Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.     

N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin

Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

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.     

Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo

The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.     

Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link.

In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed.     

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.     

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.     

Assesment of arsenic effects on cytosolic heme status using tryptophan pyrrolase as an index

Acute arsenic (As) administration produced in rat liver a decrease in the heme saturation of tryptophan pyrrolase (TP), accompanied by dose-related increases in 5-aminolevulinate synthetase (ALAS) and heme oxygenase (HO) activities, along with a corresponding decrease in cytochrome P-450 (P-450) concentration. The relationship between heme synthesis and degradation was altered as a result of As treatment. The magnitude of these effects was related to the oxidation state of arsenic, sodium arsenite (AsIII) being more potent than sodium arsenate (AsV). These results support the contention that the heme saturation of TP is sensitive to treatments that modify liver heme concentration. The increase in HO activity produced by As appears to be mediated by a mechanism largely or entirely independent of heme. The main effects of continuous exposure to AsIII were an initial decrease in the heme saturation of TP, which remained constant during the period of treatment, and an initial increase in ALAS activity, which after ten days of exposure dropped somewhat but remained above control values. No significant effects on HO or P-450 concentration were observed. These results were interpreted as indicative that a new balance between heme synthesis and degradation had been reached and that an adaptive response to the subchronic effects of AsIII was taking place.     

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.     

Characterization of the enzymatic and nonenzymatic peroxidative degradation of iron porphyrins and cytochrome P-450 heme

Both purified cytochrome P-450 (P-450) and free ferriprotoporphyrin IX are destroyed by NADPH-P-450 reductase in the presence of NADPH and O2. The process appears to be mediated by H2O2 generated by reduction of O2. Six major products were identified from the reaction of H2O2 with ferri-protoporphyrin IX-hematinic acid, methylvinylmaleimide, and four dipyrrolic propentdyopents. The structures of the propentdyopents were elucidated by mass spectrometry and 1H NMR methods. Both free ferriprotoporphyrin IX and P-450 yielded these same products in similar relative ratios. P-450 heme in rat liver microsomes was degraded in the presence of O2 and NADPH and either NaN3 (a catalase inhibitor) or Fe-ADP (which promotes lipid peroxidation); the products were primarily hematinic acid, methylvinylmaleimide, and small quantities of one propentdyopent. Only the two maleimides were detected in the destruction of microsomal P-450 heme by cumene hydroperoxide and iodosylbenzene. On the basis of the reaction of H2O2 with several metal-octaethylethylporphyrin complexes and free octaethylporphyrin, the iron chelated in ferriprotoporphyrin IX is required for degradation by H2O2. Biliverdin is not an intermediate in the formation of maleimides and propentdyopents from heme. Experiments using the tetraethylpropentdyopent produced from ferrioctaethylporphyrin suggest that propentdyopents are not further cleaved to form the maleimides. A mechanism for oxidative heme destruction consistent with these observations is proposed.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions.

Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.     

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.     

Functional reconstitution of rat liver cytochrome P-450 with mesohemin

After allylisopropylacetamide-mediated "suicide" destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin , a 2,4-diethyl heme-analog and result in a "meso-hemoprotein" with altered spectral but not functional characteristics.     

Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation

Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant.