Benzpyrene hydroxylase activity in hepatic microsomal and solubilized systems containing rabbit or rat cytochrome P-448 or P-450

Treatment of adult, male rabbits and rats with 3-methylcholanthrene results in the formation of hepatic microsomal cytochrome P-448. In the rat, this occurs coincidently with an increase in hepatic microsomal benzpyrene hydroxylase activity. In the rabbit, benzpyrene hydroxylase activity is decreased following treatment with 3-methylcholanthrene. Benzpyrene hydroxylase activity in solubilized, reconstituted mixed-function oxidase systems containing rat cytochrome P-448 is about seven times higher than in systems containing rabbit cytochrome P-448. Evidence obtained by spectral analysis suggests that rabbit P-448 is combined with a type I compound. Residual ¹⁴C-3-methylcholanthrene does not appear to be responsible for the differences observed between rat and rabbit cytochrome P-448.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAS by 3-methylcholanthrene

Liver poly(A⁺)-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.     

Correlation between cytochrome P-448 content and benzopyrene hydroxylase activity during the induction of microsomal monooxygenases using methylcholanthrene-type xenobiotics

Using antibodies against electrophoretically homogeneous cytochrome P-448 from rat liver microsomes induced by 3-methylcholanthrene, the changes in the immunologic identity and contents by cytochrome P-448 induced by 3-methylcholanthrene, 3.4-benzpyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were studied. No cytochrome P-448 was detected in the liver microsomes of control or phenobarbital-induced rats. This form of the cytochrome makes up to about 35% of the total content of the CO-binding hemoprotein during TCDD induction and up to 90% during 3-methylcholanthrene and 3,4-benzpyrene induction. On the other hand, 3-methylcholanthrene, 3,4-benzpyrene and TCDD significantly and equally activates the cytochrome P-448-dependent benzpyrene hydroxylase, since the antibodies against cytochrome P-448 inhibit benzpyrene metabolism in the microsomes by 85-90%. The possible reasons for the TCDD-induced increase in the catalytic activity of cytochrome P-448 as compared to the immunologically identical cytochrome P-448 induced by 3-methylcholanthrene and 3,4-benzpyrene, are discussed.     

Separation of two forms of cytochrome P-450 with aryl hydrocarbon hydroxylase activity from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene

Two forms of cytochrome P-450, designated P-448a and P-448b, were purified from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene. Both the cytochromes had absorption maxima at 448 nm in the carbon monoxide-reduced difference spectra. They exhibited comparable catalytic activities with benzo(a)pyrene, 7-ethoxycoumarin, and 7-ethoxyresorufin, when reconstituted with hepatic NADPH-cytochrome c reductase and phosphatidylserine. P-448a was apparently homogeneous on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its monomeric molecular weight was estimated to be 58,000. The oxidized form had absorption maxima at 416, 512 and 571 nm, indicative of the low spin state. Thus P-448a appeared to be similar to one form of P-450, which was induced in rabbit liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). On the other hand, SDS-PAGE of P-448b gave a single major protein band with a monomeric molecular weight of 55,500, indicating that P-448b can be distinguished from P-448a.     

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.     

Specificity of antisera directed against rat liver cytochrome P-448

A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b₅, NADH-cytochrome b₅ reductase, NADPH-cytochrome c reductase or epoxide hydrase.     

Studies on the substrate-binding sites of liver microsomal cytochrome P-448.

The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated with phenobarbital or 3-methylcholanthrene, and with purified forms of the cytochromes isolated from rabbit liver. The two forms of the cytochrome have different substrate specificities; cytochrome P-450 has one type 1 substrate-binding site that can accommodate a large variety of substrates, but in contrast cytochrome P-448 may possess two type 1 substrate-binding sites, one of which is different to that of cytochrome P-450 in that it shows a specificity for substrates such as safrole and 9-hydroxy-ellipticine. These findings explain why the two forms of the cytochrome have different substrate specificities and play contrasting roles in the activation and deactivation of xenobiotics.     

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.     

Induction of cytochrome P-448 isozyme(s) in primary cultured rat hepatocytes by drugs which induce different isozymes in vivo

Effect of 2-methoxy-4-aminoazobenzene (2-MeO-AAB) and 3-methylcholanthrene (MC) on the induction of microsomal cytochrome P-448 isozymes in primary cultured rat hepatocytes was examined by means of immunochemical methods such as protein A-enzyme-linked immonosorbent assay and immuno-blots using anti-rat cytochrome P-448 monoclonal antibodies and by means of bacterial mutation tests. Although 2-MeO-AAB selectively induced cytochrome P-448H and MC induced both cytochrome P-448H and a low spin form of cytochrome P-448 (P-448L) in the liver of rats, addition of these chemicals to primary cultured rat hepatocytes resulted in selective induction of cytochrome P-448L, as determined by the immunological methods. This was substantiated by the bacterial mutation test using Salmonella typhimurium TA 98 bacteria and two aromatic amine substrates with different specificities to the cytochrome P-448 isozymes. These results suggest that the responses of rat hepatocytes to cytochrome P-450 inducers are different in in vivo and in vitro.     

Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.     

Organ selective induction of cytochrome P-448 isozymes in the rat by 2-methoxy-4-aminoazobenzene and 3-methylcholanthrene

Male Sprague Dawley rats were injected intraperitoneally with 2-methoxy-4-amino-azobenzene (2-MeO-AAB) or 3-methylcholanthrene (MC), and then the expression of microsomal cytochrome P-450 isozymes in liver and extrahepatic tissues was investigated by means of immunological methods and a bacterial mutation test. The results of protein A-enzyme-linked immunosorbent assaying and immunoblotting using anti-rat cytochrome P-448 monoclonal antibodies showed that MC induced at least two microsomal cytochrome P-448 isozymes, a high spin form (cytochrome P-448H) and a low spin form (cytochrome P-448L), in liver, but that it induced only cytochrome P-448L in extrahepatic tissues such as lung, kidney, small intestine, and colon. The results also indicated that, in contrast to MC, 2-MeO-AAB selectively induced microsomal cytochrome P-448H in liver but did not induce any cytochrome P-448 isozymes in extrahepatic tissues. The activities of 9,000 X g supernatants from the individual organs, as to the mutagenic conversion of 3 aromatic amines (3-amino-1-methyl-5H-pyrido(4,3-b)indole, 2-amino-6-methyldipyrido(1,2-a: 3',2'-d)-imidazole and 3-methoxy-4-aminoazobenzene), toward Salmonella typhimurium TA 98 bacteria were dependent upon the quantity and/or quality of the microsomal cytochrome P-448 isozymes in the organs.     

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.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Monoclonal antibodies against a high spin form of rat cytochrome P-448

Ten monoclonal antibodies reactive with a high spin form of rat cytochrome P-448 (P-448-H) were obtained from hybridoma clones established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse hyperimmunized with the cytochrome. One monoclonal antibody recognized an epitope characteristic for P-448-H. Five monoclonal antibodies were cross-reactive with a low spin form of rat cytochrome P-448, but not with cytochrome P-450. Reactivity of these monoclonal antibodies with microsomes of rats pretreated with drug metabolizing inducers and Western blots of the microsomal cytochrome P-450 components are also demonstrated.     

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (lambda max: 428 nm (ox.), 447 nm (red.), Ks: 10 microM and 5 muM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (lambda max: 428 nm (ox.), 445 nm (red.), Ks: 8 microM). 9-OHE has been shown to inhibit the benzo[a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.     

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

A low-temperature EPR study of partially purified, soluble ferric cytochromes P-450 and P-448 from rat liver microsomes

Low-temperature EPR examination of rat liver microsomes from control, phenobarbital-treated, and methylcholanthrene-treated animals showed the presence of both high- and low-spin ferric cytochromes P-450 and P-448. Partially purified cytochromes P-450 (from control and phenobarbital-treated rats) and P-448 (from methylcholanthrene-treated rats) were also examined with EPR. In all cases, both high- and low-spin ferric forms of cytochromes P-450 and P-448 could be observed and were found to be essentially identical compared to the microsomal preparations. However, the level of high-spin species in the soluble P-448 preparation from methylcholanthrene-treated animals was less than could be observed in the liver microsomes from the same animals. The addition of substrates increased the concentration of the high-spin form in the soluble preparations obtained from drug-treated animals. Thus, cytochromes P-450 and P-448 exist as mixtures of high- and low-spin forms. It is concluded that the substrate specificity of these cytochromes is not predetermined by the spin state of the hemoprotein. In all liver microsomal and soluble preparations, the low-spin ferric form of the hemoprotein consisted of more than a single species as determined from the EPR examinations. Each of these species upon reduction and the addition of CO yielded an identical optical spectrum. In all cases, for the ferric protein, a mercaptide sulfur is believed to be a heme ligand while the other heme ligand is variable.