Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Immobilization of liver microsomal cytochrome P-450

Rabbit liver microsomal cytochrome P-450 was immobilized by entrapment in calcium alginate gel. Aminopyrine demethylation experiments showed that the immobilized enzyme system is highly active and exhibits an unimpaired functional stability as compared with crude microsomes. The alginate entrapped microsomes were employed in a fixed bed recirculation reactor, where aminopyrine was continuously demethylated. Such model enzyme reactor can be a useful tool for studying extracorporeal drug detoxification or preparative substrate conversion with microsomal enzyme systems.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

Reduction of hexavalent chromium in a reconstituted system of cytochrome P-450 and cytochrome b5

The reduction of hexavalent chromium (Cr(VI] by the monooxygenase components was studied. Both a reconstituted system of cytochrome P-450 (P-450) and cytochrome b5 (b5) with NADPH was capable of reducing Na2CrO4 (30 microM) provided anaerobic atmosphere. The rates were 1.29 nmol Cr.min-1 nmol P-450(-1) and 0.73 nmol Cr.min-1 nmol b5(-1). Using NADH instead of NADPH gave very low reducing activities, confirming the enzymic nature of the P-450 dependent Cr(VI) reductase reaction. Oxygen, 22% (air) and 0.1% gave 89% and 69% inhibition of Cr(VI) reducing activity, respectively. Carbon monoxide (100%) caused an inhibition of about 37% and 44% for P-450 and b5, respectively. Externally added flavin mononucleotide (FMN) (3 microM) or Fe-ADP (10 microM) to the complete system stimulated the enzymatic reaction about 2-fold and 3-fold, respectively.     

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.     

Identification of the membrane anchor of microsomal rat liver cytochrome P-450

Cytochrome P450IIB1 isolated from rat liver microsomes was incorporated into phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (10:5:1 w/w) liposomes. Trypsinolysis of proteoliposomes and sequencing of the membrane-bound domains revealed that only one peptide, comprising amino acid residues 1-21, spans the membrane. Modification of the N-terminal methionine by membrane-impermeable fluorescein isothiocyanate occurred with the protein in solution but not in proteoliposomes. We conclude that in proteoliposomes cytochrome P-450 spans the membrane only with amino acid residues 1-21, the N-terminal methionine facing the lumen.     

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

Quaternary structure of the liver microsomal cytochrome P-450

Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies a position at the vertices of a triangular antiprism with a 32 point group symmetry.     

Active site mechanics of liver microsomal cytochrome P-450

For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (Kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate complex, and the interaction energy (delta Gint) between the substrate-binding and spin-state equilibria. The para-substituents of the toluene substrates were: hydrogen, fluoro, bromo, chloro, iodo, nitro, methyl, cyano, isopropyl, and t-butyl. Linear free energy correlations were sought between the enzymatic constants and several physical constants of the individual substrate molecules. These correlations would be useful both for empirical prediction purposes and for insight into active site chemistry and mechanics. Catalytic rates were correlated by a linear combination of the Hansch pi hydrophobic constant and the Hammett sigma value. A deuterium isotope effect (DV) of 2.6 for d8-toluene compared to d0-toluene confirmed that hydrogen abstraction was partially rate-limiting with this series of substrates. Apparent dissociation constants were predicted by a linear combination of the molar volume and pi, while the spin-state interaction energies were best predicted by a linear combination of the Hansch pi hydrophobic constant and the reciprocal of the dielectric constant.     

A phenobarbital-inducible hepatic mitochondrial cytochrome P-450 immunochemically related to microsomal P-450b

We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria.