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PubMed1982

Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.

Amino Acids↗
PubMed1992

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

Aniline Compounds↗
PubMed1988

Kinetics of the interaction of the cytochrome c oxidase of Paracoccus denitrificans with its own and bovine cytochrome c.

We have devised a relatively simple method for the purification of cytochrome aa3 of Paracoccus denitrificans with three major subunits similar to those of the larger subunits of the mitochondrial cytochrome oxidase. This preparation has no c-type cytochrome. Studies were made of the oxidation of soluble cytochromes c from bovine heart and Paracoccus. The cytochrome-c oxidase activity was stimulated by low concentrations of either cytochrome c, providing an explanation for the multiphasic nature of plots of v/S versus v. Kinetics of the oxidation of bovine cytochrome c by the Paracoccus oxidase resembled those of bovine oxidase with bovine cytochrome c in every way; the Paracoccus oxidase with bovine cytochrome c can serve as an appropriate model for the mitochondrial system. The kinetics of the oxidation of the soluble Paracoccus cytochrome c by the Paracoccus oxidase were different from those seen with bovine cytochrome c, but resembled the latter if poly(L-lysine) was added to the assays. The important difference between the two species of cytochrome c is the more highly negative hemisphere on the side of the molecule way from the heme crevice in the Paracoccus cytochrome. Thus, the data emphasize the importance of all of the charged groups on cytochrome c in influencing the binding or electron transfer reactions of this oxidation-reduction system. The data also permit some interesting connotations about the possible evolution from the bacterial to the mitochondrial electron transport system.

Animals↗
PubMed1992

Induction of hepatic microsomal cytochrome P450 and drug-metabolizing enzymes by 4-benzylpyridine and its structurally related compounds in rats. Dose- and sex-related differential induction of cytochrome P450 species.

We examined the abilities of 4-, 3- and 2-benzylpyridine and 4-tert-butylpyridine to induce hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in male and female rats in order to define the effects of pyridine-containing compounds on drug metabolism. 4-Benzylpyridine (0.4 mmol/kg, for 2 consecutive days) induced total cytochrome P450 to about three times that of the controls at 24 hr, and its inducing effect was sustained for 120 hr after the treatment in male and female rats. 4-Benzylpyridine was a more potent inducer of cytochrome P450 than 3- and 2-benzylpyridine, which induced the cytochrome to 71.4 and 43.9%, respectively, of that produced by the 4-substituted isomer. 4-tert-Butylpyridine also induced cytochrome P450. Immunoblot analysis revealed that a single treatment of male rats with 4-benzylpyridine at doses ranging from 0.05 to 0.80 mmol/kg induced cytochrome P450b/e and caused a maximum increase in the level of the isozyme at the 0.2 mmol/kg dose. 4-Benzylpyridine at doses from 0.40 to 0.80 mmol/kg also induced cytochrome P450c/d in male rats. In female rats, 4-benzylpyridine induced cytochrome P450b at doses ranging from 0.1 to 0.80 mmol/kg and produced a maximum increase in the level of this isozyme at 0.40 to 0.60 mmol/kg. Induction of cytochrome P450c/d by 4-benzylpyridine in female rats was observed at a dose of 0.20 mmol/kg, and the magnitude of the induction of the isozyme was increased in a dose-dependent manner. Both 3- and 2-benzylpyridine induced cytochrome P450b/e and/or c/d depending on the increase of total cytochrome P450 without changing the induction patterns of the isozymes. 4-tert-Butylpyridine induced cytochrome P450b at doses ranging from 0.20 to 0.60 mmol/kg and slightly induced P450c/d at doses ranging from 0.10 to 0.40 mmol/kg in male rats. These results and our previous report (Matsuura et al., Biochem Pharmacol 41: 1949-1956, 1991) clearly show that the pyridine compounds having lipophilic groups at the 4- or 3-position of the ring could be inducers of cytochrome P450. The present results also revealed that 4-benzylpyridine shows dose- and sex-related differences in the induction of cytochrome P450b/e and c/d in rats.

Aminopyrine N-Demethylase↗
PubMed1997

Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release.

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.

Apoptosis↗
PubMed1993

Acidic regions of cytochrome c1 are essential for ubiquinol-cytochrome c reductase activity in yeast cells lacking the acidic QCR6 protein.

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex.

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.

Cell Fractionation↗
PubMed1992

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.

Animals↗
PubMed1987

Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family.

