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.     

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.     

Lactoferrin-mediated formation of oxygen radicals by NADPH-cytochrome P-450 reductase system.

Superoxide generation in the NADPH oxidase reaction of NADPH-cytochrome P-450 reductase, demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline-1-oxide, increased on the addition of lactoferrin. The NADPH-lactoferrin reductase activity was assessed in terms of NADPH oxidation and oxygen consumption. From Lineweaver-Burk plots, the Km and Vmax for lactoferrin were estimated to be 13 microM and 0.5 S-1, respectively. The liberation of iron from lactoferrin was proven with the use of bathophenanthroline and by the demonstration of bleomycin-dependent DNA degradation; lactoferrin was reduced by the enzyme in the presence of NADPH. During the reaction, the ESR spectrum of the spin trap adduct changed from one characteristic of DMPO-OOH to that of DMPO-OH. The conversion was ascribed to the reaction of hydrogen peroxide with reduced lactoferrin.     

Relationship between state of aggregation and catalytic activity for cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.     

Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2

Carboxyl groups of NADPH-cytochrome P-450 reductase have been modified with the water-soluble carbodiimide EDC. Although there is no significant loss in DCPIP reduction the activity with cytochrome c and cytochrome P-450 LM2 as electron acceptors was inhibited by about 60 and 85%, respectively (1 h incubation time, 20 mM EDC). The inactivation by EDC was nearly completely prevented in the presence of cytochrome P-450 LM2, but not by bovine serum albumin. These results and crosslinking studies suggest that carboxyl groups of NADPH-cytochrome P-450 reductase are involved in charge-pair interactions to cytochrome c and to at least two amino groups of cytochrome P-450 LM2.     

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.     

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

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.     

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.     

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.     

NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies.

NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent.     

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.     

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.     

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.     

EM-field effect upon properties of NADPH-cytochrome P-450 reductase with model substrates.

Artificial substrates, including ferricyanide and dichlorophenol indophenol (IP), are frequently used to model the activity of NADPH-cytochrome P-450 reductase, in the xenobiotic-metabolic pathway catalyzed by the P-450 complex. Here, the two oxidants were compared in a microsomal preparation from chicken liver. Low-energy 9.14 GHz perturbation affected both reactions similarly, though the IP reaction may be more sensitive to extremely low energy levels. The reactions of the two oxidants differed from each other in their response to the prior incubation of the microsomes with carbon monoxide and to the presence of superoxide dismutase. The mechanics of the reduction of ferricyanide and the reduction of IP are not identical and the electron-flow paths may be dissimilar. Microwave effect cannot be attributed a temperature change in the reaction medium; it appears to occur at the level of the electron-flow path across the dual-flavin reductase.     

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.