Destruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidation

Vinyl fluoride, vinyl bromide, fluroxene (2,2,2-trifluoroethyl vinyl ether), and acetylene alkylate the prosthetic heme group of cytochrome P-450 enzymes which catalyze their metabolism. The alkylated heme moiety has been identified in all four cases, after carboxyl group methylation and demetalation, as the dimethyl easier of N-(2-oxoethyl)protoporphyrin IX. The dimethyl acetal derivative of the aldehyde group in this structure is also isolated. The formation of the same prosthetic heme adduct with the four substrates requires introduction of an oxygen at the trifluoroethoxy or halide-substituted terminus of the pi bond and reaction of the unsubstituted terminus with a heme nitrogen atom. This reaction orientation is consistent with a radical intermediate, possibly formed by way of an initial pi-bond radical cation, but is difficult to reconcile with a cationic intermediate. The occurrence of a radical intermediate in the oxidation of olefins by cytochrome P-450 is thus suggested.     

Studies on the interaction of furan with hepatic cytochrome P-450

In vitro incubation of rat liver micro-somes with [¹⁴C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was observed with microsomes from phenobarbital-treated rats and a four- to five-fold increase was observed with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chemicals containing an amine group (semicarbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [¹⁴C]-furan to microsomal protein. A decrease in cytochrome P-450 (P-450) content and decreases in the activities of P-450-dependent aniline hydroxylase, 7-ethoxycoumarin-O-deethylase (BCD), and 7-ethoxyresorufin-O-deethylase (ERD) was observed 24 hours after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P-450 catalyzed metabolism could be binding with nucleophilic groups within the P-450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and BCD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P-450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after In vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P-450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P-450.     

The role of binding in inhibition of hepatic microsomal metabolism by parathion and malathion

Parathion, malathion and their oxygenated analogs bind to the reduced form of cytochrome P-450 from rats and mice producing spectra with a maximum at 421 nm and a minimum at 450 nm. Determinations of microsomal heme suggest that the Soret at 421 nm is not associated with conversion of cytochrome P-450 to cytochrome P-420. $\text{In vivo}$ pretreatment with C¹⁴-parathion indicates that this insecticide covalently binds to mouse hepatic microsomal protein. These findings suggest that the mechanism by which these insecticides and their analogs inhibit hepatic microsomal metabolism is identical to their mode of inhibiting esterases, that is, covalent binding to catalytic site.     

Tin-protoporphyrin inhibits heme oxygenase and prevents the decline in hepatic heme and cytochrome P-450 contents produced in nude mice by tumor transplantation

Heme and hemeprotein perturbations are present in nude mice bearing transplanted tumors. Hepatic microsomal heme oxygenase activity is increased 50-100% in tumor bearing nu/nu mice when compared with normal controls. This elevation in activity of the rate-limiting enzyme of heme degradation is associated with a 50% depletion of microsomal heme and cytochrome P-45 concentrations in liver. The synthetic heme analogue, Sn-protoporphyrin, a potent inhibitor of heme oxygenase, lowers the activity of heme oxygenase in tumor bearing animals to below control levels. This effect is associated with a normalization of hepatic heme and cytochrome P-450 contents. These findings might have implications for protecting normal cells during tumor growth and chemotherapy.     

Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.     

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.     

Spectroscopic and electrochemical properties of chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), a species related to the intermediate in the formation of N-alkylprotoporphyrins from the cytochrome P-450 prosthetic group

N-alkylporphyrins are formed when certain agents such as 3,5-diethoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine or ethylene interact with cytochrome P-450 in rats. It is likely that the iron protoporphyrin complex in cytochrome P-450 is first alkylated and then demetallated to form the free base N-alkylprotoporphyrins that are observed. An iron complex of N-methylprotoporphyrin IX dimethyl ester, chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), shows the following properties: a double Soret band (λ_max = 435 nm, with a shoulder at 390 nm) relatively facile reduction (E_{$\text{1}\text{2}$} for Fe(III)/Fe(II) of 0.385 V vs Ag/AgCl in acetonitrile) and facile demetallation by acid or good nucleophiles such as thiophenol. A knowledge of such properties should be useful in determining the mechanism of formation of N-alkylprotoporphyrins in vivo.     

