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Sample records for cytochrome b5 reductase

  1. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  2. Expression and characterization of a functional canine variant of cytochrome b5 reductase.

    PubMed

    Roma, Glenn W; Crowley, Louis J; Barber, Michael J

    2006-08-01

    Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 micro

  3. Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.

    PubMed

    Maeda, Shintaro; Kobori, Hiroki; Tanigawa, Minoru; Sato, Katsuya; Yubisui, Toshitsugu; Hori, Hiroshi; Nagata, Yoko

    2015-07-01

    For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 μM and 160 μmol min(-1) mg(-1), respectively, for ferricyanide and 328 μM and 500 μmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA. PMID:25829149

  4. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans. PMID:26806391

  5. Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Bork, Bettina; Bittner, Florian; Mendel, Ralf; Clement, Bernd

    2016-10-01

    The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase. PMID:27469001

  6. Induction of cytochrome P-450, cytochrome b-5, NADPH-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment.

    PubMed

    Kawamoto, T; Hobara, T; Nakamura, K; Imamura, A; Ogino, K; Kobayashi, H; Iwamoto, S; Sakai, T

    1988-12-30

    Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB. PMID:3145630

  7. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours.

    PubMed Central

    Marín, A.; López de Cerain, A.; Hamilton, E.; Lewis, A. D.; Martinez-Peñuela, J. M.; Idoate, M. A.; Bello, J.

    1997-01-01

    The level of expression of enzymes that can activate or detoxify bioreductive agents within tumours has emerged as an important feature in the development of these anti-tumour compounds. The levels of two such reductase enzymes have been determined in 19 human non-small-cell lung tumours and 20 human breast tumours, together with the corresponding normal tissue. DT-diaphorase (DTD) enzyme levels (both expression and activity) were determined in these samples. Cytochrome b5 reductase (Cytb5R) activity was also assessed. With the exception of six patients, the levels of DTD activity were below 45 nmol min(-1) mg(-1) in the normal tissues assayed. DTD tumour activity was extremely variable, distinguishing two different groups of patients, one with DTD activity above 79 nmol min(-1) mg(-1) and the other with levels that were in the same range as found for the normal tissues. In 53% of the lung tumour samples, DTD activity was increased with respect to the normal tissue by a factor of 2.4-90.3 (range 79-965 nmol min[-1] mg[-1]). In 70% of the breast tumour samples, DTD activity was over 80 nmol min(-1) mg(-1) (range 83-267 nmol min[-1] mg[-1]). DTD expression measured by Western blot correlated well with the enzyme activity measured in both tumour and normal tissues. The levels of the other reductase enzyme, Cytb5R, were not as variable as those for DTD, being in the same range in both tumour and normal tissue or slightly higher in the normal tissues. The heterogeneous nature of DTD activity and expression reinforces the need to measure enzyme levels in individual patients before therapy with DTD-activated bioreductive drugs. Images Figure 1 Figure 2 PMID:9328153

  8. NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I.

    PubMed

    Percy, M J; Aslan, D

    2008-10-01

    The development of cyanosis at birth, the so-called blue baby syndrome, alerts paediatricians to the presence of congenital heart disease. In rare cases where the arterial blood gas analysis is normal the cyanosis is a consequence of methaemoglobinaemia. There are three distinct origins of methaemoglobinaemia; the presence of a haemoglobin variant, environmental toxicity and deficiency of cytochrome b5 reductase (cb(5)r). Two children born to two sets of first-degree related parents were cyanotic from birth. Differential diagnosis eliminated cardiac and pulmonary abnormalities. Measurement of methaemoglobin levels confirmed recessive congenital methaemoglobinaemia (RCM) and treatment with ascorbic acid was commenced. In the absence of neurological defects, type I disease was diagnosed. Sequence analysis of CYB5R3 revealed two different missense mutations (one which is novel, Ile85Ser) in the two families. Neither of the mutations was located in the FAD or the NADH binding sites of cb(5)r, thus supporting a diagnosis of type I disease. PMID:18820099

  9. High expression of cytochrome b 5 reductase isoform 3/cytochrome b 5 system in the cerebellum and pyramidal neurons of adult rat brain.

    PubMed

    Samhan-Arias, A K; López-Sánchez, C; Marques-da-Silva, D; Lagoa, R; Garcia-Lopez, V; García-Martínez, V; Gutierrez-Merino, C

    2016-05-01

    Cytochrome b 5 reductase (Cb 5R) and cytochrome b 5 (Cb 5) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15 years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-Cb 5R isoform 3 and anti-Cb 5 antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of Cb 5R isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of Cb 5R isoform 3/Cb 5 system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration. PMID:25850901

  10. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

    PubMed Central

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  11. The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1998-04-10

    NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity. PMID:9535868

  12. Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous.

    PubMed

    Gutiérrez, María Soledad; Rojas, María Cecilia; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2015-01-01

    The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. PMID:26466337

  13. Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous

    PubMed Central

    Gutiérrez, María Soledad; Rojas, María Cecilia; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2015-01-01

    The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. PMID:26466337

  14. Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

    PubMed

    Kollipara, Sireesha; Tatireddy, Shivakishore; Pathirathne, Thusitha; Rathnayake, Lasantha K; Northrup, Scott H

    2016-08-25

    Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70. PMID:27059440

  15. Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

    PubMed

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

    2010-09-24

    NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain. PMID:20630863

  16. Congenital Recessive Methemoglobinemia Revealed in Adulthood: Description of a New Mutation in Cytochrome b5 Reductase Gene.

    PubMed

    Forestier, Alexandra; Pissard, Serge; Cretet, Justine; Mambie, Adeline; Pascal, Laurent; Cliquennois, Manuel; Cambier, Nathalie; Rose, Christian

    2015-01-01

    Methemoglobinemia can be acquired (oxidizing drugs or chemicals products) or inherited either by mutations affecting globin chains [M hemoglobins (M Hbs)] or by defects in the enzymatic system involved in the reduction of spontaneous Hb oxidation: nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. It is encoded by the CYB5R3 gene: there are two phenotypes of autosomal recessive congenital methemoglobinemia, in type II CYB5R deficiency is generalized and affects all cells, leading to an early onset, whereas in type I, the enzyme deficiency is restricted to erythrocytes, usually discovered in infancy but not exclusively. We report a new case of methemoglobinemia discovered in a patient from Bahrain who exhibited an unknown dyspnea at the age of 37 years without trigger events or oxidizing products. We discovered a new mutation in the CYB5R3 gene: exon 9, codon 266 (delGAG) (GLU) (CYB5R3: c.726_729delGAG) in the homozygous state. Appearance of methemoglobinemia in an adult usually suggests an acquired cause but our case illustrated that it could also reveal a type I mutation of cytochrome b5 reductase. PMID:26291966

  17. Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5.

    PubMed

    Shaw, P M; Hosea, N A; Thompson, D V; Lenius, J M; Guengerich, F P

    1997-12-01

    The development of enzyme and buffer premixes for in vitro biotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochrome b5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at -80 degrees C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different Vmax and K(m) values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that Vmax and K(m) could be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays. PMID:9390180

  18. A novel L218P mutation in NADH-cytochrome b5 reductase associated with type I recessive congenital methemoglobinemia.

    PubMed

    Arikoglu, Tugba; Yarali, Nese; Kara, Abdurrahman; Bay, Ali; Bozkaya, Ikbal O; Tunc, Bahattin; Percy, Melanie J

    2009-01-01

    The presence of central cyanosis that is unrelated to cardiopulmonary causes alerts clinicians to a possible diagnosis of methemoglobinemia. Congenital methemoglobinemia due to deficiency of nicotinamide-adenine dinucleotide (NADH)-cytochrome b5 reductase (cb(5)r) is an autosomal recessive disorder characterized by life long cyanosis. Here we report a six-year old boy who presented with central cyanosis and upon examination revealed a methemoglobin level of 19.0%. Sequencing the CYB5R3 gene identified a homozygous T-->C transition at base c.653, which changed codon 218 from leucine to proline (L218P) in cb(5)r protein. Treatment with ascorbic acid relieved the cyanosis and returned methemoglobin levels to normal. PMID:19579085

  19. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  20. Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5.

    PubMed

    Kaspera, Rüdiger; Naraharisetti, Suresh B; Evangelista, Eric A; Marciante, Kristin D; Psaty, Bruce M; Totah, Rheem A

    2011-09-15

    Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k(cat) seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K(D) and K(m,red)) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k(cat). In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k(cat) between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level. PMID:21726541

  1. Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria*

    PubMed Central

    Neve, Etienne P. A.; Nordling, Åsa; Andersson, Tommy B.; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-01-01

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b5 reductase (CYB5R), cytochrome b5 (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b5 type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b5 type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  2. Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

    PubMed

    Neve, Etienne P A; Nordling, Asa; Andersson, Tommy B; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-02-24

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  3. Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.

    PubMed Central

    Haucke, V; Ocana, C S; Hönlinger, A; Tokatlidis, K; Pfanner, N; Schatz, G

    1997-01-01

    Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems. PMID:9199337

  4. The N-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

    PubMed Central

    Jakobs, Heyka H.; Mikula, Michal; Havemeyer, Antje; Strzalkowska, Adriana; Borowa-Chmielak, Monika; Dzwonek, Artur; Gajewska, Marta; Hennig, Ewa E.; Ostrowski, Jerzy; Clement, Bernd

    2014-01-01

    The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism. PMID:25144769

  5. Cytochrome b5 reductase, a plasma membrane redox enzyme, protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics.

    PubMed

    Hyun, Dong-Hoon; Lee, Ga-Hyun

    2015-12-01

    The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD(+)/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H2O2 (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H2O2 and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD(+)/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases. PMID:26611738

  6. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

    PubMed

    Kugler, W; Pekrun, A; Laspe, P; Erdlenbruch, B; Lakomek, M

    2001-04-01

    Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. PMID:11295830

  7. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability*

    PubMed Central

    Rahaman, Md. Mizanur; Reinders, Fabio G.; Koes, David; Nguyen, Anh T.; Mutchler, Stephanie M.; Sparacino-Watkins, Courtney; Alvarez, Roger A.; Miller, Megan P.; Cheng, Dongmei; Chen, Bill B.; Jackson, Edwin K.; Camacho, Carlos J.; Straub, Adam C.

    2015-01-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μm) and ZINC39395747 (IC50 = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  8. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability.

    PubMed

    Rahaman, Md Mizanur; Reinders, Fabio G; Koes, David; Nguyen, Anh T; Mutchler, Stephanie M; Sparacino-Watkins, Courtney; Alvarez, Roger A; Miller, Megan P; Cheng, Dongmei; Chen, Bill B; Jackson, Edwin K; Camacho, Carlos J; Straub, Adam C

    2015-07-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μM) and ZINC39395747 (IC50 = 9.14 μM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  9. Analysis of cytochrome b(5) reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence.

    PubMed

    Derbyshire, Mark C; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-09-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC-MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  10. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A.

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  11. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence

    PubMed Central

    Derbyshire, Mark C.; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J.; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-01-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC–MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  12. Recombinant human erythrocyte cytochrome b5.

    PubMed

    Lloyd, E; Ferrer, J C; Funk, W D; Mauk, M R; Mauk, A G

    1994-09-27

    The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium. PMID:7918357

  13. NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

    PubMed

    Lund, Rikke R; Leth-Larsen, Rikke; Caterino, Tina Di; Terp, Mikkel G; Nissen, Jeanette; Lænkholm, Anne-Vibeke; Jensen, Ole N; Ditzel, Henrik J

    2015-11-01

    Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are

  14. Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

    PubMed Central

    Douglas, R H; Hultquist, D E

    1978-01-01

    Homogeneous preparations of two forms of soluble cytochrome b5 have been obtained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P-60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of approximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome b5 are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome b5. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation. PMID:277914

  15. The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities.

    PubMed

    Billen, M J; Squires, E J

    2009-01-01

    Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5alpha-androst-16-en-3-one (5alpha-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17alpha-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-beta synthase as well as the 17alpha-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17alpha-hydroxylase (p<0.013), a transient increase in C17,20 lyase, and an increase in andien-beta synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17alpha-hydroxylase, but did not affect the andien-beta synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1. PMID:19101629

  16. Identification of an NADH-Cytochrome b5 Reductase Gene from an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4, by Sequencing of the Encoding cDNA and Heterologous Expression in a Fungus, Aspergillus oryzae

    PubMed Central

    Sakuradani, Eiji; Kobayashi, Michihiko; Shimizu, Sakayu

    1999-01-01

    Based on the sequence information for bovine and yeast NADH-cytochrome b5 reductases (CbRs), a DNA fragment was cloned from Mortierella alpina 1S-4 after PCR amplification. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (Saccharomyces cerevisiae), bovine, human, and rat CbRs. These results suggested that this cDNA is a CbR gene. The results of a structural comparison of the flavin-binding β-barrel domains of CbRs from various species and that of the M. alpina enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds. The corresponding genomic gene, which was also cloned from M. alpina 1S-4 by means of a hybridization method with the above probe, had four introns of different sizes. These introns had GT at the 5′ end and AG at the 3′ end, according to a general GT-AG rule. The expression of the full-length cDNA in a filamentous fungus, Aspergillus oryzae, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes. The M. alpina CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity. The purified CbR preferred NADH over NADPH as an electron donor. This is the first report of an analysis of this enzyme in filamentous fungi. PMID:10473389

  17. Low reduction potential cytochrome b5 isotypes of Giardia intestinalis.

    PubMed

    Pazdzior, Robert; Yang, Zhen Alice; Mesbahuddin, Mirfath Sultana; Yee, Janet; van der Est, Art; Rafferty, Steven

    2015-10-01

    Despite lacking mitochondria and a known pathway for heme biosynthesis the micro-aerotolerant anaerobic protozoan parasite Giardia intestinalis encodes four members of the cytochrome b5 family of electron transfer proteins, three of which are small, single-domain proteins. While these are similar in size and fold to their better-known mammalian counterparts the Giardia proteins have distinctly lower reduction potentials, ranging from -140 to -171 mV compared to +6 mV for the bovine microsomal protein. This difference is accounted for by a more polar heme environment in the Giardia proteins, as mutation of a conserved heme pocket tyrosine residue to phenylalanine in the Giardia cytochrome b5 isotype-I (gCYTb5-I Y61F) raises its reduction potential by nearly 100 mV. All three isotypes have UV-visible spectra consistent with axial coordination of the heme by a pair of histidine residues, but electron paramagnetic spectroscopy indicates that the planes of their imidazole rings are nearly perpendicular rather than coplanar as observed in mammalian cytochrome b5, which may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia proteins. Although no function has yet to be ascribed to any Giardia cytochrome b5, the presence of similar sequences in many other eukaryotes indicates that these represent an under-characterized class of low reduction potential family members. PMID:26299244

  18. Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR*

    PubMed Central

    Estrada, D. Fernando; Laurence, Jennifer S.; Scott, Emily E.

    2013-01-01

    The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site. PMID:23620596

  19. Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy*

    PubMed Central

    Zhang, Meng; Huang, Rui; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-01-01

    Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners. PMID:25795780

  20. Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b₅ in parasitic adaptation.

    PubMed

    Takamiya, Shinzaburo; Hashimoto, Muneaki; Mita, Toshihiro; Yokota, Takehiro; Nakajima, Yoshitaka; Yamakura, Fumiyuki; Sugio, Shigetoshi; Fujimura, Tsutomu; Ueno, Takashi; Yamasaki, Hiroshi

    2016-04-01

    We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation. PMID:26571414

  1. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  2. A spectroscopic study of uranyl-cytochrome b5/cytochrome c interactions

    NASA Astrophysics Data System (ADS)

    Sun, Mei-Hui; Liu, Shuang-Quan; Du, Ke-Jie; Nie, Chang-Ming; Lin, Ying-Wu

    2014-01-01

    Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO22+) to interact with various proteins and disturb their biological functions. Cytochrome b5 (cyt b5) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b5-cyt c complex. In previous molecular modeling study in combination with UV-Vis studies, we found that UO22+ is capable of binding to cyt b5 at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b5 and cyt c, as well as cyt b5-cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b5 (KD = 10 μM), cyt c (KD = 87 μM), and cyt b5-cyt c complex (KD = 30 μM) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b5-cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b5/cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity.

  3. Giardia intestinalis Incorporates Heme into Cytosolic Cytochrome b5

    PubMed Central

    Pyrih, Jan; Harant, Karel; Martincová, Eva; Sutak, Robert; Lesuisse, Emmanuel; Hrdý, Ivan

    2014-01-01

    The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated. PMID:24297440

  4. Higher Plant Cytochrome b5 Polypeptides Modulate Fatty Acid Desaturation

    PubMed Central

    Kumar, Rajesh; Tran, Lam-Son Phan; Neelakandan, Anjanasree K.; Nguyen, Henry T.

    2012-01-01

    Background Synthesis of polyunsaturated fatty acids (PUFAs) in the endoplasmic reticulum of plants typically involves the fatty acid desaturases FAD2 and FAD3, which use cytochrome b5 (Cb5) as an electron donor. Higher plants are reported to have multiple isoforms of Cb5, in contrast to a single Cb5 in mammals and yeast. Despite the wealth of information available on the roles of FAD2 and FAD3 in PUFA synthesis, information regarding the contributions of various Cb5 isoforms in desaturase-mediated reactions is limited. Results The present functional characterization of Cb5 polypeptides revealed that all Arabidopsis Cb5 isoforms are not similarly efficient in ω-6 desaturation, as evidenced by significant variation in their product outcomes in yeast-based functional assays. On the other hand, characterization of Cb5 polypeptides of soybean (Glycine max) suggested that similar ω-6 desaturation efficiencies were shared by various isoforms. With regard to ω-3 desaturation, certain Cb5 genes of both Arabidopsis and soybean were shown to facilitate the accumulation of more desaturation products than others when co-expressed with their native FAD3. Additionally, similar trends of differential desaturation product accumulation were also observed with most Cb5 genes of both soybean and Arabidopsis even if co-expressed with non-native FAD3. Conclusions The present study reports the first description of the differential nature of the Cb5 genes of higher plants in fatty acid desaturation and further suggests that ω-3/ω-6 desaturation product outcome is determined by the nature of both the Cb5 isoform and the fatty acid desaturases. PMID:22384013

  5. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

  6. Reduction of Hexavalent Chromium by Human Cytochrome b5: Generation of Hydroxyl Radical and Superoxide

    PubMed Central

    Borthiry, Griselda R.; Antholine, William E.; Kalyanaraman, B.; Myers, Judith M.; Myers, Charles R.

    2007-01-01

    The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b5 and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure 53Cr(VI). When 5-Diethoxyphos-phoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO•) adduct was observed as well as a smaller signal for the superoxide (O2•−) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 μM). NADPH, Cr(VI), O2 and the PLs were all required for significant HO• generation. Superoxide dismutase eliminated the O2• − adduct and resulted in a 30% increase in the HO• adduct. Catalase largely diminished the HO• adduct signal indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that it was generated by the PLs. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO• adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethylsulfoxide (DMSO) plus the spin trap α-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO•. Quantification of the various species over time was consistent with a stoichiometric excess of HO• relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b5 in the generation of HO•. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO• at low Cr

  7. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

  8. Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5*

    PubMed Central

    Peng, Hwei-Ming; Liu, Jiayan; Forsberg, Sarah E.; Tran, Hong T.; Anderson, Sean M.; Auchus, Richard J.

    2014-01-01

    Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: 84EVLIKK89-b5: 53EQAGGDATENFEDVGHSTDAR73 and CYP17A1-R347K: 341TPTISDKNR349-b5: 40FLEEHPGGEEVLR52. Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis. PMID:25315771

  9. Reduction potential and heme-pocket polarity in low potential cytochrome b5 of Giardia intestinalis.

    PubMed

    Yang, Zhen Alice; Pazdzior, Robert; Yee, Janet; Rafferty, Steven

    2016-05-01

    Although it lacks mitochondria and the ability to synthesize heme, the protozoan parasite Giardia intestinalis encodes several heme proteins. This includes four members of the cytochrome b5 family, three of which are of similar size to mammalian cytochromes b5 but with reduction potentials that are 140 to 180mV lower. While no structures have yet been determined for any of these proteins, homology modeling points to an increase in heme pocket polarity as a reason for their low potentials. To test this we measured the reduction potentials of four mutants of Giardia cytochrome b5 isotype-I (gCYTB5-I) in which polar residues at two candidate positions (C84, Y51) in the heme pocket were changed to nonpolar ones (C84A, C84F; Y51L, Y51F). All mutants were expressed with comparable levels of heme incorporation and had UV-visible spectra consistent with low spin bis-histidyl coordination. These mutations increased the reduction potential by 18 to 57mV and highlight the influence of C84, which is a residue unique to gCYTB5-I and whose mutation to alanine caused the largest increase. The influence of these two residues plus that of Y61 reported previously accounts for much of the reduction potential difference between gCYTB5-I and microsomal cytochrome b5. A complementary triple mutant of the latter with the hydrophilic residues found in gCYTB5-I bound heme less effectively but nonetheless had a reduction potential that was 135mV lower than wild type. PMID:27048807

  10. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells

    PubMed Central

    Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.

    2016-01-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  11. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells.

    PubMed

    Sondhi, Varun; Owen, Bryn M; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A; Hughes, Beverly A; Arlt, Wiebke; Mangelsdorf, David J; Auchus, Richard J

    2016-04-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  12. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  13. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    NASA Astrophysics Data System (ADS)

    Gnedenko, O. V.; Yablokov, E. O.; Usanov, S. A.; Mukha, D. V.; Sergeev, G. V.; Bulko, T. V.; Kuzikov, A. V.; Moskaleva, N. E.; Shumyantseva, V. V.; Ivanov, A. S.; Archakov, A. I.

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  14. Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

    PubMed

    Rivera, M; Barillas-Mury, C; Christensen, K A; Little, J W; Wells, M A; Walker, F A

    1992-12-01

    The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver. PMID:1333795

  15. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  16. The action of cytochrome b(5) on CYP2E1 and CYP2C19 activities requires anionic residues D58 and D65.

    PubMed

    Peng, Hwei-Ming; Auchus, Richard J

    2013-01-01

    The capacity of cytochrome b(5) (b(5)) to influence cytochrome P450 activities has been extensively studied and physiologically validated. Apo-b(5) enhances the activities of CYP3A4, CYP2A6, CYP2C19, and CYP17A1 but not that of CYP2E1 or CYP2D6, suggesting that the b(5) interaction varies among P450s. We previously showed that b(5) residues E48 and E49 are required to stimulate the 17,20-lyase activity of CYP17A1, but these same residues might not mediate b(5) activation of other P450 reactions, such as CYP2E1-catalyzed oxygenations, which are insensitive to apo-b(5). Using purified P450, b(5), and reductase (POR) in reconstituted assays, the D58G/D65G double mutation, of residues located in a hydrophilic α-helix of b(5), totally abolished the ability to stimulate CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. In sharp contrast, the D58G/D65G double mutation retained the full ability to stimulate the 17,20-lyase activity of CYP17A1. The D58G/D65G double mutation competes poorly with wild-type b(5) for binding to the CYP2E1·POR complex yet accepts electrons from POR at a similar rate. Furthermore, the phospholipid composition markedly influences P450 turnover and b(5) stimulation and specificity, particularly for CYP17A1, in the following order: phosphatidylserine > phosphatidylethanolamine > phosphatidylcholine. The D58G/D65G double mutation also failed to stimulate CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation, whereas the E48G/E49G double mutation stimulated these activities of CYP2C19 and CYP2E1 equivalent to wild-type b(5). We conclude that b(5) residues D58 and D65 are essential for the stimulation of CYP2E1 and CYP2C19 activities and that the phospholipid composition significantly influences the b(5)-P450 interaction. At least two surfaces of b(5) differentially influence P450 activities, and the critical residues for individual P450 reactions cannot be predicted from sensitivity to apo-b(5) alone. PMID:23193974

  17. The action of cytochrome b5 on both CYP2E1 and CYP2C19 activities requires the anionic residues D58 and D65

    PubMed Central

    Peng, Hwei-Ming; Auchus, Richard J.

