Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Inverse relationship between cytochrome P-450 phosphorylation and complexation with cytochrome b5

Cytochrome P-450 LM2 purified from rabbit liver microsomes has been shown to be a substrate for cAMP-dependent protein kinase. Cytochrome b5, in contrast, was a very poor substrate for cAMP-dependent protein kinase, although it stimulated the activity of the kinase toward histone. When purified rabbit cytochrome b5 was mixed with purified LM2, phosphorylation of LM2 by cAMP-dependent protein kinase was inhibited approximately 80-90%. Recently, a functional covalent complex of cytochrome b5 and LM2 was prepared and purified to homogeneity (P.P. Tamburini and J.B. Schenkman (1987) Proc. Natl. Acad. Sci. USA 84, 11-15). When present as a covalent complex with cytochrome b5, the phosphorylation of LM2 in the complex by cAMP-dependent protein kinase was also inhibited about 80-90% relative to an equivalent amount of LM2 alone. On the other hand, when the LM2 was phosphorylated prior to interaction with cytochrome b5, the ability of the latter to perturb the spin equilibrium of LM2 and oxidation of p-nitroanisole by the LM2 was diminished to an extent comparable to the degree of phosphorylation. The results suggest either that the phosphorylation site on LM2 may be within the cytochrome b5 binding site or that phosphorylation and cytochrome b5 cause mutually exclusive conformational changes in LM2. In addition, eight different forms of cytochrome P-450 from the rat (RLM2, RLM3, fRLM4, RLM5, RLM5a, RLM5b, RLM6, and PBRLM5) were examined as potential substrates for cAMP-dependent protein kinase under the same conditions. Maximal phosphorylation of about 20 mol% was obtained with LM2, and about half as much with PBRLM5. The low extent of phosphorylation of LM2 was not due to the prior presence of phosphate on the enzyme since LM2, as isolated, contains less than 0.1 mol phosphate/mol of enzyme. The other forms of cytochrome P-450 tested showed little or no phosphorylation in vitro despite the presence of a cAMP-dependent protein kinase phosphorylation sequence on at least two of them.     

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.     

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450 LM2 reduction. Substrate effects on the rate of reductase-LM2 association

The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

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.     

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.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.     

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].     

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

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.     

Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups

The role of cytochrome b5 heme propionate groups in the functional interactions between cytochromes P-450 RLM5 and b5 has been investigated by comparing the capacity of RLM5 to interact with both native b5 and a b5 derivative in which the native heme was replaced with ferric protoporphyrin IX dimethyl ester (DME-b5). Both forms of b5 interacted with RLM5 causing an increase in the RLM5 spin state from 28 to 68% high-spin RLM5 at saturation, as judged using uv-visible spectrophotometry. However, DME-b5 exhibited a 7-fold weaker affinity for RLM5. The apparent dissociation constant (Kd) for the interaction between RLM5 and b5 was also shown to be a strong function of ionic strength, in a manner consistent with the involvement of electrostatic attraction in complex formation. Reconstitution of b5 into an RLM5-dependent monooxygenase system stimulated the p-nitroanisole demethylase rate about 25-fold and 7-ethoxycoumarin deethylase about 6-fold. DME-b5, however, produced only 30% of the stimulation of RLM5-dependent turnover of p-nitroanisole observed at equivalent concentrations of native b5 without a change in Km. With 7-ethoxycoumarin, turnover was 50% diminished. The diminished capacity of DME-b5 to stimulate RLM5-dependent substrate turnover was shown not to be due to impairment of electron flow between NADPH-cytochrome P-450 reductase and DME-b5, since the Km of reductase for DME-b5 is 2.5-fold lower, and the Vmax is actually increased, but rather to an impairment of some aspect of functional interaction between the DME-b5 and RLM5. The data show that complex formation between cytochrome P-450 and b5 involves electrostatic attraction mediated in part by cytochrome b5 heme propionate groups.     

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.     

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.     

Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes

Water-soluble carbodiimide-catalyzed cross-linking of purified cytochrome P-450 LM2, cytochrome b5, and NADPH-cytochrome P-450 reductase was used to identify stable complexes formed between these proteins. High yields of P-450-b5 and P-450 reductase-b5 dimers, and lower yields of P-450 reductase-LM2 dimers were obtained. Substitution of native b5 and P-450 reductase with fully amidinated derivatives showed that LM2 and b5 were cross-linked exclusively through their respective amino and carboxyl groups. However, there appeared to be two complexation sites on the reductase which cross-link to b5 through amino groups and to LM2 through carboxyl groups respectively. A heterotrimer could not be identified following incubation of all three proteins together with EDC.     

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.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.