Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals.

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high-valent iron-oxo species which are responsible for both peroxidase-like and cytochrome P450-like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2* radicals formed by the oxidation of nitrite by high-valent iron-oxo species. This new activity of MP8 opens a new access to nitro-aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO-derived species with biomolecules under oxidative stress conditions.     

Formation of nitric oxide by cytochrome P450-catalyzed oxidation of aromatic amidoximes.

Rat liver microsomes catalyze the oxidation of para-hexyloxy-benzamidoxime 1 to the corresponding arylamide 2 and NO2-, by NADPH and O2. Involvement of cytochromes P450 as catalysts of this reaction was shown by the strong inhibitory effects of CO and miconazole and the spectacular increase of the activity upon treatment of rats with dexamethasone, a specific inducer of cytochromes P450 of the 3A subfamily. Formation of NO during oxidation of 1 was shown by detection of the formation of cytochrome P450- and cytochrome P420-Fe(II)-NO complexes by visible and EPR spectroscopy. The formation of these complexes should be responsible, at least in part, for the fast decrease of the rate of microsomal oxidation of 1 with time. These results suggest that exogenous compounds containing amidine or amidoxime functions could act as precursors of NO in vivo after in situ oxidation by cytochromes P450.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: the origin of double Soret spectra.

Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.     

Resonance Raman investigation of lysine and N-acetylmethionine complexes of ferric and ferrous microperoxidase

In order to evaluate the steric and electronic effects of mixed axial ligations on the heme c structure, lysine (Lys) and N-acetylmethionine (AcMet) complexes of ferric and ferrous microperoxidase-8 (MP8(III) and MP8(II), respectively) are characterized by absorption and resonance Raman (RR) spectroscopies. Spectrophotometric titrations establish that MP8(III) binds one molecule of exogenous ligand while MP8(II) forms mono(ligated) and bis(ligated) compounds. The Soret-excited RR spectra of the six-coordinated low-spin MP8(III) complexes show that the macrocycle can adopt different structures between planar and ruffled conformations. The ferriheme c conformation is primarily determined by the ionization state of the His side chain of MP8(III) and, secondarily, by the bonding and nonbonding heme-ligand interactions. As far as the RR spectra of the MP8(II) complexes are concerned, they permit us to conclude that the mixed His/Lys and His/AcMet coordinations induce a nonplanar heme conformation, the extent of deformation again depending on the ionization state of the endogenous His ligand. In contrast, the RR spectra of the bis(Lys) and bis(AcMet) compounds are associated with a planar heme structure. When the His of MP8 is bound to heme c, the stabilization of distorted heme conformations is thus associated with constraints exerted by the Cys-Ala-Gln-Cys-His-peptide on the porphyrin macrocycle. More generally, the spectroscopic data obtained in this study can be used to predict both the axial coordination and the structure of heme in c-type cytochromes. Received: 19 January 1998 / Revised version: 23 March 1998 / Accepted: 27 March 1998     

Interaction of nitric oxide with cytochrome P450 BM3

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.     

Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.     

Mammalian cytochromes P450—Importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

Mammalian cytochromes P450--importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix

When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.     

Orientation of cytochromes P450 in the endoplasmic reticulum

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.     

Interaction of aromatic cytokinins with human liver microsomal cytochromes P450

Aromatic cytokinins (ortho-topolin riboside, 6-benzylaminopurine riboside and 6-(2-hydroxy-3-methoxybenzyla mino)purine riboside) were tested for their possible interaction with human liver microsomal cytochromes P450 by absorption difference spectroscopy. All three compounds were shown to bind to the CYP enzymes producing a high to low spin shift of the heme iron yielding a Soret absorption band shift to approximately 425 nm. As this type of spectral change means that the substance is able to bind directly to the heme iron, the results obtained open the possibility of an interaction of these compounds with metabolism of other drugs or, in general, with other substrates of cytochromes P450.     

Crystal structure of the Mycobacterium tuberculosis P450 CYP121-fluconazole complex reveals new azole drug-P450 binding mode

Azole and triazole drugs are cytochrome P450 inhibitors widely used as fungal antibiotics and possessing potent antimycobacterial activity. We present here the crystal structure of Mycobacterium tuberculosis cytochrome P450 CYP121 in complex with the triazole drug fluconazole, revealing a new azole heme ligation mode. In contrast to other structurally characterized cytochrome P450 azole complexes, where the azole nitrogen directly coordinates the heme iron, in CYP121 fluconazole does not displace the aqua sixth heme ligand but occupies a position that allows formation of a direct hydrogen bond to the aqua sixth heme ligand. Direct ligation of fluconazole to the heme iron is observed in a minority of CYP121 molecules, albeit with severe deviations from ideal geometry due to close contacts with active site residues. Analysis of both ligand-on and -off structures reveals the relative position of active site residues derived from the I-helix is a key determinant in the relative ratio of on and off states. Regardless, both ligand-bound states lead to P450 inactivation by active site occlusion. This previously unrecognized means of P450 inactivation is consistent with spectroscopic analyses in both solution and in the crystalline form and raises important questions relating to interaction of azoles with both pathogen and human P450s.     

The active sites of cytochromes P450 IA1, IIB1, IIB2, and IIE1. Topological analysis by in situ rearrangement of phenyl-iron complexes

The reactions of cytochromes P450 IA1, IIB1, IIB2, and IIE1 with phenyldiazene yield complexes with absorption maxima at either 474 or 480 nm. Anaerobic extraction of the prosthetic group from the phenyldiazene-treated proteins followed by its exposure to oxygen and strong acid produces an equal mixture of the four possible N-phenylprotoporphyrin IX regioisomers. Exposure of the anaerobically extracted heme complexes to oxygen in the absence of strong acid results in their decomposition to heme and products other than N-phenylprotoporphyrin IX. These results show that the 474/480 nm absorption maxima are due to sigma phenyl-iron complexes. Treatment of the intact hepatic cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 474/480 nm band. Extraction of the prosthetic group then yields only the two N-phenylprotoporphyrin IX regioisomers with the N-phenyl group on pyrrole rings A and D. The same regioisomer pattern is obtained if the cytochrome P450IA1 phenyl-iron complex is simply warmed to 37 degrees C, but this thermal rearrangement occurs much less readily, if at all, with the complexes of the other isozymes. The regioisomers with the N-phenyl on pyrrole rings A and D are obtained in a 2:1 ratio with isozyme IA1, 1:1 with IIB2, 1:1.7 with IIB1, and 1:2 with IIE1. These results establish that the active sites of these cytochrome P450 isozymes have a common architecture despite their gross differences in substrate specificity. In this architecture, the region directly above pyrrole rings A and D is relatively open whereas that above pyrrole rings B and C is occluded by protein residues.     

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.     

Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.     

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.