    2013-01-01

    The capacity of cytochrome b5 (b5) to influence cytochrome P450 activities has been extensively studied and physiologically validated. Apo-b5 enhances the activities of CYP3A4, CYP2A6, CYP2C19, and CYP17A1 but not of CYP2E1 or CYP2D6, suggesting that the b5 interaction varies amongst P450s. We previously showed that b5 residues E48 and E49 are required to stimulate the 17,20-lyase activity of CYP17A1, but these same residues might not mediate b5 activation of other P450 reactions, such as CYP2E1-catalyzed oxygenations, which are insensitive to apo-b5. Using purified P450, b5, and reductase (POR) in reconstituted assays, mutation D58G+D65G, residues located in a hydrophilic α-helix of b5, totally abolished the ability to stimulate CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. In sharp contrast, the D58G+D65G mutation retained full capability to stimulate the 17,20 lyase activity of CYP17A1. Mutation D58G+D65G competes poorly with wild-type b5 for binding to the CYP2E1•POR complex yet accepts electrons from POR at a similar rate. Furthermore, the phospholipid composition markedly influences P450 turnover and b5 stimulation and specificity, particularly for CYP17A1, in the order phosphatidylserine > phosphatidylethanolamine > phosphatidylcholine. Mutation D58G+D65G also failed to stimulate CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation, whereas mutation E48G+E49G stimulated these activities of CYP2C19 and CYP2E1 equivalent to wild-type b5. We conclude that b5 residues D58 and D65 are essential for the stimulation of CYP2E1 and CYP2C19 activities and that phospholipid composition significantly influences the b5-P450 interaction. At least two surfaces of b5 differentially influence P450 activities, and the critical residues for individual P450 reactions cannot be predicted from sensitivity to apo-b5 alone. PMID:23193974

  18. High level expression of peptides and proteins using cytochrome b5 as a fusion host.

    PubMed

    Mitra, Ashima; Chakrabarti, Kalyan Sundar; Shahul Hameed, M S; Srinivas, Kalyan V; Senthil Kumar, Ganesan; Sarma, Siddhartha P

    2005-05-01

    A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa). PMID:15802225

  19. Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

    DOEpatents

    Craft, David L.; Madduri, Krishna M.; Loper, John C.

    2003-01-01

    A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

  20. Purification and characterization of two forms of cytochrome b5 from an arachidonic acid-producing fungus, Mortierella hygrophila.

    PubMed

    Kouzaki, N; Kawashima, H; Chung, M C; Shimizu, S

    1995-06-01

    Two forms of cytochrome b5 have been purified from the microsomes of an arachidonic acid-producing fungus, Mortierella hygrophila IFO 5941, after detergent solubilization. They have monomeric molecular masses of about 16 kDa and 19 kDa. Their absorption spectra are similar to those of mammalian cytochrome b5s. Their amino acid compositions show some similarity to those of mammalian cytochrome b5s, but the contents of some amino acids (glycine, alanine, aspartic acid + asparagine, glutamic acid + glutamine, arginine, proline, histidine, leucine and lysine) are unique to the cytochrome b5s of M. hygrophila. Some of their internal peptide sequences also show close homology with those of some mammals (approx. 65 to 67%), while some others show no or little homology. The addition of various acyl-CoAs to NADH-reduced microsomes caused an abrupt shiftdown of the steady state reduction level of cytochrome b5. This indicates the increased utilization of electrons for the desaturation process and may suggest that the cytochrome b5s of this fungus actually take part in its microsomal desaturation system for polyunsaturated fatty acid biosynthesis as electron carriers. PMID:7786894

  1. High-resolution crystal structures of the solubilized domain of porcine cytochrome b 5

    PubMed Central

    Hirano, Yu; Kimura, Shigenobu; Tamada, Taro

    2015-01-01

    Mammalian microsomal cytochrome b 5 has multiple electron-transfer partners that function in various electron-transfer reactions. Four crystal structures of the solubilized haem-binding domain of cytochrome b 5 from porcine liver were determined at sub-angstrom resolution (0.76–0.95 Å) in two crystal forms for both the oxidized and reduced states. The high-resolution structures clearly displayed the electron density of H atoms in some amino-acid residues. Unrestrained refinement of bond lengths revealed that the protonation states of the haem propionate group may be involved in regulation of the haem redox properties. The haem Fe coordination geometry did not show significant differences between the oxidized and reduced structures. However, structural differences between the oxidized and reduced states were observed in the hydrogen-bond network around the axial ligand His68. The hydrogen-bond network could be involved in regulating the redox states of the haem group. PMID:26143928

  2. A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*

    PubMed Central

    Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stéphanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

  3. Cytochrome-P450-Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors

    PubMed Central

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Ahuja, Shivani; Huang, Rui; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein-protein complex. To this end, here we report the experimentally measured backbone amide-15N CSA tensors for a membrane-bound 16.7-kDa full-length rabbit cytochrome-b5 (cytb5), in complexation with a 55.8-kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The 15N-CSAs, determined using the 15N CSA/15N-1H dipolar coupling transverse cross-correlated rates, for free cytb5 are compared with that for the cytb5 bound to cytP4502B4. An overall increase in backbone amide-15N transverse cross-correlated rates for the cytb5 residues in the cytb5-cytP450 complex was observed as compared to the free cytb5 residues. Due to fast spin-spin relaxation (T2) and subsequent broadening of the signals in the complex, we were able to measure amide-15N CSAs only for 48 residues of cytb5 as compared to 84 residues of free cytb5. We observed a change in 15N CSA for most residues of cytb5 in the complex, when compared to free cytb5, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb5 and cationic cytP450. The mean values of 15N CSA determined for residues in helical, sheet and turn regions of cytb5 in the complex are −184.5, −146.8, and −146.2 ppm, respectively, with an overall average value of −165.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb5, which is similar to our previously reported values for the free cytb5. PMID:24107224

  4. Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

    PubMed Central

    Benveniste, I; Lesot, A; Hasenfratz, M P; Durst, F

    1989-01-01

    Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals. Images Fig. 5. PMID:2499315

  5. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  6. The carboxyl-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of tung tree (Vernicia fordii) tail-anchored cytochrome b5 (Cb5) target specifically to endoplasmic reti...

  7. Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Le Clair, Stéphanie V.; Huang, Rui; Ahuja, Shivani; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-02-01

    Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.

  8. The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells*

    PubMed Central

    Plitzko, Birte; Ott, Gudrun; Reichmann, Debora; Henderson, Colin J.; Wolf, C. Roland; Mendel, Ralf; Bittner, Florian; Clement, Bernd; Havemeyer, Antje

    2013-01-01

    The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system. PMID:23703616

  9. Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

    PubMed

    Duggal, Ruchia; Liu, Yilin; Gregory, Michael C; Denisov, Ilia G; Kincaid, James R; Sligar, Stephen G

    2016-08-19

    Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to

  10. Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production.

    PubMed

    Peng, Hwei-Ming; Im, Sang-Choul; Pearl, Naw May; Turcu, Adina F; Rege, Juilee; Waskell, Lucy; Auchus, Richard J

    2016-08-01

    Human cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (∼50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products. PMID:27426448

  11. Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.

    PubMed

    Hatakeyama, Mayumi; Kitaoka, Takuya; Ichinose, Hirofumi

    2016-07-01

    Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways. PMID:27233123

  12. Structure of Physarum polycephalum cytochrome b{sub 5} reductase at 1.56 Å resolution

    SciTech Connect

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-04-01

    The structure of P. polycephalum cytochrome b{sub 5} reductase, an enzyme which catalyzes the reduction of cytochrome b{sub 5} by NADH, was determined at a resolution of 1.56 Å. Physarum polycephalum cytochrome b{sub 5} reductase catalyzes the reduction of cytochrome b{sub 5} by NADH. The structure of P. polycephalum cytochrome b{sub 5} reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b{sub 5} reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

  13. The Desulfuromonas acetoxidans Triheme Cytochrome c7 Produced in Desulfovibrio desulfuricans Retains Its Metal Reductase Activity

    PubMed Central

    Aubert, Corinne; Lojou, Elisabeth; Bianco, Pierre; Rousset, Marc; Durand, Marie-Claire; Bruschi, Mireille; Dolla, Alain

    1998-01-01

    Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7. PMID:9546165

  14. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  15. Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

    PubMed

    Jeřábek, Petr; Florián, Jan; Martínek, Václav

    2016-04-18

    Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex. PMID:26918755

  16. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions. PMID:27238232

  17. Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

    PubMed

    Hedison, Tobias M; Hay, Sam; Scrutton, Nigel S

    2015-11-01

    Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins. PMID:26307151

  18. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  19. Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

    PubMed Central

    Huang, Wei-Cheng; Ellis, Jacqueline; Moody, Peter C.E.; Raven, Emma L.; Roberts, Gordon C.K.

    2013-01-01

    Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. PMID:23911089

  20. Quantum Chemical Calculations of Amide-15N Chemical Shift Anisotropy Tensors for a Membrane-Bound Cytochrome b5

    PubMed Central

    Pandey, Manoj Kumar; Ramamoorthy, Ayyalusamy

    2013-01-01

    There is considerable interest in determining amide-15N chemical shift anisotropy (CSA) tensors from biomolecules and understanding their variation for structural and dynamics studies using solution and solid-state NMR spectroscopy and also by quantum chemical calculations. Due to the difficulties associated with the measurement of CSA tensors from membrane proteins, NMR-based structural studies heavily relied on the CSA tensors determined from model systems, typically single crystals of model peptides. In the present study, the principal components of backbone amide-15N CSA tensor have been determined using density functional theory for a 16.7-kDa membrane-bound paramagnetic heme containing protein, cytochrome b5 (cytb5). All the calculations were performed by taking residues within 5Å distance from the backbone amide-15N nucleus of interest. The calculated amide-15N CSA spans agree less well with our solution NMR data determined for an effective internuclear distance rN-H = 1.023 Å and a constant angle β = 18° that the least shielded component (δ11) makes with the N-H bond. The variation of amide-15N CSA span obtained using quantum chemical calculations is found to be smaller than that obtained from solution NMR measurements, whereas the trends of the variations are found to be in close agreement. We believe that the results reported in this study will be useful in studying the structure and dynamics of membrane proteins and heme-containing proteins, and also membrane-bound protein-protein complexes such as cytochromes-b5-P450. PMID:23268659

  1. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

    NASA Astrophysics Data System (ADS)

    Bredt, David S.; Hwang, Paul M.; Glatt, Charles E.; Lowenstein, Charles; Reed, Randall R.; Snyder, Solomon H.

    1991-06-01

    Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.

  2. Distinct roles of cytochrome P450 reductase in mitomycin c redox cycling and cytotoxicity

    PubMed Central

    Wang, Yun; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-01-01

    Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, CHO cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in cytotoxicity of this chemotherapeutic agent. PMID:20501808

  3. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence for interactions across the dimeric interface

    PubMed Central

    Judd, Evan T.; Youngblut, Matthew; Pacheco, A. Andrew; Elliott, Sean J.

    2013-01-01

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer. PMID:23210513

  4. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONANZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-Cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14a-demethylase. esistance is restored through complementation by the plasmid-born...

  5. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  6. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. PMID:20561902

  7. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  8. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy

    PubMed Central

    Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-01-01

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy. PMID:26439985

  9. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

    PubMed

    Shao, Fang-Yuan; Du, Zhi-Yun; Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-Dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-10-13

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy. PMID:26439985

  10. The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

    PubMed Central

    Vaz, W L; Austin, R H; Vogel, H

    1979-01-01

    A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer. PMID:262426

  11. Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes

    PubMed Central

    Zhang, Haifeng; Gao, Na; Liu, Tingting; Fang, Yan; Qi, Bing; Wen, Qiang; Zhou, Jun; Jia, Linjing; Qiao, Hailing

    2015-01-01

    Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities. PMID:26046844

  12. Structures of complexes of octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens with sulfite and cyanide.

    PubMed

    Trofimov, Anton A; Polyakov, Konstantin M; Boyko, Konstantin M; Tikhonova, Tamara V; Safonova, Tatyana N; Tikhonov, Alexey V; Popov, Alexandre N; Popov, Vladimir O

    2010-10-01

    The structures of complexes of octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens (TvNiR) with the substrate sulfite (1.4 Å resolution; R(cryst) = 0.126) and the inhibitor cyanide (1.55 Å resolution; R(cryst) = 0.148) have been established. The complex with sulfite was prepared by the reduction of the protein crystal with sodium dithionite. The sulfite ion is bound to the iron ion of the catalytic haem through the S atom. The Fe-S distance is 2.24 Å. The structure of the cyanide complex with full occupancy of the ligand site was established for the first time for cytochrome c nitrite reductases. The cyanide ion is bound to the catalytic haem iron through the C atom. The Fe-C distance is 1.91 Å and the Fe-C-N angle is 171°. The sulfite reductase activity of TvNiR was measured at different pH values. The activity is 0.02 µmol of HS(-) per minute per milligram at pH 7.0; it decreases with increasing pH and is absent at pH 9.0. PMID:20944237

  13. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Mancinelli, R. L.; Cronin, S.; Hochstein, L. I.

