Study of the structural organization of cytochrome P-450 oligomers using immobilized isoenzyme LM2

Subunit interactions of highly purified hexameric cytochrome P-450 LM 2 has been studied using covalent binding of one of the six protomers to an insoluble matrix. Immobilized cytochrome was catalytically active in monooxygenase reactions and retained the spectral characteristics of cytochrome P-450 LM 2. High ionic strength, large scale pH changes and addition of guanidine chloride were without effect on the aggregation state of the immobilized hemoprotein. However, several detergents induced hexamer dissociation. The crucial role of hydrophobic forces in hexamer subunit interaction was demonstrated. Incubation of the immobilized cytochrome P-450 LM 2 with sonicated liposomes composed of various phospholipids did not result in oligomer dissociation and protein translocation from the matrix to the lipid phase, although the catalytic activity of the immobilized cytochrome significantly increased in the presence of liposomes. The data suggest that cytochrome P-450 LM 2 may be of hexameric structure in the native membranes.     

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.     

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Immobilized cytochrome P-450LM2: Dissociation and reassociation of oligomers

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

ESR-spectroscopy of cytochrome P-450 LM2 in the soluble and membrane-bound state

Influence of microenvironment on the structure of spin-labeled cytochrome P-450 LM2 was studied. The distance between the spin-label which is covalently bound to cysteine-152 and heme ferrum in soluble protein is 17 A and 23 A in the membrane-bound one. The spin-label was exposed in water solution in both cases. It was shown that solubilized cytochrome P-450 LM2 in water solution forms the aggregates consisting of 4-6 monomers.     

Modification of cytochrome P-450 with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.     

The study of the active site of cytochrome P-450 LM2 using the chemical modification of tyrosine residues by tetranitromethane

Selective chemical modification of the hemoprotein by tetranitromethane was used in order to elucidate the functional role of tyrosine residues in the cytochrome P-450 LM2 molecule. It was shown that the degree of cytochrome P-450 LM2 modification can be determined, using the second derivative of the UV absorption spectra. Modification of one tyrosine residue resulted in the inactivation of cytochrome P-450 LM2. Nitration of the cytochrome was accompanied by changes in the spectral properties of the hemoprotein with the formation of spectra typical of hyperporphyrin structures, thus suggesting the involvement of tyrosine residues in the formation of the active center of cytochrome P-450 LM2.     

Inactivation of sodium dithionite reduced cytochromes P-450 of different origins

The inactivation of five dithionite reduced soluble cytochrome P-450 isoforms has been studied. The inactivation of microsomal rabbit liver isoform LM2 and bacterial linalool cytochrome P-450 is followed by its conversion into cytochrome P-420. Microsomal rabbit liver isoform LM4, bacterial camphor and p-cymene cytochromes P-450 were not inactivated under these conditions. The inactivation of linalool cytochrome P-450 and LM2 isoform is a first order reaction; the rate constants for linalool cytochrome P-450 and LM2 are 0.3 and 0.1 min-1, respectively. In the case of linalool cytochrome P-450 its carboxycomplex (Fe2+-CO) is inactivated, while in the case of LM2 the inactivation affects its oxycomplex (Fe2+-O2). The amino acid residues of linalool cytochrome P-450 are probably modified due to a direct electron transfer in its carboxycomplex. The amino acid residues of LM2 isoform are modified, presumably due to oxidation by oxygen active species which are released during the oxycomplex decay.     

cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6

A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.     

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.     

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.     

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.     

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.