Reconstitution of the monooxygenase system in a solution and in an immobilized phospholipid layer

To clarify the molecular organization of NADH- and NADPH-dependent microsomal redox systems their isolated purified carriers were incorporated into immobilized azolectin layer with a higher viscosity than that of the liposomes. It was shown that the NADH-cytochrome c reductase activity characterizing the NADH-cytochrome b5 reductase and cytochrome b5 interaction sharply decreased in the immobilized system as compared to that in solution. However, the activity of hydroxylase reactions catalyzed by immobilized NADPH-cytochrome P-450 reductase and cytochrome P-450 was the same as in solution. This, the reconstitution in the immobilized phospholipid layer allowed to characterize NADH-cytochrome b5 reductase as a system operating on occasional collisions of its components. On the contrary, the diffusion of the NADPH-dependent redox chain carriers was not the rate-limiting step of the reaction.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

Identification of delta 5,7-sterol-delta 7-reductase in higher plant microsomes

A microsomal preparation from seedlings of Zea mays catalyzed the NADPH dependent reduction of the delta 7-bond of delta 5,7-cholestadienol (1) giving the first in vitro evidence for the intermediacy of delta 5,7-sterols in plant sterol biosynthesis. Using a GC assay developed to detect the cholesterol (2) produced, the properties of the microsomal enzyme have been established with respect to cofactor requirements and kinetics. The potent in vitro inhibition of the plant delta 5,7-sterol-delta 7-reductase by the ammonium-ion containing fungicides, tridemorph2 (3), fenpropimorph (4) and AY 9944 (5) was demonstrated. The high affinities observed for these derivatives, especially for (4) (I50 = 8 x 10(-8) M, I50/Km = 2 x 10(-4)), are in full accordance with the previously proposed cationic mechanism involved in this reduction reaction.     

Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) linked to NADPH-cytochrome P450 reductase by different membrane-binding segments

Reconstitution of the enzymatic activities using purified microsomal cytochrome P450s (P450) requires the presence of a membrane-binding segment in the mammalian flavoprotein, NADPH--cytochrome P450 reductase (CPR), and the hemeprotein, cytochrome b(5) (b(5)). The mechanism(s) by which the membrane-binding segments of these proteins exert such a critical role in influencing the reconstitution of the NADPH-supported activity of a P450 remains undefined. In the present work we describe the construction, expression, and purification of four different types of recombinant flavocytochromes containing rat b(5) and rat CPR linked by various membrane-binding segments. The physical properties of these artificial fusion proteins have been studied to determine their ability to serve as electron transfer agents. These studies are a prelude to the subsequent study (accompanying paper) evaluating the functional roles of the hydrophobic (membrane-binding) sequences of b(5) and CPR in the reconstitution of P450 activities. The present study shows that the purified recombinant fusion proteins can serve as active electron transport carriers from NADPH to cytochrome c as well as b(5) by intramolecular as well as intermolecular reactions. It is shown here that the electron transport properties of these purified fusion proteins are influenced by high concentrations of KCl, suggesting a role for charged amino acids in protein-protein interactions. The present study illustrates the application of artificial recombinant flavocytochromes as useful proteins for the study of intramolecular electron transport reactions for comparison with intermolecular interactions.     

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.     

Solubilization and partial purification of a microsomal 3-ketosteroid reductase of cholesterol biosynthesis

The rat liver microsomal enzyme that catalyzes NADPH-dependent reduction of 3-ketosteroid intermediates of cholesterol biosynthesis from lanosterol has been solubilized. Although the specific activity has been enhanced only modestly, 24-fold, the solubilized and partially purified reductase can be obtained free of 4-methyl sterol oxidase (also NAD(P)H dependent) and 4α-steroidoic acid decarboxylase (NAD dependent) that are the other two constitutive enzymes of microsomal sterol 4-demethylation. In addition, the isolated protein can be incorporated into artificial phospholipid membranes with retention of activity. Thus, the partially purified 3-ketosteroid reductase is suitable for reconstitution with other enzymes and electron carriers to achieve the 10-step oxidative removal of the 4-gem-dimethyl group of sterols. Both the solubilized and microsomalbound enzyme are essentially inactive with NADH. Also, similar sterol substrate specificities with 4α-monomethyl- and 4,4-dimethyl-3-ketosteroids, pH optima, and other properties of microsomal-bound and solubilized 3-ketoreductase are observed. As observed for other microsomal enzymes the K_m of the solubilized enzyme is significantly lower than that of the membrane-bound enzyme. Membrane-bound 3-ketosteroid reductase is stimulated two- to- threefold by cytosolic Z protein (fatty acid binding protein), but stimulatory activity is lost after solubilization of the microsomal enzyme. Stimulation could not be restored by incorporating the partially purified reductase into an artificial membrane. Stimulation can be reversed by titration of Z-protein with either fatty acids or anti-Z-protein immunoglobulin. Thus, Z protein may modulate several microsomal enzymic activities of sterol biosynthesis in concert by exhibiting affinities for the membrane as well as low-molecular-weight cofactors, substrates, and metabolic effectors.     

Evidence for involvement of cytochrome P-450-linked oxygenase system in the conversion of C21-steroids to delta 16-C19-steroids catalyzed by pig testicular microsomes

Formation of androstadienone or androstadienol, a delta 16-C19-steroid, from progesterone or pregnenolone is catalyzed by the so-called delta 16-C19-steroid synthesizing enzyme in the pig testicular microsomes. The enzyme activity was also present in the testicular microsomes prepared from neonatal pig. The enzyme activity was considerably inhibited by CO, and such cytochrome P-450 inhibitors as SU 8000, SU 10603, and metyrapone. delta 16-C19-Steroid synthesizing enzyme activity was extracted from the testicular microsomes by sodium cholate in potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol, and the solubilized enzyme activity was partially purified by DEAE-cellulose column chromatography. It was shown by reconstitution of the enzyme activity that delta 16-C19-steroid synthesizing enzyme is a cytochrome P-450-linked oxygenase system dependent on cytochrome P-450-reductase and cytochrome b5. In particular, cytochrome b5 was an essential component for the activity of delta 16-C19-steroid synthesizing enzyme.     

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.     

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.     

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.     

Evidence for the formation of 26-hydroxycholesterol by cytochrome P-450 in pig kidney mitochondria

Pig kidney mitochondria were found to catalyze the formation of 26-hydroxycholesterol, an inhibitor of cholesterol biosynthesis. The cholesterol 26-hydroxylase was purified 600-fold. It was present in a mitochondrial enzyme fraction enriched in cytochrome P-450. The cytochrome P-450 fraction required NADPH, mitochondrial ferredoxin and ferredoxin reductase for 26-hydroxylase activity. The mitochondria and the purified 26-hydroxylase preparation also catalyzed 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and intermediate in cholic acid biosynthesis, and of 25-hydroxyvitamin D3. The role of extra-hepatic formation of 26-hydroxycholesterol is discussed.     

Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.     

Soluble NADH-cytochrome b5 reductase from rabbit liver cytosol: partial purification and characterization.

A soluble form of NADH-cytochrome b5 reductase (NADH: ferricytochrome b5 oxidoreductase, EC 1.6.2.2) was found in the cytosolic fraction of rabbit liver. The partially purified enzyme was strictly specific for NADH. It catalyzed the reduction of several substrates such as the methemoglobin-ferrocyanide complex (Hegesh, E. and Avron, M. (1967) Biochim. Biophys. Acta 146, 91-101) (apparent Km: 8 micrometer), potassium ferricyanide (apparent Km: 10 micrometer) and ferricytochrome b5 (apparent Km: 15 micrometer). Upon acrylamide gel isoelectro-focusing followed by specific staining, the enzyme was resolved into four bands (isoelectric pH: 7.05, 6.70, 6.50 and 6.30). The optimum pH of activity with ferricytochrome b5 as a substrate was 6.5. The estimated molecular weight was 25 000--30 000. The enzyme was unsensitive to cyanide. It was strongly inhibited by p-hydroxymercuribenzoate. The cytosolic liver cytochrome b5 reductase was immunologically related to the soluble cytochrome b5 reductase from human and rabbit red-cells, and to the microsomal cytochrome b5 reductase from rabbit liver.     

Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Intermembrane electron transport in the absence of added water-soluble carriers.

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems "intact mitochondria - mersalyzed microsomes" and "mersalyzed mtiochondria - untreated microsomes". No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

Comparison between the nitric oxide reductase family and its aerobic relatives,the cytochrome oxidases

The denitrification pathway has been studied in the hyperthermophilic archaeon Pyrobaculum aerophilum. In contrast with Gram-negative bacteria, all four denitrification enzymes are membrane-bound. P. aerophilum is also the only denitrifyer identified so far in which menaquinol is the electron donor to all four denitrification reductases. The NO reductase (NOR) of P. aerophilum belongs to the superfamily of haem-copper oxidases and is of the qNOR (quinol-dependent) type. Three types of NOR have been purified so far: cNOR (cytochrome c/pseudoazurin-dependent), qNOR and qCu(A)NOR (qNOR that contains Cu(A) at the electron entry site). It is proposed that the NORs and the various cytochrome oxidases have evolved by modular evolution, in view of the structure of their electron donor sites. qNOR is further proposed to be the ancestor of all NORs and cytochrome oxidases belonging to the superfamily of haem-copper oxidases.