    1986-01-01

    Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

  14. Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

    PubMed Central

    Aigrain, Louise; Pompon, Denis; Moréra, Solange; Truan, Gilles

    2009-01-01

    Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo- and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 Å resolution crystal structure of a functionally competent yeast–human chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 Å. PMID:19483672

  15. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. PMID:19444989

  16. Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase.

    PubMed

    Mothersole, Robert G; Meints, Carla E; Louder, Alex; Wolthers, Kirsten R

    2016-09-15

    Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp(634), Ala(635), Arg(636) and Asn(637); human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp(679)) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c(3+) turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp(677) and slower release of NADP(+). In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c(3+) reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg(636) influences catalysis is discussed. PMID:27461959

  17. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed

    Fujiwara, T; Fukumori, Y

    1996-04-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  18. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed Central

    Fujiwara, T; Fukumori, Y

    1996-01-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  19. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. PMID:117798

  20. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed Central

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-01-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. Images Fig. 3. PMID:117798

  1. Iron stimulation of chemiluminescence by microsomes and purified NADPH-cytochrome P-450 reductase

    SciTech Connect

    Puntarulo, S.; Clejan, L.; Palakodety, R.; Cederbaum, A.I.

    1987-05-01

    Low level chemiluminescence (CL) was measured as an assay of the steady state level of production of oxygen radicals during microsomal electron transfer. In the presence of an NADPH-generating system, antioxidant-sensitive CL was observed with isolated rat liver microsomes. Depending on the nature of the chelating agent, ferric iron markedly affected this CL. For example, ferric-EDTA inhibited, whereas ferric-ADP stimulated CL. This response to iron chelators was identical to that found when measuring microsomal lipid peroxidation, but was opposite to the catalytic effectiveness of ferric-chelates in stimulating microsomal generation of hydroxyl radicals. Similar studies were conducted with purified NADPH-cytochrome P-450 reductase in the presence of t-butyl hydroperoxide (t-BOOH). No CL was observed in the absence of added iron. The addition of ferric-EDTA or ferric-detapac stimulated production of CL, whereas ferric chloride or ferric-ATP has little or no effect. This pattern of response to iron chelates is opposite to that found with the microsomes. CL was inhibited by catalase and OH scavengers such as ethanol and DMSO but not by superoxide dismutase. Thus, CL by the reductase system appears to involve the generation of OH via a Fenton-type of reaction, and subsequent interaction of OH with t-BOOH to produce excited species.

  2. A third subunit in ancestral cytochrome c-dependent nitric oxide reductases.

    PubMed

    Bricio, C; Alvarez, L; San Martin, M; Schurig-Briccio, L A; Gennis, R B; Berenguer, J

    2014-08-01

    Reduction of NO to N2O by denitrifiying bacteria is catalyzed either by a monomeric quinol-nitric oxide reductase (qNor) or by a heterodimeric cytochrome c-dependent nitric oxide reductase (cNor). In ancient thermophilic bacteria belonging to the Thermales and Aquificales phylogenetic groups, the cluster encoding the cNor includes a small third gene (norH), in addition to those encoding homologues to the subunits of a typical cNor (norC and norB). We show in Thermus thermophilus that the three genes are cotranscribed in a single mRNA from an inducible promoter. The isolation of individual nor mutants and the production in vivo of His-tagged NorH protein followed by immobilized-metal affinity chromatography (IMAC) allowed us to conclude that NorH constitutes a third subunit of the cNor from T. thermophilus, which is involved in denitrification in vivo, likely allowing more efficient electron transport to cNor. PMID:24907324

  3. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval)

    PubMed Central

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it’s much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

  4. Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries

    PubMed Central

    Peng, Hwei-Ming; Auchus, Richard J.

    2014-01-01

    Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23–42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type > E48G/E49G > D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4. PMID:24256945

  5. Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries.

    PubMed

    Peng, Hwei-Ming; Auchus, Richard J

    2014-01-01

    Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl]>50mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type>E48G/E49G>D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4. PMID:24256945

  6. Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c*

    PubMed Central

    Huang, Rui; Zhang, Meng; Rwere, Freeborn; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-01-01

    Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis. PMID:25512382

  7. The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase

    SciTech Connect

    Liu, M.-C.; Peck, H.D., Jr.

    1981-12-01

    Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal M/sub r/=66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced..cap alpha..-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase,however, is able to transfer electrons from H/sub 2/ to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially reoxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57/sup o/C. Purified nitrite reductase also has hydroxylamine reductase activity, and the K/sub m/ for nitrite was determined to be 1.14 mM.

  8. Regulation of cytochrome b5 gene transcription by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells.

    PubMed

    Huang, Ningwu; Dardis, Andrea; Miller, Walter L

    2005-08-01

    Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances. PMID:15831526

  9. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOEpatents

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  10. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene. PMID:25504221

  11. Potential Biological Functions of Cytochrome P450 Reductase-dependent Enzymes in Small Intestine

    PubMed Central

    D'Agostino, Jaime; Ding, Xinxin; Zhang, Peng; Jia, Kunzhi; Fang, Cheng; Zhu, Yi; Spink, David C.; Zhang, Qing-Yu

    2012-01-01

    NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine. PMID:22453923

  12. Excess NO Predisposes Mitochondrial Succinate-Cytochrome c Reductase to Produce Hydroxyl Radical†

    PubMed Central

    Chen, Jingfeng; Chen, Chwen-Lih; Alevriadou, B. Rita; Zweier, Jay L.; Chen, Yeong-Renn

    2011-01-01

    Mitochondria–derived oxygen free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O2•− generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/•OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H2O2 dependent Fenton reaction or O2•−–dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77 °K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce •OH. In H9c2 cells, SCR dependent oxygen free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR mediated oxygen free radical production was also detected in cultured vascular endothelial cells. PMID:21406178

  13. Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays

    PubMed Central

    Silveira, Célia M.; Besson, Stéphane; Moura, Isabel; Moura, José J. G.; Almeida, M. Gabriela

    2010-01-01

    The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force. PMID:20689707

  14. Structure of octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens in a complex with phosphate

    SciTech Connect

    Trofimov, A. A.; Polyakov, K. M.; Boiko, K. M.; Filimonenkov, A. A.; Dorovatovskii, P. V.; Tikhonova, T. V.; Popov, V. O.; Koval'chuk, M. V.

    2010-01-15

    Octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR) catalyzes the reduction of nitrite and hydroxylamine to ammonia. The structures of the free enzyme and of the enzyme in complexes with the substrate (nitrite ion) and the inhibitor (azide ion) have been solved previously. In this study we report the structures of the oxidized complex of TvNiR with phosphate and of this complex reduced by europium(II) chloride (1.8- and 2.0-A resolution, the R factors are 15.9 and 16.7%, respectively) and the structure of the enzyme in the complex with cyanide (1.76-A resolution, the R factor is 16.5%), which was prepared by soaking a crystal of the oxidized phosphate complex of TvNiR. In the active site of the enzyme, the phosphate ion binds to the iron ion of the catalytic heme and to the side chains of the catalytic residues Arg131, Tyr303, and His361. The cyanide ion is coordinated to the heme-iron ion and is hydrogen bonded to the residue His361. In the structure of reduced TvNiR, the phosphate ion is bound in the same manner as in the structure of oxidized TvNiR, and the nine{sub c}oordinated europium ion is located on the surface of one of the crystallographically independent monomers of the enzyme.

  15. Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes.

    PubMed

    Olbrich, Matthias; Weigl, Kevin; Kahler, Elke; Mihara, Katsuhiro

    2016-10-01

    1. The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated. 2. Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC. 3. The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors. 4. Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19. 5. A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity. 6. Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man. 7. In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation. PMID:26796435

  16. Identification of ubiquinol cytochrome c reductase hinge (UQCRH) as a potential diagnostic biomarker for lung adenocarcinoma

    PubMed Central

    Gao, Feng; Liu, Qicai; Li, Guoping; Dong, Feng; Qiu, Minglian; Lv, Xiaoting; Zhang, Sheng; Guo, Zheng

    2016-01-01

    Ubiquinol cytochrome c reductase hinge (UQCRH) is a novel protein that localizes in the mitochondrial membrane and induces mitochondrial reactive oxygen species (ROS) generation. It had a high expression rate of 87.10% (108/124) in lung adenocarcinoma. Moreover, serum UQCRH level in patients with lung adenocarcinoma was significantly increased compared with that of pneumonia patients (p < 0.0001) and normal control subjects (p < 0.0001). Receiver operating characteristic curve analysis using an optimal cut-off value of 162.65 pg ml−1 revealed sensitivity and specificity for the diagnosis of lung adenocarcinoma of 88.7% and 85.7%, respectively, with an area under the curve of 0.927 (95% CI: 0.892 to 0.962, p < 0.0001). Serum UQCRH discriminates lung adenocarcinoma patients from the population without cancer with considerable sensitivity and specificity, but it does not distinguish between heavy smokers and lung adenocarcinoma patients. Serum UQCRH could be a potential diagnostic biomarker for lung adenocarcinoma. PMID:27358292

  17. A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane.

    PubMed

    Zhang, Zhiqiang; Xia, Siqing; Leonard, Didier; Jaffrezic-Renault, Nicole; Zhang, Jiao; Bessueille, François; Goepfert, Yves; Wang, Xuejiang; Chen, Ling; Zhu, Zhiliang; Zhao, Jianfu; Almeida, M Gabriela; Silveira, Célia M

    2009-02-15

    A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples. PMID:18804367

  18. Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor).

    PubMed

    McQuarters, Ashley B; Wirgau, Nathaniel E; Lehnert, Nicolai

    2014-04-01

    Denitrifying bacteria and fungi efficiently detoxify the toxic metabolite nitric oxide (NO) through reduction to nitrous oxide (N2O) using nitric oxide reductase (NOR) enzymes. In fungi, for example Fusarium oxysporum, NO is reduced by a Cytochrome P450 NOR (P450nor). This enzyme contains a heme b center coordinated to a proximal cysteinate ligand in the active site. In the proposed mechanism of P450nor, the ferric heme binds NO first to form a ferric heme-nitrosyl complex, which is subsequently reduced by NAD(P)H to generate a ferrous HNO species as the next key intermediate. Recently, key progress has been made in our understanding of the electronic structures and fundamental reactivity of these important intermediates, using suitable model complexes. In this review, model complexes of ferric heme-nitrosyls with varied axial anionic ligands (such as N-donors, O-donors, and S-donors) are discussed first. Then, the generation and reactivity of ferrous heme-HNO complexes is summarized and related back to the mechanism of P450nor. PMID:24658055

  19. Controlling the formation of a monolayer of cytochrome P450 reductase onto Au surfaces

    NASA Astrophysics Data System (ADS)

    Convery, J. H.; Smith, C. I.; Khara, B.; Scrutton, N. S.; Harrison, P.; Farrell, T.; Martin, D. S.; Weightman, P.

    2012-07-01

    The conditions necessary for the formation of a monolayer and a bilayer of a mutated form (P499C) of human cytochrome P450 reductase on a Au(110)/electrolyte interface have been determined using a quartz crystal microbalance with dissipation, atomic force microscopy, and reflection anisotropy spectroscopy (RAS). The molecules adsorb through a Au-S linkage and, for the monolayer, adopt an ordered structure on the Au(110) substrate in which the optical axes of the dipoles contributing to the RAS signal are aligned roughly along the optical axes of the Au(110) substrate. Differences between the absorption spectrum of the molecules in a solution and the RAS profile of the adsorbed monolayer are attributed to surface order in the orientation of dipoles that contribute in the low energy region of the spectrum, a roughly vertical orientation on the surface of the long axes of the isoalloxazine rings and the lack of any preferred orientation in the molecular structure of the dipoles in the aromatic amino acids. Our studies establish an important proof of principle for immobilizing large biological macromolecules to gold surfaces. This opens up detailed studies of the dynamics of biological macromolecules by RAS, which have general applications in studies of biological redox chemistry that are coupled to protein dynamics.

