Evidence for lipid involvement in the high-affinity interaction between cimetidine and rat liver cytochrome P-450

The spectral interaction between cimetidine and a cytochrome P-450 fraction isolated from liver of untreated rats has been shown to be markedly affected by dilauroylphosphatidylcholine. In the absence of the lipid the pigment preparation yielded a binding curve characteristic of a single isoenzyme species with low affinity for the drug, whereas its inclusion led to the observation of a much stronger interaction with a dissociation constant close to that obtained for the high-affinity component(s) of the parent microsomes; material with lower affinity was also observed. Gel chromatography and partition equilibrium studies yielded results which precluded interpretation of this finding either in terms of incomplete incorporation of the pigment into the phospholipid or of disproportionate solvation of cimetidine into the lipid phase of the reconstituted phospholipid complex. In contrast, phospholipid caused only a minor change in the strength of cimetidine binding by the predominant liver cytochrome P-450 from phenobarbitone-pretreated rats. Pronounced lipid sensitivity of cimetidine-binding affinity is thus not a general feature of the microsomal cytochrome P-450 system but rather a specific characteristic of individual isoenzyme species.     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces changes in the heme spin state of microsomal cytochrome P-450

In vitro studies on the nature of interaction of the neurotoxin MPTP with hepatic microsomal cytochrome P-450 were carried out. Spectral perturbation studies showed nitrogenous ligand type binding between MPTP and cytochrome P-450 with a peak at 423 nm and a broad trough at 400 nm. Scatchard analysis of MPTP-cytochrome P-450 binding suggested that MPTP binds to at least 2 species of cytochrome P-450--a high affinity binding species with an apparent spectral dissociation constant (Ks) of 372 microM and a low affinity species with Ks of 37.6 mM. EPR studies confirmed that MPTP is a type II substrate for the forms of cytochrome P-450 with which it interacts and causes a shift from the high spin state of cytochrome P-450 to the low spin state. MPTP is, thus, likely to be an effective inhibitor of cytochrome P-450.     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

Interaction of cytochrome P-450 with hydrocarbons

Hydrocarbons of different structures interact with microsomal and solubilized cytochrome P-450 from liver of phenobarbital-pretreated rats forming a high spin enzyme-substrate type complex. The affinity of cytochrome P-450 for hydrocarbons increases with increasing lipophilicity independently of the chemical structure. The binding capacity of microsomal P-450 for aliphatic hydrocarbons is generally higher than for aromates. Mutual influence in binding of two different hydrocarbons by microsomal P-450 is stronger among aromatic than among aliphatic hydrocarbons; in both cases it appears to be effected rather by specific interaction of both substances with the binding site than by a nonspecific influence on the microsomal membrane. Only one fraction of low spin form of solubilized cytochrome P-450 from rat liver interacts with hydrocarbons. The binding capacity for aromatic and aliphatic substances corresponds to that found in microsomes. The affinity for the most lipiphilic substrate, perhydrophenanthrene, is equal in microsomal and solubilized preparation; with decreasing lipophilicity the affinity of solubilized P-450 decreases faster than in microsomes. The LM2 fraction of cytochrome P-450 from phenobarbital-pretreated rabbits interacts only with aliphatic hydrocarbons with wide variation of the binding capacity. The affinity is generally one order of magnitude lower than in microsomes. Active fractions of solubilized P-450 from both species are rapidly converted to P-420 by dithionite. The extent of this conversion is strongly reduced by saturation with substrate.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae. Substrate binding, spectral and kinetic data

Saccharomyces cerevisiae, brewer's yeast, produces a microsomal benzo(a)pyrene hydroxylase when grown at high glucose concentrations of which the haemoprotein, cytochrome P-450 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating) EC 1.14.14.1) is a component. We report here kinetic data derived from Lineweaver-Burk plots of benzo(a)pyrene hydroxylation. The Michaelis constant was decreased by growth of the yeast in the presence of benzo(a)pyrene showing the induction of a form of the enzyme more specific for this compound. NADPH or cumene hydroperoxide could be used as cofactors by this enzyme, although with different Km and V values for benzo(a)pyrene. A solubilised and a solubilised, immobilised enzyme preparation were capable of benzo(a)pyrene hydroxylation, using cumene hydroperoxide but not NADPH as the cofactor. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. The interaction of benzo(a)pyrene with cytochrome P-450 was investigated further by means of an equilibrium gel filtration technique. There appeared to be 20 binding sites per mol ofcytochrome P-450 for benz(a)pyrene, in both yeast and rat liver microsomes.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