Lauric acid omega-hydroxylase (cytochrome P-450LA omega) was purified from livers of rats that had been given the hypolipidemic drug clofibrate. Immunoblot analysis with anti-cytochrome P-450LA omega antibody revealed the presence of two clofibrate-induced cytochrome P-450 proteins in both liver and kidney. This antibody was used to screen a lambda gt11 expression library constructed from liver mRNA isolated from rats given clofibrate. The clone, pP-450LA omega, was isolated and found to contain 2062 base pairs with an open reading frame of 509 amino acids (Mr 58,222). The pP-450LA omega cDNA insert was placed in the yeast expression vector pAAH5, and the resultant plasmid expressed a protein of the same size and activity as cytochrome P-450LA omega. The mechanism of the regulation of the cytochrome P-450LA omega gene by hypolipidemic agents was examined. The rate of cytochrome P-450LA omega gene transcription was increased as early as 1 h after administration of clofibrate, and this increase was followed by an elevation of cytochrome P-450LA omega mRNA, immunochemically detectable protein, and cytochrome P-450LA omega activity. In contrast, no increase in transcriptional activity was detected after clofibrate administration for the cytochrome P-450PCN or P-450b/e genes. The cytochrome P-450LA omega has several structural features in common with other cytochrome P-450s, including a conserved cysteine-containing heme-binding fifth ligand fragment and a hydrophobic amino terminus. Overall, cytochrome P-450LA omega shared less than 35% cDNA nucleotide and amino acid similarity with cytochromes P-450c, P-450d, P-450e, and P-450PCN, indicating that it is a member of a new cytochrome P-450 gene family. Southern blot analysis indicates that this family contains two or three genes.

Amino Acid Sequence↗
PubMed1983

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase.

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.

Anaerobiosis↗
PubMed1993

Benzene metabolism by reconstituted cytochromes P450 2B1 and 2E1 and its modulation by cytochrome b5, microsomal epoxide hydrolase, and glutathione transferases: evidence for an important role of microsomal epoxide hydrolase in the formation of hydroquinone.

Benzene metabolism was investigated using two purified rat hepatic MFO systems containing either cytochrome P450 2B1 or cytochrome P450 2E1. Studies performed over a wide substrate concentration range indicate that cytochrome P450 2B1 represents a relatively low-affinity form of cytochrome P450 with respect to benzene metabolism while cytochrome P450 2E1 is substantially more efficient at low benzene concentrations (apparent Km value 0.17 mM). Cytochrome b5 stimulated benzene metabolism by both cytochromes P450 2B1 and P450 2E1. With cytochrome P450 2E1 the stimulation of benzene metabolism by cytochrome b5 was very pronounced (up to 6-fold) at low concentrations of benzene and was most effective (up to 15-fold) with respect to formation of hydroquinone. The metabolites observed in these studies were phenol and hydroquinone. Cytochrome P450 2E1 metabolized phenol with an affinity and capacity comparable to those of benzene. Hydroquinone was the major product formed at all substrate concentrations, while some catechol was formed at all substrate concentrations, while some catechol was formed at higher concentrations of phenol. Phenol metabolism was also stimulated by cytochrome b5. The metabolism of benzene by cytochrome P450 2E1 in the presence of the major microsomal epoxide hydrolase, mEHb, yielded phenol, hydroquinone, and benzene dihydrodiol. Interestingly, the addition of mEHb did not lead to a decrease of the toxicologically important metabolite hydroquinone as might be expected from sequestration of the intermediate benzene oxide to the vicinal dihydrodiol pathway but rather led to a marked (more than 4-fold) increase in the formation of hydroquinone, suggesting catalysis by mEHb of a predominant attack at the homoallylic position rather than at a carbon atom which forms the epoxide ring of benzene oxide. The addition of glutathione transferases plus glutathione did not yield GSH conjugates during benzene metabolism. However, metabolism of phenol by cytochrome P450 2E1 in the presence of glutathione yielded a nonenzymatically formed glutathione conjugate derived from hydroquinone or from an oxidative product of hydroquinone.

Animals↗
PubMed1984

On the function of cytochrome b5 in the cytochrome P-450-dependent oxygenase system.