  20. A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization.

    PubMed

    Besson, S; Carneiro, C; Moura, J J; Moura, I; Fauque, G

    1995-08-01

    Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments. PMID:16887530

  1. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  2. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

  3. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450. PMID:26045289

  4. Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome cd1 Biosynthesis in Pseudomonas stutzeri

    PubMed Central

    Heikkilä, Mari P.; Honisch, Ulrike; Wunsch, Patrick; Zumft, Walter G.

    2001-01-01

    By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N2O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification. PMID:11160097

  5. Molecular population genetics of the NADPH cytochrome P450 reductase (CPR) gene in Anopheles minimus.

    PubMed

    Srivastava, Hemlata; Huong, Ngo Thi; Arunyawat, Uraiwan; Das, Aparup

    2014-08-01

    Development of insecticide resistance (IR) in mosquito vectors is a primary huddle to malaria control program. Since IR has genetic basis, and genes constantly evolve with response to environment for adaptation to organisms, it is important to know evolutionary pattern of genes conferring IR in malaria vectors. The mosquito Anopheles minimus is a major malaria vector of the Southeast (SE) Asia and India and is susceptible to all insecticides, and thus of interest to know if natural selection has shaped variations in the gene conferring IR. If not, the DNA fragment of such a gene could be used to infer population structure and demography of this species of malaria vector. We have therefore sequenced a ~569 bp DNA segment of the NADPH cytochrome P450 reductase (CPR) gene (widely known to confer IR) in 123 individuals of An. minimus collected in 10 different locations (eight Indian, one Thai and one Vietnamese). Two Indian population samples were completely mono-morphic in the CPR gene. In general, low genetic diversity was found with no evidence of natural selection in this gene. The data were therefore analyzed to infer population structure and demography of this species. The 10 populations could be genetically differentiated into four different groups; the samples from Thailand and Vietnam contained high nucleotide diversity. All the 10 populations conform to demographic equilibrium model with signature of past population expansion in four populations. The results in general indicate that the An. minimus mosquitoes sampled in the two SE Asian localities contain several genetic characteristics of being parts of the ancestral population. PMID:25038863

  6. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. PMID:27243445

  7. The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

    PubMed Central

    Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

    2012-01-01

    Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

  8. Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus.

    PubMed

    Ranghino, G; Scorza, E; Sjögren, T; Williams, P A; Ricci, M; Hajdu, J

    2000-09-12

    The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fülöp, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fülöp, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fülöp, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis. PMID:10998232

  9. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b/sub 5/ reductase

    SciTech Connect

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-06-01

    A cDNA coding for human liver NADH-cytochrome b/sub 5/ reductase was cloned from a human liver cDNA library constructed in phage lambdagt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b/sub 5/ reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb/sub 5/R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb/sub 5/R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  10. A Missense Mutation in the Human Cytochrome b5 Gene causes 46,XY Disorder of Sex Development due to True Isolated 17,20 Lyase Deficiency

    PubMed Central

    Idkowiak, Jan; Randell, Tabitha; Dhir, Vivek; Patel, Pushpa; Shackleton, Cedric H. L.; Taylor, Norman F.; Krone, Nils

    2012-01-01

    Context: Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). Objective: We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. Design: We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. Results: All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. Conclusion: We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations. PMID:22170710

  11. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  12. Molecular dynamics simulations give insight into the conformational change, complex formation, and electron transfer pathway for cytochrome P450 reductase

    PubMed Central

    Sündermann, Axel; Oostenbrink, Chris

    2013-01-01

    Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies. PMID:23832577

  13. Prognostic significance of ubiquinol-cytochrome c reductase hinge protein expression in patients with clear cell renal cell carcinoma

    PubMed Central

    Liu, Wei-Si; Liu, Yi-Dong; Fu, Qiang; Zhang, Wei-Juan; Xu, Le; Chang, Yuan; Xu, Jie-Jie

    2016-01-01

    Ubiquinol-cytochrome c reductase hinge protein (UQCRH), as a connecter between cytochrome c1 with cytochrome c in complex III of respiratory chain, is top-ranked hypermethylated gene in clear cell renal cell carcinoma (ccRCC). This study aims to evaluate the impact of UQCRH on recurrence and survival of 424 ccRCC patients enrolled retrospectively from a single institution after surgical resection using immunohistochemistry method. UQCRH was specifically downregulated in ccRCC, compared with papillary and chromophobe RCC. Moreover, patients with low UQCRH were prone to possess high T stage and TNM stage and associated with poor survival and early recurrence. UQCRH remained an independent favorable prognosticator for OS (Hazard rate [HR]: 0.510, 95% CI: 0.328-0.795, p=0.003) and RFS (HR: 0.506, 95% CI: 0.334-0.767, p=0.001) adjusting with other well-established factors using backward Cox model. Furthermore, in stratified subgroups, patients with low UQCRH had an increased risk of recurrence (HR: 0.452, 95% CI: 0.261-0.783, p=0.005) and mortality (HR: 0.386, 95% CI: 0.205-0.726, p=0.003) in subgroup of early TNM stage. Taken together, UQCRH is a potential independent favorable prognostic factor for recurrence and survival of patients with ccRCC after nephrectomy. PMID:27186431

  14. A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 1,1-diphenyl-2-picrylhydrazyl.

    PubMed

    Yim, Sung-Kun; Yun, Su-Jung; Yun, Chul-Ho

    2004-09-30

    NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring A(520) reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was 4.09mM(-1) cm(-1). DPPH reduction followed classical Michaelis-Menten kinetics (K(m) = 28 microM, k(cat) = 1690 min(-1)). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive. PMID:15479629

  15. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes

    PubMed Central

    Silveira, Célia M.; Quintas, Pedro O.; Moura, Isabel; Moura, José J. G.; Hildebrandt, Peter; Almeida, M. Gabriela; Todorovic, Smilja

    2015-01-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1. PMID:26091174

  16. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

    PubMed

    Silveira, Célia M; Quintas, Pedro O; Moura, Isabel; Moura, José J G; Hildebrandt, Peter; Almeida, M Gabriela; Todorovic, Smilja

    2015-01-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1. PMID:26091174

  17. Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

    SciTech Connect

    Stiborova, Marie Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva

    2008-02-01

    Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by {sup 32}P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

  18. Proton-Detected 2D Radio Frequency Driven Recoupling Solid-state NMR Studies on Micelle-associated Cytochrome-b5

    PubMed Central

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Yamamoto, Kazutoshi; Im, Sangchoul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-01-01

    Solid-state NMR spectroscopy is increasingly used in the high-resolution structural studies of membrane-associated proteins and peptides. Most such studies necessitate isotopically labeled (13C, 15N and 2H) proteins/peptides, which is a limiting factor for some of the exciting membrane-bound proteins and aggregating peptides. In this study, we report the use of a proton-based slow magic angle spinning (MAS) solid-state NMR experiment that exploits the unaveraged 1H-1H dipolar couplings from a membrane-bound protein. We have shown that the difference in the buildup rates of cross peak intensities against the mixing time - obtained from 2D 1H-1H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) experiments on a 16.7-kDa micelle-associated full-length rabbit cytochrome-b5 (cytb5) - can provide insights into protein dynamics and could be useful to measure 1H-1H dipolar couplings. The experimental buildup curves compare well with theoretical simulations and are used to extract relaxation parameters. Our results show that due to fast exchange of amide protons with water in the soluble heme-containing domain of cyb5, coherent 1H-1H dipolar interactions are averaged out for these protons while alpha and side chain protons show residual dipolar couplings that can be obtained from 1H-1H RFDR experiments. The appearance of resonances with distinct chemical shift values in 1H-1H RFDR spectra enabled the identification of residues (mostly from the transmembrane region) of cytb5 that interact with micelles. PMID:24657390

  19. Proton-detected 2D radio frequency driven recoupling solid-state NMR studies on micelle-associated cytochrome-b5

    NASA Astrophysics Data System (ADS)

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Yamamoto, Kazutoshi; Im, Sangchoul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-05-01

    Solid-state NMR spectroscopy is increasingly used in the high-resolution structural studies of membrane-associated proteins and peptides. Most such studies necessitate isotopically labeled (13C, 15N and 2H) proteins/peptides, which is a limiting factor for some of the exciting membrane-bound proteins and aggregating peptides. In this study, we report the use of a proton-based slow magic angle spinning (MAS) solid-state NMR experiment that exploits the unaveraged 1H-1H dipolar couplings from a membrane-bound protein. We have shown that the difference in the buildup rates of cross-peak intensities against the mixing time - obtained from 2D 1H-1H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) experiments on a 16.7-kDa micelle-associated full-length rabbit cytochrome-b5 (cytb5) - can provide insights into protein dynamics and could be useful to measure 1H-1H dipolar couplings. The experimental buildup curves compare well with theoretical simulations and are used to extract relaxation parameters. Our results show that due to fast exchange of amide protons with water in the soluble heme-containing domain of cyb5, coherent 1H-1H dipolar interactions are averaged out for these protons while alpha and side chain protons show residual dipolar couplings that can be obtained from 1H-1H RFDR experiments. The appearance of resonances with distinct chemical shift values in 1H-1H RFDR spectra enabled the identification of residues (mostly from the transmembrane region) of cytb5 that interact with micelles.

  20. NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

    PubMed Central

    Rana, Satiander; Lattoo, Surrinder K.; Dhar, Niha; Razdan, Sumeer; Bhat, Wajid Waheed; Dhar, Rekha S.; Vishwakarma, Ram

    2013-01-01

    Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis

  1. Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.

    PubMed

    Arnesano, F; Banci, L; Bertini, I; Koulougliotis, D

    1998-12-01

    One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (

  2. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    PubMed

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-01

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking. PMID:7703255

  3. Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069.

    PubMed Central

    Patterson, A. V.; Saunders, M. P.; Chinje, E. C.; Talbot, D. C.; Harris, A. L.; Strafford, I. J.

    1997-01-01

    P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069. Images Figure 1 PMID:9374381

  4. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  5. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  6. RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

    PubMed Central

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F.; Potter, Michael F.; Palli, Subba R.

    2012-01-01

    Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs. PMID:22347424

  7. Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

    PubMed

    Pedroso, Humberto A; Silveira, Célia M; Almeida, Rui M; Almeida, Ana; Besson, Stéphane; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

    2016-09-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552. PMID:27133504

  8. The role of cytochromes p450 and aldo-keto reductases in prognosis of breast carcinoma patients.

    PubMed

    Hlaváč, Viktor; Brynychová, Veronika; Václavíková, Radka; Ehrlichová, Marie; Vrána, David; Pecha, Václav; Trnková, Markéta; Kodet, Roman; Mrhalová, Marcela; Kubáčková, Kateřina; Gatěk, Jiří; Vážan, Petr; Souček, Pavel

    2014-12-01

    Metabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and survival of breast carcinoma patients. Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting. AKR1A1 was significantly overexpressed and AKR1C1-4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro. Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in

  9. Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system.

    PubMed

    Hlavica, P; Golly, I; Wolf, J

    1987-09-01

    N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2. PMID:3113486

  10. NADPH-cytochrome P-450 reductase, cytochrome P-450 2C11 and P-450 1A1, and the aryl hydrocarbon receptor in livers of rats fed methyl-folate-deficient diets.

    PubMed

    Zhang, J; Henning, S M; Heber, D; Choi, J; Wang, Y; Swendseid, M E; Go, V L

    1997-01-01

    We investigated three hepatic cytochrome P-450 isozymes and the aryl hydrocarbon (Ah) receptor in rats fed one of the following three diets for 15 months: a diet containing the AIN vitamin mixture (control), the control diet devoid of choline and folate (CFD), or the CFD diet devoid of niacin (CFND). Hepatic tumors developed in all CFD- and CFND-fed rats. Western blot analyses of nontumor hepatic tissue showed that NADPH-cytochrome P-450 reductase (P-450 reductase) increased significantly in the CFD and CFND groups compared with the control group. Hepatic cytochrome P-450 2C11 (CYP2C11) was not detectable in the CFD and CFND groups compared with the control group. Ah receptor and cytochrome P-450 1A1 (CYP1A1) were detected in higher amounts in livers of both deficient groups. CYP1A1 is an enzyme associated with bioactivation of exogenous genotoxins. To our knowledge, this is the first time it has been shown that CYP1A1 and the Ah receptor are induced by dietary deficiencies. PMID:9290122

  11. Improved free energy profile for reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR).

    PubMed

    Blomberg, Margareta R A; Siegbahn, Per E M

    2016-07-15

    Quantum chemical calculations play an essential role in the elucidation of reaction mechanisms for redox-active metalloenzymes. For example, the cleavage and the formation of covalent bonds can usually not be described only on the basis of experimental information, but can be followed by the calculations. Conversely, there are properties, like reduction potentials, which cannot be accurately calculated. Therefore, computational and experimental data has to be carefully combined to obtain reliable descriptions of entire catalytic cycles involving electron and proton uptake from donors outside the enzyme. Such a procedure is illustrated here, for the reduction of nitric oxide (NO) to nitrous oxide and water in the membrane enzyme, cytochrome c dependent nitric oxide reductase (cNOR). A surprising experimental observation is that this reaction is nonelectrogenic, which means that no energy is conserved. On the basis of hybrid density functional calculations a free energy profile for the entire catalytic cycle is obtained, which agrees much better with experimental information on the active site reduction potentials than previous ones. Most importantly the energy profile shows that the reduction steps are endergonic and that the entire process is rate-limited by high proton uptake barriers during the reduction steps. This result implies that, if the reaction were electrogenic, it would become too slow when the gradient is present across the membrane. This explains why this enzyme does not conserve any of the free energy released. © 2016 Wiley Periodicals, Inc. PMID:27130561

  12. Effects of various compounds on lipid peroxidation mediated by detergent-solubilized rat liver NADPH-cytochrome C reductase.