The metabolism in vitro and hepatic microsomal interactions of some enantiomeric drug substrates

1. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (-)-amphetamine, (+)-benzphetamine and (-)-benzphetamine were studied with hepatic microsomes from phenobarbitone-pretreated male rabbits. 2. (+)-Benzphetamine was N-demethylated 30-35% faster than (-)-benzphetamine, but the apparent Michaelis constants for the two enantiomers were similar. 3. (-)-Amphetamine was deaminated about 200% faster than (+)-amphetamine. 4. The benzphetamine enantiomers gave qualitatively and quantitatively identical type I microsomal difference spectra (peak, 390nm; trough, 425nm) indicating identical apparent binding affinities for microsomes and identical spectral changes at maxima (DeltaE(max.) values). 5. The amphetamine enantiomers gave qualitatively identical type II microsomal difference spectra (peak, 433nm; trough, 395nm). However, the type II spectral data indicated that (+)-amphetamine had a markedly higher apparent binding affinity than (-)-amphetamine for microsomes. The amphetamine enantiomers gave identical DeltaE(max.) values. 6. The benzphetamine enantiomers (0.5mm) enhanced the rate of microsomal cytochrome P-450 reduction by NADPH by 400-500%, (+)-benzphetamine enhancing the rate 20-25% more than (-)-benzphetamine. 7. The amphetamine enantiomers decreased the rate of microsomal cytochrome P-450 reduction by NADPH. At a concentration of 2mm, (+)-amphetamine decreased the rate more than (-)-amphetamine. 7. All four enantiomers enhanced microsomal NADPH oxidation.     

Retinoic acid metabolism by a system reconstituted with cytochrome P-450

Feeding rats with a diet containing a hundred times the normal amount of vitamin A resulted, within 2 to 3 weeks, in an increase in total hepatic microsomal cytochrome P-450 content. This was associated, in isolated microsomes, with an enhanced conversion of all-trans-retinoic acid to polar metabolites, including a two- to threefold increased production of 4-hydroxy- and 4-oxo-retinoic acid, whether expressed per microsomal protein or per cytochrome P-450. Unlike effects of other inducers (e.g., phenobarbital or methylcholanthrene), activities of benzphetamine, aminopyrine, and ethylmorphine demethylases or benzopyrene hydroxylase were not increased. Furthermore, the CO-reduced difference spectral peak was shifted towards 449 nm. On sodium dodecyl sulfate-gel electrophoresis, one band was increased with electrophoretic mobility identical to that of cytochrome P-450f, a recently isolated new form which has a CO-reduced difference spectral peak at 448 nm. In a system reconstituted with NADPH-cytochrome P-450 reductase, NADPH, and phospholipid, purified cytochromes P-450f and b were discovered to promote conversion of retinoic acid to polar metabolites, including 4-hydroxy-retinoic acid.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450LM.

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to catalyze electron transfer to cytochrome P-450, whereas the trypsin-solubilized reductase does not. In the present study, treatment of the highly purified detergent-solubilized rabbit liver enzyme (m.w. 77,700) with trypsin was shown to yield a small peptide (m.w. 6,100) as well as the large peptide (m.w. 71,200) which retains the flavin prosthetic groups. The small peptide, which is hydrophobic in nature as shown by its amino acid composition and solubility properties, is apparently the moiety in the native reductase involved in binding to cytochrome P-450 and to the microsomal membrane. The C-terminal amino acid sequences of the native reductase and large fragment are identical [-Trp-(Leu, Val)-Asp-Ser-COOH], thereby indicating that the hydrophobic peptide is located in the N-terminal region of the enzyme.     

Specificity of steroid binding to testicular microsomal cytochrome P-450. Relation of steroid structure to type-I spectral responses after correction for hydrophobic association with the membrane

On the basis of the concept that steroids accumulate in the lipid phase of endoplasmic reticulum membranes and approach the active sites of steroidogenic cytochromes P-450 from a hydrophobic environment, we describe a procedure that allows calculation of spectral dissociation constants Ks for steroid interaction with testicular microsomal cytochrome P-450 after correction for hydrophobic association of ligand with the membrane. Maximal type-I spectral responses, apparent Ks, and partition into microsomal lipids were determined for 36 steroids, and corrected Ks values were derived from these primary data. Partition coefficients range from 60 to 62,000, and corrected Ks range from 60 microM to 25 mM steroid concentration in the lipid phase. Full spectral properties depend on a side-chain (1-3 carbon atoms) at the C17-position which may be hydrophobic or may bear a 20-oxo or 20 beta-hydroxy, but not a 20 alpha-hydroxy group. Binding constants are especially sensitive towards modifications of ring A structure (aromatization or 5 beta-, but not 5 alpha-reduction) and of the side-chain length. Androgens, with the exception of those bearing a 17 beta-acetoxy or 17 beta-propionyloxy group, are poorly accommodated by this cytochrome P-450.     

Studies on the decrease of liver cytochrome P-450 content by triiodothyronine

In the rat liver, the microsomal content of cytochrome P-450 decreased by 50% after triiodothyronine (T3) administration. The molecular basis for the decreased cytochrome P-450 levels was investigated. The activities of the enzymes involved in heme synthesis or degradation were not altered by thyroid hormone administration. The incorporation of 3H-delta-aminolaevulinate into the liver microsomal heme was markedly reduced in T3-treated rats. The latter appeared not to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation showed a decrease in apocytochrome P-450. It is suggested that the amount of the apocytochrome may be the primary event affected in the formation of cytochrome P-450, by triiodothyronine treatment of thyroidectomized rats.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450.

A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Interaction of the complex copper compound Cu-2 with liver monooxygenases

It has been shown that the antitumour drug Cu-2 (copper complex compound) inhibited the activity of liver monooxygenases in male CBA mice. The in vivo experiments have revealed a considerably increased duration of sleep in mice treated with hexenal after the administration of different Cu-2 doses. In vitro, after the incubation of intact mouse liver microsomal fractions with different concentrations of Cu-2 the level of cytochrome Y-450 was decreased and a non-active form of hemoprotein--cytochrome P-420--appeared. At the same time, after the incubation of Cu-2 with liver microsomal fractions stabilized by 20% glycerol type I spectral changes (Ks 330 microM) were registered. This shows the possible metabolism of Cu-2 by cytochrome P-450. The role of the revealed interaction of Cu-2 with liver microsomes is being discussed for the chemotherapy of cancer.     

Mechanistic studies with purified components of the liver microsomal hydroxylation system: spectral intermediates in reaction of cytochrome P-450 with peroxy compounds.

Recent investigations in this laboratory on the mechanism of action of liver microsomal cytochrome P-450 (P-450 LM) and its interaction with other components of the hydroxylation system are presented. Two electrophoretically homogeneous forms of the cytochrome, phenobarbital-inducible P-450 LM2 and 5,6-benzoflavone-inducible P-450 LM4, so designated according to their relative electrophoretic mobilities, were used in these studies. Phosphatidylcholine is required in the reconstituted enzyme system for rapid electron transfer from NADPH to P-450 LM, catalyzed by NADPH-cytochrome P-450 reductase, as well as for maximal hydroxylation activity with either molecular oxygen or a peroxy compound serving as oxygen donor to the substrate. The phospholipid facilitates the binding of both substrate and reductase to P-450 LM and apparently causes a structural change in the cytochrome as shown by an increase in alpha-helical content, determined by circular dichroic spectrometry. P-450LM3 and LM4 are one-electron acceptors under anaerobic conditions, in accord with previous potentiometric titrations and product yield data, but in disagreement with previous titrations with reducing agents. The cause for the discrepancy between the present and earlier results is not yet fully understood. Stopped flow spectrophotometry was employed to detect intermediates in the reaction of peroxy compounds with P-450LM2. With m-chloroperbenzoic acid the intermediate formed has absorption maxima at 375, 425, and 540 nm in the absolute spectrum and at 370, 436, and 540 nm in the difference spectrum (intermediate minus oxidized form). A study of the magnitude of the spectral change at various peracid concentrations indicated that with this oxidant the reaction shows a dependence resembling a binding curve. These and other experiments with various oxidants, including cumente hydroperoxide, suggest a reversible two-step mechanism according to the reaction: P-450 LM + oxidant equilibrium C equilibrium D, where C may be an enzyme-oxidant complex and D is a spectral intermediate of unknown structure. A scheme is proposed for the mechanism of action of P-450 LM based on these and earlier studies, including evidence from deuterium isotope experiments for the formation of a substrate carbon radical prior to oxygen transfer.     

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.