Complex formation between the phenobarbital-inducible form of rabbit liver microsomal cytochrome P-450 incorporated into phosphatidylcholine and detergent-solubilized cytochrome b5 is associated with a low-to-high spin transition of the former pigment. It is concluded that the proteins combine in a 1:1 molar ratio. CD spectral analysis in the far uv region reveals that interaction of the cytochromes results in a conformational change of one or both hemoproteins. Such a cytochrome b5-induced structural alteration of the reconstituted enzyme system is accompanied by an increase in affinity of 4-chloroaniline for cytochrome P-450, as measured in terms of cumene hydroperoxide-supported N-oxidation of the arylamine; the maximum velocity of the catalytic process remains unchanged. Similarly, incorporation into the assay media of cytochrome b5 decreases the apparent Kd values of both the amine substrate and the oxygen donor, as determined by optical titration. Stopped-flow spectrophotometric studies on the influence of cytochrome b5 on the kinetics of binding to cytochrome P-450 of 4-chloroaniline and/or cumene hydroperoxide show that the rates of formation and decay of the adducts change as the molar ratio of cytochrome b5 to cytochrome P-450 varies. Moreover, cytochrome b5 modifies the activation energies required for production of the substrate-bound oxy complex. These findings suggest that cytochrome b5, apart from its well-known role as an electron carrier, might exert an effector function in the cytochrome P-450 system.

Aniline Compounds↗
PubMed2004

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes.

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

Amino Acid Sequence↗
PubMed1982

Interaction and electron transfer between cytochrome b5 and cytochrome P-450 in the reconstituted p-nitroanisole O-demethylase system.

The interaction and electron transfer between cytochrome b5 and cytochrome P-450B1 were investigated using the reconstituted p-nitroanisole O-demethylase system. Apocytochrome b5 was prepared from detergent-solubilized cytochrome b5 by the acid-butanone method. The apocytochrome b5 thus obtained has been substituted with several metalloporphyrin derivatives. The reconstituted system containing cytochrome b5 substituted with heme derivatives such as proto-, meso-, and deuteroheme exhibited demethylation activity at the maximum turnover rates of 94, 58, 30%, respectively, compared to that containing the native cytochrome b5, while neither apocytochrome b5 nor cobaltic protoporphyrin-cytochrome b5 displayed the activity. Kinetic analysis showed the formation of a 1:1 complex between cytochrome P-450B1 and each of these substituted cytochrome b5's, except for cobaltic protoporphyrin-cytochrome b5; the affinities differed with the cytochrome b5 species used. The synergistic effect with the addition of the NADH-linked electron transport system was more remarkable at the lower reduction levels of cytochrome b5 in the steady state. Interaction between the components involved in NADH- and NADPH-linked electron transport systems was modulated by the existence of Triton X-100. The optimal concentration in the reconstituted system for the demethylation was observed at around 0.03% of Triton X-100, where the reduction rates for cytochrome b5 and cytochrome P-450B1 by the respective reductases were maximal. These results indicate that the two electron transport systems are closely coupled and exhibit the demethylase activity.

Animals↗
PubMed1990

[Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5].

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.

Animals↗
PubMed2001

Site-directed mutagenesis of cytochrome P450scc. II. Effect of replacement of the Arg425 and Arg426 residues on the structural and functional properties of the cytochrome P450scc.

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and site-directed mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.

Adrenodoxin↗
PubMed1987

Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis.

The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.

Animals↗
PubMed1978

Surface differences and similarities in two homologous proteins. Cytochrome b5 and cytochrome b2 core.

From previous work (Guiard, B., Groudinsky, O. and Lederer, F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2539-2543) it is now clear that the overall secondary and tertiary structure of cytochrome b2 core is very similar to that of cytochrome b5. We present here a direct comparison of circular dichroism spectra and low-temperature absorption spectra which bring further evidence about this structural similarity. Cytochrome b2 core reacts only sluggishly with cytochrome b5 reductase, showing a lack of correspondence with the reductase binding area in cytochrome b5. On the other hand, literature data indicate similar electron transfer rates between cytochrome c on one hand, cytochrome b5 and cytochrome b2 core on the other hand. A structural inspection of cytochrome b2 core suggests that the mouth of the heme crevice in the latter is the most likely region for interaction with cytochrome c, with perhaps ionic bonds slightly different from those proposed by Salemme (Salemme, F.R. (1976) J. Mol. Biol. 102, 563--568) for the cytochrome c-cytochrome b5 interaction. In view of this partial surface similarity, the lack of immunological cross-reactivity between the two hemoprotein cores is attributed to their close similarity with the cytochrome b5 of the antibody-producing rabbit.

Animals↗