    PubMed

    Kamataki, T; Sugita, O; Naminohira, S; Kitagawa, H

    1978-12-01

    A reconstituted lipid peroxidation system containing NADPH-cytochrome c reductase isolated from detergent-solubilized rat liver microsomes was used to determine the effects of several compounds, including drugs, on the lipid peroxidation activity. EDTA and ferrous ion were essential requirements for reconstitution of the activity. The addition of 1,10-phenanthroline to the system containing both EDTA and ferrous ion further enhanced the activity. Pyrocatecol, thymol, p-aminophenol, imipramine, p-chloromercuribenzoate (PCMB) and alpha-tocopherol exhibited strong inhibition, aniline, N-monomethylaniline, aminopyrine, benzphetamine, SKF 525-A and NADP exhibited moderate inhibition, and phenol, benzoic acid, acetanilide and nicotinamide exhibited less or no inhibition at the concentrations lower than 1000 micron M. Metal ions such as Hg+, Hg2+, Co2+, Cu2+, Mn2+ and U6+ inhibited lipid peroxidation strongly. In addition, Cd2+, St2+ and Ca2+ exhibited less potent to moderate inhibition, and Ba2+ and Mg2+ were without effects on the activity. Among sulfhydryl compounds tested, dithiothreitol inhibited lipid peroxidation to a greater extent than did the other three compounds, glutathione, cysteine and mercaptoethanol. PMID:106178

  13. Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures.

    PubMed

    Schwarz, H; Liu, B; Peters, S; Barillas, W; Beerhues, L

    2009-05-01

    Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation. PMID:19470102

  14. The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

    PubMed

    Ascenzi, Paolo; Sbardella, Diego; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo

    2016-06-01

    Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc. PMID:27010463

  15. FMN binding site of yeast NADPH-cytochrome P450 reductase exposed at the surface is highly specific

    PubMed Central

    Ivanov, Alexis S.; Gnedenko, Oksana V.; Molnar, Andrey A.; Archakov, Alexander I.; Podust, Larissa M.

    2010-01-01

    NADPH-cytochrome P450 reductase (CPR) transfers two reducing equivalents derived from NADPH via FAD and FMN to microsomal P450 monooxygenases in one-electron transfer steps. The crystal structure of yeast CPR (yCPR) contains a surface-exposed FMN binding site (FMN2 site) at the interface of the FMN binding and connecting domains, in addition to the single buried site that has been observed in rat CPR. This finding provides a testable hypothesis of how intramolecular (between FAD and FMN) and intermolecular (between FMN and P450) electron transfer may occur in CPR. To verify that occupancy of the FMN2 site is not an artifact of crystallization, a surface plasmon resonance (SPR) biosensor technique has been applied to probe the selectivity of this site under functional conditions. A series of kinetic and equilibrium binding experiments involving yCPR immobilized on different sensor chip surfaces was performed using FMN and FAD, as well as FMN-derived compounds, including riboflavin, dimethylalloxazine, and alloxazine, and other molecules which resemble the planar isoalloxazine ring structure. Only FMN and FAD showed stoichiometric binding responses. Binding affinity for FMN was in the submicromolar range, thirty times higher than that for FAD. Association kinetic rates for the yCPR/FMN complex were up to 60-fold higher than for the yCPR/FAD complex. Taken together, these data indicate that (i) the surface-exposed site in yCPR is highly selective toward binding flavins, (ii) binding of FMN in this site is notably favored, and finally, (iii) both the phosphate group and the isoalloxazine ring of FMN are essential for binding. PMID:20557022

  16. Six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase: insights from density functional theory studies.

    PubMed

    Bykov, Dmytro; Neese, Frank

    2015-10-01

    In this Forum Article, an extensive discussion of the mechanism of six-electron, seven-proton nitrite reduction by the cytochrome c nitrite reductase enzyme is presented. On the basis of previous studies, the entire mechanism is summarized and a unified picture of the most plausible sequence of elementary steps is presented. According to this scheme, the mechanism can be divided into five functional stages. The first phase of the reaction consists of substrate binding and N-O bond cleavage. Here His277 plays a crucial role as a proton donor. In this step, the N-O bond is cleaved heterolytically through double protonation of the substrate. The second phase of the mechanism consists of two proton-coupled electron-transfer events, leading to an HNO intermediate. The third phase involves the formation of hydroxylamine, where Arg114 provides the necessary proton for the reaction. The second N-O bond is cleaved in the fourth phase of the mechanism, again triggered by proton transfer from His277. The Tyr218 side chain governs the fifth and last phase of the mechanism. It consists of radical transfer and ammonia formation. Thus, this mechanism implies that all conserved active-site side chains work in a concerted way in order to achieve this complex chemical transformation from nitrite to ammonia. The Forum Article also provides a detailed discussion of the density functional theory based cluster model approach to bioinorganic reactivity. A variety of questions are considered: the resting state of enzyme and substrate binding modes, interaction with the metal site and with active-site side chains, electron- and proton-transfer events, substrate dissociation, etc. PMID:26237518

  17. Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase.

    PubMed Central

    Knight, Kirsty; Scrutton, Nigel S

    2002-01-01

    The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+). The second and third steps represent further reduction of the enzyme flavins and NADP(+) release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP(+) release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+) is modestly slower in calmodulin-depleted enzyme. Our studies indicate the

  18. Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system.

    PubMed

    Fujii, H; Kakinuma, K

    1991-11-12

    Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils. PMID:1659905

  19. Conformational change induced by electron transfer in a monolayer of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface

    NASA Astrophysics Data System (ADS)

    Weightman, P.; Smith, C. I.; Convery, J. H.; Harrison, P.; Khara, B.; Scrutton, N. S.

    2013-09-01

    The reflection anisotropy spectroscopy profiles of a variant of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface depend on the sequence of potentials applied to the Au(110) electrode. It is suggested that this dependence arises from changes in the orientation of the isoalloxazine ring structures in the protein with respect to the Au(110) surface. This offers a method of monitoring conformational change in this protein by measuring variations in the reflection anisotropy spectrum arising from changes in the redox potential.

  20. Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies.

    PubMed

    Dell'acqua, Simone; Pauleta, Sofia R; Monzani, Enrico; Pereira, Alice S; Casella, Luigi; Moura, José J G; Moura, Isabel

    2008-10-14

    The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center. PMID:18803407

  1. The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

    PubMed

    Stiborová, Marie; Černá, Věra; Moserová, Michaela; Mrízová, Iveta; Arlt, Volker M; Frei, Eva

    2015-01-01

    Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models. PMID:25547492

  2. Accommodating a Non-Conservative Internal Mutation by Water-Mediated Hydrogen-Bonding Between β-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5

    PubMed Central

    Parthasarathy, Sudharsan; Altuve, Adriana; Terzyan, Simon; Zhang, Xuejun; Kuczera, Krzysztof; Rivera, Mario; Benson, David R.

    2015-01-01

    Mammalian type B (mitochondrial) cytochromes b5 exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-stranded β-sheet, attributable to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen-bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of water access to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic and redox properties. The results provide new insight into the factors stabilizing the cytochrome b5 fold. PMID:21574570

  3. Elastic rotation of Escherichia coli F{sub O}F{sub 1} having ε subunit fused with cytochrome b{sub 562} or flavodoxin reductase

    SciTech Connect

    Oka, Hideyuki; Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi; Dunn, Stanley D.; Futai, Masamitsu; Iwamoto-Kihara, Atsuko

    2014-04-18

    Highlights: • Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase was observed using a small bead probe. • Carboxyl-terminus of the ε subunit was fused to cytochrome b{sub 562} or flavodoxin reductase. • The F{sub O}F{sub 1} showed continual rotation with similar rate to the wild-type enzyme. • The intra-molecular rotation is flexible and elastic. - Abstract: Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of F{sub O}F{sub 1} with the ε subunit connected to a globular protein [cytochrome b{sub 562} (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.

  4. Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd1 nitrite reductase: regulation of the NO-bound end product.

    PubMed

    Lopes, H; Besson, S; Moura, I; Moura, J J

    2001-01-01

    The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation. PMID:11191223

  5. Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

    PubMed

    Cheng, Xingguo; Zhang, Youcai; Klaassen, Curtis D

    2014-10-01

    NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice. PMID:25034404

  6. Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH-cytochrome P450 reductase.

    PubMed Central

    Zhukov, A; Ingelman-Sundberg, M

    1999-01-01

    Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 microM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in approximately 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule. PMID:10333489

  7. Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone.

    PubMed

    Rwere, Freeborn; Xia, Chuanwu; Im, Sangchoul; Haque, Mohammad M; Stuehr, Dennis J; Waskell, Lucy; Kim, Jung-Ja P

    2016-07-01

    NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochromes P450 via its FAD and FMN. To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air stability of the neutral blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The ΔGly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral blue semiquinone and hydroquinone. The ΔGly-141 and ΔG141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c In the ΔGly-141 mutants, the backbone amide of Glu/Asn-142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of ΔG141/E142N was slightly more stable than that of ΔGly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ΔGly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (ΔGly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function. PMID:27189945

  8. Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

    NASA Astrophysics Data System (ADS)

    Smith, C. I.; Convery, J. H.; Harrison, P.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2014-08-01

    Changes observed in the reflection anisotropy spectroscopy (RAS) profiles of monolayers of cytochrome P450 reductase adsorbed at Au(110)-electrolyte interfaces at 0.056 V following the addition of nicotinamide adenine dinucleotide phosphate (NADP+) are explained in terms of a simple model as arising from changes in the orientation of an isoalloxazine ring located in the flavin mononucleotide binding domain of the protein. The model also accounts for the changes observed in the RAS as the potential applied to the Au(110) surface is varied and suggests that differences in the dependence of the RAS profile of the adsorbed protein on the potential applied to the electrode in the absence and presence of NADP+ are explicable as arising from a competition between the applied potential acting to reduce the protein and the NADP+ to oxidize it.

  9. Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784.

    PubMed

    Nodate, Miho; Kubota, Mitsutoshi; Misawa, Norihiko

    2006-07-01

    Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450. PMID:16195793

  10. Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

    SciTech Connect

    Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.; Nidetzky, Bernd

    2005-01-11

    Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.

  11. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins. PMID:8552660

  12. Chlorate reductase is cotranscribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans.

    PubMed

    Hellberg Lindqvist, Miriam; Nilsson, Thomas; Sundin, Pontus; Rova, Maria

    2015-03-01

    The chlorate-respiring bacterium Ideonella dechloratans is a facultative anaerobe that can use both oxygen and chlorate as terminal electron acceptors. The genes for the enzymes chlorate reductase (clrABDC) and chlorite dismutase, necessary for chlorate metabolism and probably acquired by lateral gene transfer, are located in a gene cluster that also includes other genes potentially important for chlorate metabolism. Among those are a gene for cytochrome c (cyc) whose gene product may serve as an electron carrier during chlorate reduction, a cofactor biosynthesis gene (mobB) and a predicted transcriptional regulator (arsR). Only chlorate reductase and chlorite dismutase have been shown to be expressed in vivo. Here, we report the in vivo production of a single polycistronic transcript covering eight open reading frames including clrABDC, cyc, mobB and arsR. Transcription levels of the cyc and clrA genes were compared to each other by the use of qRT-PCR in RNA preparations from cells grown under aerobic or chlorate reducing anaerobic conditions. The two genes showed the same mRNA levels under both growth regimes, indicating that no transcription termination occurs between them. Higher transcription levels were observed at growth without external oxygen supply. Implications for electron pathway integration following lateral gene transfer are discussed. PMID:25673284

  13. Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous

    PubMed Central

    Alcaíno, Jennifer; Barahona, Salvador; Carmona, Marisela; Lozano, Carla; Marcoleta, Andrés; Niklitschek, Mauricio; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor

    2008-01-01

    Background The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) → geranyleranyl pyrophosphate (GGPP) → phytoene → lycopene → β-carotene → astaxanthin. Recently, it has been published that the conversion of β-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family. Results In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating β-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its β-carotene content is increased. Conclusion In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of β-carotene to astaxanthin. PMID:18837978

  14. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  15. Study of the Individual Cytochrome b[subscript 5] and Cytochrome b[subscript 5] Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

    SciTech Connect

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R.; Battaile, Kevin P.; Lovell, Scott; Benson, David R.; Zhu, Hao

    2010-09-22

    NADH cytochrome b{sub 5} oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b{sub 5} (b{sub 5}), CHORD-SGT1 (CS), and cytochrome b{sub 5} reductase (b{sub 5}R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b{sub 5} and b{sub 5}R domains (Ncb5or-b{sub 5} and Ncb5or-b{sub 5}R, respectively) and compared them with human microsomal b{sub 5} (Cyb5A) and b{sub 5}R (Cyb5R3). A 1.25 {angstrom} x-ray crystal structure of Ncb5or-b{sub 5} reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His{sup 89} and His{sup 112}, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b{sub 5} family shown to have such a heme environment. Like other b{sub 5} family members, Ncb5or-b{sub 5} has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b{sub 5} differs from Cyb5A with respect to location of the second heme ligand (His{sup 112}) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b{sub 5}R to Ncb5or-b{sub 5} is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b{sub 5} and b{sub 5}R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b{sub 5}R domains suggest that the CS domain facilitates docking of the b{sub 5} and b{sub 5}R domains. Trp{sup 114} is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b{sub 5}R domain to the b{sub 5} domain.

  16. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

    PubMed Central

    Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang

    2015-01-01

    Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that

  17. Molecular Cloning, Expression Pattern and Polymorphisms of NADPH-Cytochrome P450 Reductase in the Bird Cherry-Oat Aphid Rhopalosiphum padi (L.).

    PubMed

    Wang, Kang; Peng, Xiong; Zuo, Yayun; Li, Yuting; Chen, Maohua

    2016-01-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, Rhopalosiphum padi, is unavailable. In the current study, a full-length cDNA (2,476 bp) of CPR (RpCPR) encoding 681 amino acids was cloned from R. padi. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, Acyrthosiphon pisum. The mRNA of RpCPR was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. RpCPR expression could also be induced by low concentrations (LC30) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of RpCPR from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that RpCPR may play an important role in the P450-mediated insecticide resistance of R. padi to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, RpCPRmaintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters. PMID:27124302

  18. Molecular Cloning, Expression Pattern and Polymorphisms of NADPH-Cytochrome P450 Reductase in the Bird Cherry-Oat Aphid Rhopalosiphum padi (L.)

    PubMed Central

    Zuo, Yayun; Li, Yuting

    2016-01-01

    NADPH–cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, Rhopalosiphum padi, is unavailable. In the current study, a full-length cDNA (2,476 bp) of CPR (RpCPR) encoding 681 amino acids was cloned from R. padi. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, Acyrthosiphon pisum. The mRNA of RpCPR was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. RpCPR expression could also be induced by low concentrations (LC30) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of RpCPR from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that RpCPR may play an important role in the P450-mediated insecticide resistance of R. padi to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, RpCPRmaintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters. PMID:27124302

  19. Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

    SciTech Connect

    Pandey, Amit V.; Flueck, Christa E.; Mullis, Primus E.

    2010-09-24

    Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

  20. Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase.

    PubMed

    Price, Claire L; Warrilow, Andrew G S; Parker, Josie E; Mullins, Jonathan G L; Nes, W David; Kelly, Diane E; Kelly, Steven L

    2015-05-15

    Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide. PMID:25746994

  1. The influence of the structure of the Au(110) surface on the ordering of a monolayer of cytochrome P450 reductase at the Au(110)/phosphate buffer interface

    PubMed Central

    Smith, C. I.; Convery, J. H.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2016-01-01

    The reflection anisotropy spectra (RAS) observed initially from Au(110)/phosphate buffer interfaces at applied potentials of −0.652 and 0.056 V are very similar to the spectra observed from ordered Au(110) (1 × 3) and anion induced (1 × 1) surface structures respectively. These RAS profiles transform to a common profile after cycling the potential between these two values over 72 h indicating the formation of a less ordered surface. The RAS of a monolayer of a P499C variant of the human flavoprotein cytochrome P450 reductase adsorbed at 0.056 V at an ordered Au(110)/phosphate buffer interface is shown to arise from an ordered layer in which the optical dipole transitions are in a plane that is orientated roughly normal to the surface and parallel to either the [11̄0] or [001] axes of the Au(110) surface. The same result was found previously for adsorption of P499C on an ordered interface at −0.652 V. The adsorption of P499C at the disordered surface does not result in the formation of an ordered monolayer confirming that the molecular ordering is strongly influenced by both the local structure and the long range macroscopic order of the Au(110) surface.

  2. Quantifying immunohistochemical staining of phospho-eIF2alpha, heme oxygenase-2 and NADPH cytochrome P450 reductase in oligodendrocytes during experimental autoimmune encephalomyelitis.

    PubMed

    Chakrabarty, Anuradha; Fleming, Kandace K; Marquis, Janet G; LeVine, Steven M

    2005-06-15

    As a consequence of inflammation associated with multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), stress responses are induced in many cells within the CNS, however, those that occur within the primary pathological target, the oligodendrocyte, are not fully established. Recently, we found that phosphorylated eukaryotic initiation factor-2alpha (eIF2alpha), an inhibitor of protein translation associated with the stress response, is expressed in a greater number of oligodendrocytes in EAE animals compared to controls. However, since numerous oligodendrocytes in control animals also expressed phospho-eIF2alpha, a method was developed to detect expression levels within oligodendrocytes that did not rely on the number of oligodendrocytes that were stained. This method utilized a high dilution of the primary antibody so that the staining density was kept below a maximum plateau which could eliminate expression differences. Furthermore, the staining density within oligodendrocytes, as determined by image analysis, was corrected by the background density or that within neurons. In either case, the density of staining was greater in oligodendrocytes from EAE animals versus controls. The expression of heme oxygenase-2 and NADPH cytochrome P450 reductase also were examined, but unlike phospho-eIF2alpha, neither was increased in oligodendrocytes from EAE animals compared to controls. In summary, a protocol involving a high dilution of primary antibody and image analysis revealed that the expression of phospho-eIF2alpha within oligodendrocytes was increased in EAE animals compared to control animals. PMID:15910982

  3. Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

    PubMed Central

    Price, Claire L.; Warrilow, Andrew G. S.; Parker, Josie E.; Mullins, Jonathan G. L.; Nes, W. David

    2015-01-01

    Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide. PMID:25746994

  4. NCB5OR Is a Novel Soluble NAD(P)H Reductase Localized in the Endoplasmic Reticulum*S

    PubMed Central

    Zhu, Hao; Larade, Kevin; Jackson, Timothy A.; Xie, Jianxin; Ladoux, Annie; Acker, Helmut; Berchner-Pfannschmidt, Utta; Fandrey, Joachim; Cross, Andrew R.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bunn, H. Franklin

    2011-01-01

    The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and cytochrome b5 reductase domains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = ~0.05 and ~1 s−1, respectively. The redox potential at the heme center of NCB5OR is −108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse. PMID:15131110

  5. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  6. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae).

    PubMed

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  7. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. PMID:26454083

  8. Analysis of the complex formation, interaction and electron transfer pathway between the "open" conformation of NADPH-cytochrome P450 reductase and aromatase.

    PubMed

    Dai, Yuejie; Zhen, Jing; Zhang, Xiuli; Zhong, Yonghui; Liu, Shaodan; Sun, Ziyue; Guo, Yue; Wu, Qingli

    2015-09-01

    The complex structure of human aromatase (CYP19) and the open form of ΔTGEE mutant NADPH-cytochrome P450 reductase (mCPR) was constructed using template-based protein alignment method. Dynamic simulation of formed complex was performed on NAMD 2.9, in which CHARMm all 27_prot_lipid_na force field and an explicit TIP3P water solvent model were applied. The result showed mCPR in its open conformation could steadily combine with aromatase from the proximal face. Data analysis indicates hydrogen bonds and four salt bridges on the binding surface enhance the interaction between the two protein molecules. Amino acid, Lys108 plays a key role in aromatase activity through the formation of a salt bridge with Asp147 and two hydrogen bonds with Asp147 and Gln150 in mCPR. The optimal pathway for the first electron transfer from CPR to aromatase was revealed and calculated using HARLEM software. The rates for solvent mediated and non-solvent mediated electron transfer from FMNH2 to heme were determined as 1.04×10(6)s(-)(1) and 4.86×10(5)s(-)(1) respectively, which indicates the solvent water can facilitate the electron transfer from FMNH2 to heme. This study presents a novel strategy for the study of the protein-protein interactions based on the template-based protein alignment, which may help new aromtase development targeting the electron transfer between mCPR and aromatase. PMID:26087061

  9. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    PubMed

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants. PMID:11513247

  10. Genetic variation in aldo-keto reductase 1D1 (AKR1D1) affects the expression and activity of multiple cytochrome P450s.

    PubMed

    Chaudhry, Amarjit S; Thirumaran, Ranjit K; Yasuda, Kazuto; Yang, Xia; Fan, Yiping; Strom, Stephen C; Schuetz, Erin G

    2013-08-01

    Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network. PMID:23704699

  11. Exploring the mechanistic aspects of mitomycin antibiotic bioactivation in Chinese hamster ovary cells overexpressing NADPH:cytochrome C (P-450) reductase and DT-diaphorase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1998-01-01

    We have directly demonstrated the involvement of human NADPH: cytochrome c (P-450) reductase in the aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin in living cells by varying only this enzyme in a transfected cell line. In the same manner, we have implicated rat DT-diaphorase in the aerobic and hypoxic activation of mitomycin C, but found only a minor role for this enzyme in the aerobic activation of porfiromycin. DT-Diaphorase does not cause the production of an aerobic/hypoxic differential toxicity by mitomycin C, but rather activates this agent through an oxygen insensitive pathway. The evidence suggests that DT-diaphorase activates mitomycin C more effectively than porfiromycin, with porfiromycin being preferentially activated through a one-electron reductive pathway. The therapeutic potential of mitomycin antibiotics in the treatment of cancer can be envisioned to be enhanced for those tumors containing elevated levels of the bioreductive enzymes. However, cytogenetic heterogeneity within the tumor cell population and the various environmental factors which impact on bioreductive enzyme function, including pH and oxygen tension, may subvert this approach. Moreover, if high tumor levels of a drug activating enzyme reflect high levels in the normal tissues of the patient, normal tissue damage may also be enhanced with possibly no improvement in the therapeutic ratio. Approaches utilizing gene therapy, whereby a specific bioreductive catalyst is introduced into the tumor cell population via a targeting vehicle to activate a particular prodrug, may be more effective in that not only will the prodrug of choice be specifically activated in the tumor, but the source of the catalyst, be it bacterial, rodent, or human, will not be important. In fact, in the case of DT-diaphorase and mitomycin C, the rat form of the enzyme could be advantageous because it is more effective in activating mitomycin C than is the human form of this enzyme. Assuming

  12. A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca(2+) /CAM-binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Qin, Zhiwei; Yang, Kejun; Chen, Chao; Yu, Qingyue; Zhu, Yanming

    2016-06-01

    Although research has extensively illustrated the molecular basis of plant responses to salt and high-pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca(2+) /CAM-dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N-terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII-containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N-terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress. PMID:27121031

  13. Experimental approaches to evaluate activities of cytochromes P450 3A

    PubMed Central

    Bořek-Dohalská, Lucie; Hodek, Petr; Hudeček, Jiří; Stiborová, Marie

    2008-01-01

    Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro. PMID:21218106

  14. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  15. A novel point mutation in a 3{prime} splice site of the NADH-cytochrome b{sub 5} reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia

    SciTech Connect

    Shirabe, Komie; Takeshita, Masazumi; Landi, M.T.

    1995-08-01

    Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b{sub 5} reductase (b{sub 5}R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G{r_arrow}T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b{sub 5}R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b{sub 5}R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of generalized type. 37 refs., 5 figs., 2 tabs.

  16. Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system.

    PubMed

    Kuznetsov, Vadim Yu; Ivanov, Yuri D; Archakov, Alexander I

    2004-08-01

    The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm. PMID:15274134

  17. Pantothenic acid (Vitamin B5)

    MedlinePlus

    Pantothenic acid is a vitamin, also known as vitamin B5. It is widely found in both plants and animals ... Vitamin B5 is commercially available as D-pantothenic acid, as well as dexpanthenol and calcium pantothenate, which ...

  18. Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.

    PubMed

    Ichinose, Hirofumi; Michizoe, Junji; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

    2004-06-22

    Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media. PMID:15986701

  19. The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.

    PubMed

    Nawathean, P; Maslov, D A

    2000-08-01

    By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs. PMID:10975258

  20. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected]. PMID:26300313

  1. Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

    PubMed Central

    Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; Layer, Gunhild

    2013-01-01

    The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS. PMID:23683062

  2. The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

    PubMed Central

    Zeldin, D C; Plitman, J D; Kobayashi, J; Miller, R F; Snapper, J R; Falck, J R; Szarek, J L; Philpot, R M; Capdevila, J H

    1995-01-01

    Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. Images PMID:7738183

  3. Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

    PubMed

    Akingbasote, James A; Foster, Alison J; Wilson, Ian; Sarda, Sunil; Jones, Huw B; Kenna, J Gerry

    2016-04-01

    Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment. PMID:25820915

  4. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

    PubMed

    Crawford, N M; Smith, M; Bellissimo, D; Davis, R W

    1988-07-01

    The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase. PMID:3393528

  5. Iron(II)/reductant(DH2)-induced activation of dioxygen for the hydroxylation and ketonization of hydrocarbons; mimics for the cytochrome P-450 hydroxylase/reductase system.

    PubMed

    Sawyer, D T; Liu, X; Redman, C; Chong, B

    1994-12-01

    Several metal complexes [(FeII(DPAH)2 (DPAH2 = 2,6-dicarboxyl pyridine), FeII(PA)2 (PAH = picolinic acid), FeII(bpy)2(2+), FeII(OPPh3)4(2+), (Cl8TPP)FeIIIX (X = Cl, OH, SCH2Ph) [Cl8TPP = tetrakis (2,6-dichlorophenyl)porphyrin], (TPP) FeIIICl (TPP = tetraphenylporphyrin), and CuI(tpy)2+ (typ = 2,2'-6,2"-terpyridine)] in combination with one of several reductants [DH2; PhNHNHPh (mimic of dihydroflavin), PhNHNH2, ascorbic acid (H2asc), and PhCH2SH (model ligand for cysteine residue)] catalytically activate O2 (1 atm) for the hydroxylation of saturated hydrocarbons (e.g. c-C6H12-->c-C6H11OH). This chemistry closely parallels that of cytochrome P-450 proteins, and both appear to involve a Fenton-like reactive intermediate), [LxFeOOH(DH)]. With cyclohexane as the substrate the dominant product is its ketone (as well as significant amounts of its hydroperoxide). 1,4-Cyclohexadiene (with two double-allylic carbon centers) undergoes dehydrogenation to give benzene, but also yields substantial amounts of phenol via ketonization of an allylic carbon. The 1:1 FeII(bpy)2(2+)/(PhNHNH2 or H2asc), FeII(PA)2/H2asc, and (Cl8TPP)FeIIICl/PhNHNH2 combinations initiate the autoxidation of 1,4-cyclohexadiene with turnover numbers (moles of product per mole of reductant) from 71 to 26, respectively (alpha-tocophenol reduces the turnover numbers by 20-80%). With respect to aerobic biology, the present results indicate that dysfunctional transition metals (degradation products of metalloproteins) in combination with biological reductants activate O2 for reaction with organic substrates. The level of activation is similar to that for Fenton reagents and cytochrome P-450 hydroxylases. Hence, dysfunctional transition metals, reductants, and O2 are a hazardous combination within a biological matrix. PMID:7788301

  6. Cloning and enhanced expression of the cytochrome P450nor gene (nicA; CYP55A5) encoding nitric oxide reductase from Aspergillus oryzae.

    PubMed

    Kaya, Masahiko; Matsumura, Kengo; Higashida, Katsuya; Hata, Yoji; Kawato, Akitsugu; Abe, Yasuhisa; Akita, Osamu; Takaya, Naoki; Shoun, Hirofumi

    2004-10-01

    We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml. PMID:15502348

  7. Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

    PubMed

    Cartron, Michaël L; Roldán, M Dolores; Ferguson, Stuart J; Berks, Ben C; Richardson, David J

    2002-12-01

    NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the

  8. Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

    PubMed Central

    Cartron, Michaël L; Roldán, M Dolores; Ferguson, Stuart J; Berks, Ben C; Richardson, David J

    2002-01-01

    NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the

  9. Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

    SciTech Connect

    Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, Jim K.; Bigelow, Diana J.; Squier, Thomas C.

    2011-10-14

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent

  10. Targeted protein degradation of outer membrane decaheme cytochrome MtrC metal reductase in Shewanella oneidensis MR-1 measured using biarsenical probe CrAsH-EDT(2).

    PubMed

    Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, James K; Bigelow, Diana J; Squier, Thomas C

    2011-11-15

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic Gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC (MtrC*) which is dependent on the presence of a functional type 2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC* undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 h(-1) that is insensitive to O(2) concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 h(-1)) that are

  11. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

  12. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase

    SciTech Connect

    Cummings, J.; Bartoszek, A.; Smyth, J.F. )

    1991-04-01

    An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When (14C)doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA, and the highest levels were recorded with oligonucleotides. The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM (14C)doxorubicin. Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.

  13. Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

    PubMed

    Stringer, Rowan A; Ferreira, Suzie; Rose, Jonathan; Ronseaux, Sebastien

    2016-08-01

    The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor. PMID:27271368

  14. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  15. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  16. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    PubMed

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes. PMID:17915953

  17. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

  18. Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves 1

    PubMed Central

    Aslam, Muhammad; Oaks, Ann

    1976-01-01

    A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH2-nitrate reductase and NO3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO3− was supplied. WO4= suppressed the induction of NADH- and FMNH2-nitrate reductase activities in root tips and leaves. The NO3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO4=. In leaves, on the other hand, there was a marked superinduction of the NO3−-induced NADH-cytochrome c reductase in the presence of WO4=. The half-life values of NADH-nitrate reductase and FMNH2-nitrate reductase measured by removing NO3− and adding WO4= to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO3− in the induction medium together with WO4= gave partial protection of NADH-nitrate reductase and FMNH2-nitrate reductase activities in both root tips and leaves with a t0.5 of 6 and 8 hours, respectively. NO3− also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO3−-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO3− protected the loss of NO3−-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID:16659529

  19. 7 CFR 15b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Assurances required. 15b.5 Section 15b.5 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 15b.5 Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit...

  20. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  1. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  2. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  3. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 4 2012-01-01 2012-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where...

  4. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 4 2014-01-01 2014-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where...

  5. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 4 2013-01-01 2013-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where...

  6. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where the agency...

  7. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Voting. 242b.5 Section 242b.5 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS GENERAL PROCEDURES AND DELEGATIONS OF THE BOARD OF REGENTS OF THE UNIFORMED SERVICES UNIVERSITY OF THE HEALTH SCIENCES § 242b.5 Voting. (a) The...

  8. Cytochrome f

    SciTech Connect

    Soriano, G.M.; Smith, J.L.; Cramer, W.A.

    2001-07-17

    Cytochrome f (f, folium, leaf), a c-type cytochrome with a characteristic CysXXCysHis amino acid sequence for heme ligation, is the largest of the four major protein subunits of the membrane-embedded cytochrome b{sub 6}{sup f} complex of oxygenic photosynthesis. It contains 285-86 amino acids, consisting of a soluble 250-residue domain on the p-side (positive-side) or lumen-side of the membrane, a single trans-membrane 20-residue {alpha}-helix, and an n- or stromal-side segment consisting of 15 residues. These domains contain, respectively, the heme prosthetic group and intraprotein electron transfer pathway, the membrane anchor and a short segment that is important in the assembly of the b{sub 6}{sup f} complex. The function of the cytochrome f in oxygenic photosynthesis is to act as the terminal electron acceptor in the membrane-embedded cytochrome b{sub 6}{sup f} complex that provides the electron transport connection between the photosystem II and photosystem I reaction centers. Electron transfer through the complex is coupled to proton translocation and generation of a proton electrochemical potential that is utilized to drive the synthesis of ATP through the proton-motive ATP synthase. These functions of the cytochrome b{sub 6}{sup f} complex are analogous to those of the multisubunit cytochrome bc{sub 1} complex (ubiquinol:cytochrome c oxidoreductase) of the mitochondrial respiratory chain and photosynthetic bacteria. Both complexes contain four redox centers with very similar redox and structural properties: a covalently bound c-type heme in cytochrome f or c{sub 1}, the 2Fe-2S cluster of the Rieske ISP, and the two noncovalently bound hemes of cytochrome b. The structure properties have been defined in 3.0-3.1 {angstrom} structures of the b{sub 6}{sup f} complex from a thermophilic cyanobacterium and a green alga. These structures also defined a fifth redox prosthetic group, a novel covalently bound heme, tentatively called heme x. With the exception of

  9. Reductive metabolism of carbon tetrachloride by human cytochromes P-450 reconstituted in phospholipid vesicles: mass spectral identification of trichloromethyl radical bound to dioleoyl phosphatidylcholine.

    PubMed Central

    Trudell, J R; Bösterling, B; Trevor, A J

    1982-01-01

    It has been proposed that covalent binding of reactive metabolites to liver membrane constituents may be responsible for the hepatoxicity of carbon tetrachloride. This study demonstrates that trichloromethyl free radical is the major reductive metabolite of carbon tetrachloride by cytochrome P-450 and that this free radical is capable of binding to double bonds of fatty acyl chains of the phospholipids in the membrane surrounding cytochrome P-450. The structural identification of the reactive free radical metabolite and the product of its addition to phospholipids was accomplished by use of a reconstituted system of human cytochromes P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5 in phospholipid vesicles. The reconstituted vesicles contained a mixture of dioleoyl phosphatidylcholine and egg phosphatidylethanolamine that served as both structural components and targets for trichloromethyl free radical binding. After incubation of these vesicles under a N2 atmosphere in the presence of NADPH with 14CCl4, the phospholipids were extracted and then separated by high-pressure liquid chromatography. The dioleoyl phosphatidylcholine fraction was transesterified and the resulting single 14C-labeled fatty acid methyl ester was purified by reverse-phase chromatography. Desorption chemical ionization mass spectrometry with ammonia as reagent gas as well as desorption electron-impact mass spectrometry permitted identification of the molecular structure as a mixture of 9- and 10-(trichloromethyl)stearate methyl esters. PMID:6953422

  10. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  11. Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

    PubMed

    Satoh, T; Fujita, K I; Munakata, H; Itoh, S; Nakamura, K; Kamataki, T; Itoh, S; Yoshizawa, I

    2000-11-15

    To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information. PMID:11067738

  12. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  13. 15 CFR 8b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Assurances required. 8b.5 Section 8b.5... Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit an assurance, on a form specified by the Secretary, that the program or activity will...

  14. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Legal guardians. 3b.5... INFORMATION General § 3b.5 Legal guardians. For the purposes of this part, the parent of any minor, or the legal guardian of any individual who has been declared to be incompetent due to physical or...

  15. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Legal guardians. 3b.5... INFORMATION General § 3b.5 Legal guardians. For the purposes of this part, the parent of any minor, or the legal guardian of any individual who has been declared to be incompetent due to physical or...

  16. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Personal notes. 806b.5 Section 806b.5 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE ADMINISTRATION PRIVACY ACT PROGRAM... notes on individuals used as memory aids. Personal notes may become Privacy Act records if they...

  17. Magnetic Circular Dichroism Studies XXV. A Preliminary Investigation of Microsomal Cytochromes*

    PubMed Central

    Dolinger, Peter M.; Kielczewski, Michael; Trudell, James R.; Barth, