IA1 by the presence of reductase suggests a possible transient association of P-450IA1 with the reductase.     

Artifacts in isoelectric focusing of the microsomal enzymes cytochrome P-450 and NADPH-cytochrome P-450 reductase.

Highly-purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase (NADPH-ferricytochrome oxidoreductase, EC 1.6.2.4) preparations gave rise to a large number of bands under a variety of isoelectric focusing conditions, as observed after staining for either zymogen or protein. The binding patterns were not independent of sample concentration and position of application, and eluted bands did not refocus as expected. The artifactual heterogeneity is attributed to strong protein-protein interactions and perhaps to complexation of proteins with carrier ampholytes. These findings suggest caution in using isoelectric focusing to resolve mixtures of membrane proteins.     

Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase

A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface.     

Purification and properties of human placental NADPH-cytochrome P-450 reductase

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes.

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Differences in the spectral interactions between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 enzymes

The interaction between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 isozymes was investigated using UV-visible spectrophotometry. In the absence of substrate the interactions between the reductase and RLM3, RLM5, and RLM5a were tight, exhibiting sub-micromolar dissociation constants and resulted in type I spectra of varying magnitude from which the following increases in the proportion of high spin hemoprotein were calculated; RLM3 (7%), RLM5 (36%), RLM5a (6%), LM2 (29%), RLM2 (0%). Preincubation of LM2 with its type I substrate benzphetamine increased the affinity of the cytochrome for the reductase. Using initial estimates of the P-450 spin states in the absence of reductase in conjunction with the spectral binding data and equations relating these parameters to the microequilibria for the association of reductase with high or low spin P-450, RLM3, RLM5, RLM5a and LM2 were shown to bind significantly more tightly to high spin P-450. The relevance of this data to the understanding of spin state influence on P-450 reduction is discussed.     

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.     

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome $\text{c}$ as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min.     

Kinetic properties of guinea pig liver microsomal NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.     

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Comparative study of the reaction kinetics of cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase and dithionite

The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes.