Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b₅ is the main electron acceptor of cytochrome b₅ reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b₅ reductase flavin autofluorescence that was dependent upon the presence of cytochrome b_5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b₅ reductase redox potential in presence of cytochrome b₅ was also measured. These experiments suggest that the FAD group of cytochrome b₅ reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.     

The conformation and thermal stability of soluble cytochrome P-450 and cytochrome P-450 incorporated into liposomal membranes

The α-helix content of the cytochrome P-450 incorporated into the liposomal membranes of either phosphatidylcholine or microsomal phospholipid insignificantly differed from that of the soluble one. The binding of both type I and type II substrates with cytochrome P-450 incorporated into phosphatidylcholine and microsomal phospholipid membranes did not change the conformation of the polypeptide chain. In contrast to this the type II substrates increased the α-helix content of soluble hemoprotein about 3–5%. Dithionite reduction of the cytochrome P-450 haem increased the degree of α spiralization up to 10% for soluble hemoprotein and up to 5% for the membrane-bound enzyme only. The investigation of the thermal stability of the soluble and liposomal forms of cytochrome P-450 showed that the enzyme incorporated into phospholipid vesicles was highly stable. The heating of the enzyme was followed by a slightly pronounced cooperative transition in contrast to the well-pronounced transition for the soluble cytochrome P-450. Hence, the incorporation of the soluble cytochrome P-450 into phospholipid bilayer does not result in significant change of α-helix content, but the increasing of rigidity and thermal stability of the membrane-bound hemoprotein molecule is observed.     

Participation du cytochrome P450 dans l'oxydation des alcanes chez Candida tropicalis

Candida tropicalis strain 101 possesses a hydroxylase system when grown on tetradecane as the carbon source which is active towards hydrocarbons and fatty acids. This system including cytochrome P₄₅₀ and NADPH-cytochrome c reductase has been localized exclusively in the microsomal fraction.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Mode of binding of cytochrome b5 to phospholipid bilayers in lamellar structure

X-ray diffraction studies were made on the multilamellar systems produced by incubation of phospholipid bilayers and the membrane protein, cytochrome b₅, or non-membrane proteins (albumin, ovalbumin and β-lactoglobulin A) at pH 8.1 in aqueous 5 mM CaCl₂ solutions.Detergent-extracted cytochrome b₅ (soluble aggregate) forms two types of lamellar phase with dipalmitoyl phosphatidylcholine bilayers, depending upon the incubation temperature. One type, which has a repeat distance of 114Å, is formed above 34°C, where the binding of cytochrome b₅ to the bilayers is hydrophobic. The other type, with a repeat distance of 153 Å, is formed below 34°C, where the binding is electrostatic. It is also suggested that cytochrome b₅ is monomeric in the former phase but remains aggregated in the latter phase.When dimyristoyl phosphatidylcholine is used, the boundary temperature for the two types shifts to 12°C. These boundary temperatures coincide with the thermal pretransition points of hydrated dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, respectively.Trypsin-treated cytochrome b₅ (monomeric) and the three non-membrane proteins exhibit only binding of the electrostatic type to the bilayers, independently of the incubation temperature. The observed repeat distances suggest that in these cases two layers of protein molecules are incorporated between the bilayers.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

The molecular cloning of cytochrome P-450c information

Clones containing the information for cytochrome P-450c were produced by transfecting Escherichia coli HB101 with a hybrid mRNA_P-450-c:cDNA_P-450c that had been annealed to PstI-linearized pBR322 by the A-T tailing method. Over 250 tetracycline-resistant, ampicillin-sensitive clones were obtained from which several were selected on the basis of positive hybridization to cDNA_P-450c. pEB163 and pEB339 contained DNA inserts of 0.5 and 1.0 kb in length, respectively. When poly(A)⁺-RNA that had been prepared from the livers of 3-methylcholanthrene-treated rats was hybridized to nitrocellulose-immobilized, denatured HindIII-linearized pEB163 and pEB339 DNA, a mRNA could be eluted which coded exclusively for cytochrome P-450c production in a cell-free reticulocyte assay system. The clones now make possible further studies on the cytochrome P-450c gene in the rat.     

Age-dependent exression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2α-, 2β-, 15α-, 16α-, and 16β-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7α-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible form) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6β-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Quantitative determination of cytochrome P-450 in rat liver homogenate

Cytochrome P-450 in whole liver homogenates, which contain an appreciable amount of hemoglobin, is detected by dithionite-difference spectroscopy of CO-bubbled homogenates. The molar extinction difference of cytocrhome P-450 by this method was determined to be 104 mm^?1cm^?1 by comparative observations of the absorbance change in the dithionite- and CO-difference spectra of the membrane-bound hemoprotein. The content of cytochrome P-450 in normal rat liver was estimated to be 50 nmol/g wet weight of liver, and increased significantly after pretreatment of the animals with either phenobarbital or 3-methylcholanthrene.     

Photoreduction of cytochrome b559 in a Photosystem-II subchloroplast particle

A Photosystem-II reaction-center particle derived from spinach chloroplasts by Triton treatment contains only one kind of cytochrome, namely, cytochrome b₅₅₉, in the amount of slightly more than 2 per 100 total chlorophyll molecules. Cytochrome b₅₅₉ is present in the oxidized form, has a standard redox potential of 58 mV, and undergoes photoreduction.     

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N′-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c₁, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c₁ complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c₁ complex. The binding of [¹⁴C]DCCD to the isolated b-c₁ complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c₁ complex are discussed.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (M_r 120 000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843–850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450_BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450_BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its M_r of 38 000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH₂-terminal amino acid is proline; the penultimate NH₂-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450_BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450_BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450_BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450_BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K_m for pisatin was relatively low, less than 5 μm. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mm) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.     

Electron transfer through the isolated mitochondrial cytochrome b-c1 complex

(1) A kinetic analysis of electron donation into and through the cytochrome b-c₁ complex isolated from bovine heart mitochondria has been undertaken, using trimethylquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.     

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b₅₅₉ (cyt b₅₅₉) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP^red) forms of cyt b₅₅₉ exhibit superoxide scavenging activity and interconversion between IP and HP^red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O₂⁻) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O₂⁻ scavengers prevented the light-induced conversion of IP ↔ HP^red cyt b₅₅₉ confirmed that IP and HP^red cyt b₅₅₉ are reduced and oxidized by O₂⁻, respectively. Redox changes in cyt b₅₅₉ by an exogenous source of O₂⁻ reconfirmed the superoxide oxidase and reductase activity of cyt b₅₅₉. Furthermore, the light-induced conversion of IP to HP^red form of cyt b₅₅₉ was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His²² residue generates a favorable condition for the oxidation and reduction of O₂⁻, respectively.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H₂O₂, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450_LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450_LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.