Carbon radicals in the metabolism of alkyl hydrazines

The metabolism of phenelzine (2-phenylethylhydrazine) by rat liver microsomes yields phenylacetaldehyde, 2-phenylethanol, and ethylbenzene. A carbon radical is formed during the oxidative metabolism of phenelzine that reacts with the prosthetic heme of cytochrome P-450 and irreversibly inactivates the enzyme. The radical has been spin-trapped, isolated, and shown by mass spectrometry to be the 2-phenylethyl radical. The metal-free pophyrin derived from the prosthetic heme group has been isolated and identified as N-(2-phenylethyl)protoporphyrin IX. The metabolism of phenelzine, an alkyl hydrazine, thus yields a carbon radical that inactivates cytochrome P-450, is converted to a hydrocarbon by hydrogen atom abstraction, and reacts with spin traps or (presumably) alternative cellular targets.     

Incorporation of radioactive- -aminolevulinic acid into microsomal cytochrome P 450 : selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl₄ to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P₄₅₀ within 1 hr. The amount of cytochrome b₅ and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl₄ administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b₅ and NADPH cytochrome c reductase. By using ³H-δ-aminolevulinic acid to label microsomal cytochrome P₄₅₀ heme, the effect of AIA and CCl₄ on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P₄₅₀ heme accumulate in the liver after AIA or CCl₄ treatment.     

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

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-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.     

Reversible transfer of heme between different molecular species of microsome-bound cytochrome P-450 in rat liver

Incorporation of newly synthesized heme into microsome-bound cytochrome P-450 in rat liver was not affected by cycloheximide administration to the animals, indicating that the heme incorporation into cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When the heme of microsomal cytochrome P-450 had been labeled in vivo with delta-[14C]aminolevulinic acid, and then the animals were treated with phenobarbital (PB) or 3-methylcholanthrene (MC), PB-induced or MC-induced form of cytochrome P-450 was found to contain labeled heme derived from preexistent cytochrome P-450. These observations indicated that the heme of microsome-bound cytochrome P-450 is not tightly associated with the protein portion, and exchanges reversibly between different molecular species of cytochrome P-450 in vivo.     

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.     

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [¹⁴C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [¹⁴C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [¹⁴C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 $\text{in vivo}$, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Model systems for the coordination chemistry of cytochrome P-450.

Studies on model complexes have supported the presence of a mercaptide as the fifth ligand of cytochrome P-450 monooxygenases. When alcohol or thiol ligands are added to the sixth coordination position of a five-coordinated 4-nitrobenzene thiolate complex of FeIII protoporphyrin IX dimethyl ester chloride low spin complexes with optical and EPR-spectra very similar to cytochrome P-450 are obtained. From a comparison with all ligands of cytochrome P-450 and the model complexes it is concluded that a hard ligands must occupy the sixth coordination position of cytochrome P-450. An imidazole group is less likely, also in view of the ligand field parameters. The significance of the fifth and sixth ligand of cytochrome P-450 is discussed with respect to the monooxygenase mechanism.     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Calculation of the electronic structure and spectra of model cytochrome P450 compound I

The electronic structure and spectra of the oxyferryl (Fe=O) compound I P450 heme species, the transient putative active intermediate of cytochrome P450s, have been calculated employing a full protoporphyrin IX heme model representation. The principal aim of this work was to compare the computed spectra of this species with the observed transient spectra attributed to it. Computations were made using both nonlocal density functional theory (DFT) and semiempirical INDO/CI methods to characterize the electronic structure of the compound I P450 species. Both methods resulted in a similar antiferromagnetic doublet as the ground state with a ferromagnetic quartet excited state partner, slightly higher in energy. The INDO/ROHF/CI semiempirical method was used to calculate the spectrum of the protoporphyrin IX P450 compound I heme species in its lowest energy antiferromagnetic doublet state at the DFT optimized geometry. As a reference, the spectrum of the ferric resting form of the protoporphyrin IX P450 heme species was also calculated. The computed shifts in the Soret and Q bands of compound I relative to the resting state were both in good agreement with the corresponding experimentally observed shifts in the transient spectra of cytochrome P450cam (Biochem. Biophys. Res. Commun. 201 (1994) 1464) and chloroperoxidase (Biochem. Biophys. Res. Commun. 94 (1980) 1123) both ascribed to their common compound I heme site. This consistency provides additional, independent support for the assignment of compound I as the origin of the reported observed transient spectra.     

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel

When Cd (3.58 mg CdCl₂·H₂O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphoneN-demethylase (EMND) (26%), and aminopyrineN-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b₅ (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomalp-nitroanisoleO-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl₂·6H₂O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%),p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b₅ (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b₅ (26%), and microsomal heme (30%) compared to those of controls. In the case ofp-NAOD activity, the influence was in favor of Ni, i.e, the inhibition was about 61% by the combined treatment. These results reveal that:

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 influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.