Amino acid sequence of COOH-terminal 20K Da fragment from pig liver microsomal NADPH-cytochrome P-450 reductase

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.     

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.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..     

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.     

Photochemically induced dynamic nuclear polarization study on microsomal NADPH-cytochrome P-450 reductase

Sedimentation equilibrium experiments with NADPH-cytochrome P-450 reductase showed that increasing 1-O-n-octyl-beta-D-glucopyranoside levels promoted disaggregation of the flavoprotein. The reductase was monomeric at a molar ratio of detergent to protein above 10(3). Addition of N3-carboxymethyllumiflavin to the flavoprotein in the presence of 1-O-n-octyl-beta-D-glucopyranoside results in photochemically induced dynamic nuclear polarization (CIDNP) signals in the aromatic region. The CIDNP spectrum of the holoprotein shows sharp resonances due to histidine residues. On removal of FMN from the protein, CIDNP signals originating from a tyrosine residue appeared, suggesting that the tyrosine residue is exposed to solvent after the depletion of FMN. However, this tyrosine residue appears to become inaccessible to the external dye after full incubation of FMN-depleted reductase with FMN. This suggests that the tyrosine residue could be located in the vicinity of the FMN-binding domain which constitutes the active center of the reductase.     

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.     

Fluorescence probing of the function-specific cysteines of rat microsomal NADPH-cytochrome P-450 reductase

Titration of NADPH-cytochrome P-450 reductase with a fluorigenic maleimide suggests that approximately four cysteines are initially accessible and in close proximity to four tryptophans. Perturbation of the cysteines and/or tryptophans results in concomitant decreases in enzymic activity. These cysteines were correlated with functional components by binding studies and subsequent tryptic peptide mapping on the acid mobile phase-reverse phase HPLC. Adenine nucleotides and cytochrome c block labelling of the more hydrophilic peptides, while detergents facilitate labelling of the more hydrophobic peptides. The more hydrophobic peptides contain the microsomal binding site of cytochrome P-450. Removal of the prosthetic flavins exposes more cysteines in the more hydrophilic and hydrophobic regions of the peptide map, associating the former with FAD and the latter with FMN binding sites.     

Purification and some properties of NADPH-cytochrome P-450 reductase from crab-eating monkey liver microsomes

NADPH-cytochrome P-450 reductase was purified to 30.8 units/mg from monkey liver microsomes. The purified reductase showed one major protein band (78,000) and two minor ones (58,000 and 20,000) on analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monkey, rat, and guinea pig reductases were not immunochemically identical to each other judged from Ouchterlony double diffusion analysis and immunotitration with regard to NADPH-cytochrome c reductase activity.     

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.     

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.     

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes.

NADPH-cytochrome P-450 reductase has been purified to electrophoretic homogeneity from rabbit liver microsomes by a procedure that may be used in conjunction with the isolation of the major forms of cytochrome P-450. The purified reductase is active in a reconstituted hydroxylation system containing P-450LM₂ or P-450LM₄. The enzyme contains one molecule each of FMN and FAD per polypeptide chain having an apparent minimal molecular weight of 74,000. Immunological techniques provided evidence for only a single form of the reductase; lower molecular weight forms occasionally seen are believed to be due to degradation by contaminating microsomal or bacterial proteases. Upon anaerobic photochemical reduction, the rabbit liver reductase undergoes spectral changes highly similar to those previously described by Vermilion and Coon for the rat liver enzyme; the fully reduced rabbit liver enzyme is converted to the three-electron-reduced form by the addition of NADP and then to the stable one-electron-reduced form by exposure to oxygen. The CD spectra of the fully oxidized enzyme, one-electron-reduced form (air-stable semiquinone), three-electron-reduced form, and fully reduced form are presented. The results obtained provide evidence that the FMN and FAD are in highly different environments in the enzyme, as also indicated by the different redox potentials and oxygen reactivities of the flavins.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome $\text{c}$ per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.     

Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin

The basis for our previous observations [Kaminsky, L.S., Guengerich, F.P., Dannan, G.A. & Aust, S.D. (1983) Arch. Biochem. Biophys. 225, 398-404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F greater than P-450PB-D greater than or equal to P-450UT-A greater than or equal to P-450BNF/ISF-G greater than P-450PB/PCN-E greater than P-450PB-B greater than or equal to P-450PB-C greater than or equal to P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations.     

Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems

A series of equilibrium and kinetic measurements involving the oxidation-reduction properties of purified rat liver NADPH-cytochrome P-450 reductase and eight different purified rat liver cytochromes P-450 (P-450s) were carried out. Apparent spin states of P-450 iron were determined in the absence and presence of a number of known substrates by using second-derivative and conventional near-UV absorbance spectroscopy. Many of the substrates examined did not produce significant changes in the apparent iron spin state, even when binding could be demonstrated with equilibrium dialysis. Further, the spin state was not correlated to catalytic activity of the P-450s in reconstituted enzyme systems. The oxidation-reduction potentials were determined for the ferric/ferrous couples of each of the eight P-450s in the presence and absence of known substrates, as well as other proteins suspected of altering the potentials. The midpoint potential (Em,7) ranged from -350 to -289 mV for the P-450s under these conditions. In some cases Em,7 was raised with the addition of substrates, but the extent of the increase was no greater than +33 mV. The Em,7 of one P-450 (P-450 beta NF/ISF-G) was not changed significantly when the fraction of high-spin iron varied between 11 and 67%. Steady-state spectral studies provided evidence for the accumulation of an oxygenated ferrous intermediate (or a derived product) of one P-450 (P-450PB-B) in the presence of a substrate, cyclohexane. Studies on the donation of electrons from cytochrome b5 and a series of dyes to this complex suggest that it has an effective Em,7 (for reduction) of approximately +50 mV. In studies with one of the P-450s, steady-state spectral studies indicated that the three-electron-reduced form of NADPH-P-450 reductase accumulates, consistent with the view that this form of the reductase is involved in the reduction of P-450 from the ferric to the ferrous state.     

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)