The optical interconversion of the P-450 and P-420 forms of neuronal nitric oxide synthase: effects of sodium cholate, mercury chloride and urea

We investigated whether or not neuronal nitric oxide synthase (nNOS) (EC 1.14.13.39) was converted to the P-420 form on exposure to sodium cholate, mercury chloride or urea, and the reconversion of the P-420 to the P-450 form. Sodium cholate and mercury chloride induced the conversion of nNOS from the P-450 to the P-420 form in concentration- and incubation time-dependent manners, and the nNOS activity decreased. In the presence of glycerol, L-arginine and/or tetrahydrobiopterin, the sodium cholate-treated P-420 form could be reconverted to the P-450 form under constant experimental conditions, and the nNOS activity could also be restored. The mercury chloride-treated P-420 form of nNOS could be reconverted to the P-450 form on incubation with reduced glutathione (GSH) or L-cysteine, and the nNOS activity was recovered. However, no reconversion of the mercury chloride-treated P-420 form to the P-450 form was observed in the presence of glycerol, L-arginine, or tetrahydrobiopterin. Urea (4.0 M) dissociated nNOS into its subunits, but nNOS remained in the P-450 form. The nNOS monomer was more susceptible to sodium cholate. After removing the urea by dialysis, and supplementation of the nNOS solution with glycerol, L-arginine or BH(4), the P-420 was reconverted to the P-450 form, and the reassociation of nNOS monomers was also observed. These results suggested that nNOS was more stable as to exposure to sodium cholate, mercury chloride or urea in comparison to microsomal cytochrome P-450, which may be due to the different heme environment and protein structure.     

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.     

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.     

Modulation in cytochrome P-420 and P-450 content in Saccharomyces cerevisiae according to physiological conditions and genetic background

The diploid strain D5 of Saccharomyces cerevisiae, relative to other strains of yeast, has a large amount of cytochrome P-450 present during the logarithmic phase of growth and a low amount of cytochrome P-420. As the stationary phase of growth is approached, an increasing intensity of absorbance is observed at 420 nm. If the cells are suspended in buffer during mid-logarithmic growth, the absorbance at 450 nm disappears and absorbance at 420 nm is increased after the cells have been held in buffer for 24 h. At late logarithmic growth, the absorbance at 450 nm is still retained after the cells have been held in buffer for 24 h. Within 44 h of the time of harvest, the absorbance at 450 nm disappears completely and the absorbance at 420 nm is intense. Cytoplasmic petite variants of strain D5 have less of both cytochromes P-450 and P-420 than does the grande D5 strain; the absorbance at 450 and 420 nm are retained up to 96 h when the cells are held in buffer. Haploid spores of strain D5 exhibit absorbances at 450 and 420 nm during the logarithmic phase of growth, and these absorbances are retained after the cells are held in buffer for 24 h.

An hypothesis is proposed which states that cytochrome P-450 is the membrane-bound form and cytochrome P-420 is free in the cytosol; the cytochromes interconvert and are active in either state until the associated enzymes disassociate.     

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by alpha-naphthoflavone

Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents. alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5 degrees C increase in thermal stability.     

Interaction of cytochrome P-450 with hydrocarbons

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

Surface enhanced resonance Raman scattering as a probe of the spin state of structurally related cytochromes P-450 from rat liver

Surface enhanced resonance Raman scattering (SERRS) was observed from structurally related drug-induced rat liver cytochromes P-450 adsorbed on a silver colloid. Careful control of pH and the sequence of addition of components to the so1 is required to prevent protein denaturation at the surface due to conversion to P-450's biologically inactive form P-420 or haem loss. A low-spin P-450 (PB3a), a mixed low- and high-spin P-450 (PB3b) and a predominantly high-spin P-450 (MC1a) were investigated. Spectra recorded in the 1300-1700 cm-1 frequency region, containing the oxidation state marker v4 at 1375 cm-1 (Fe3+) and spin state markers v10 (1625 cm-1, high-spin; 1633 cm-1, low-spin) and v19 (1575 cm-1, high-spin; 1585 cm-1, low-spin) were used to differentiate between the spin states of the various forms of cytochrome P-450. As well as the established spin state marker bands, the intensity of a band at 1400 cm-1 appeared to depend on the high-spin content. Thus, with this method SERRS from silver colloids can be used to determine spin states of related cytochromes P-450 in dilute solution (10(-8)M) and may be of value in studies of protein-substrate interactions.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

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.     

Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.     

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.     

In vitro effects of trans-4-hydroxy-2-alkenals on mouse liver cytochrome P-450

Under in vitro conditions, trans-4-hydroxy-2-hexenal (t-4HH), trans-4-hydroxy-2-nonenal (t-4-HN) and trans-2-hexenal (t-2H) significantly reduced the levels of mouse liver microsomal cytochrome P-450. Incubation of trans-4-hydroxy-alkenals, under anaerobic conditions in the absence of an NADPH-generating system indicated that these compounds were converting cytochrome P-450 to cytochrome P-420. Prior activation by the mixed function oxidase system was not required for trans-4-hydroxy-alkenals to alter cytochrome P-450 concentrations. trans-4-Hydroxy-alkenals and non-hydroxylated alpha,beta-unsaturated aldehydes may be exerting their effects on cytochrome P-450 by binding to sulfhydryl groups in a similar manner as reported for sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate.     

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.     

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

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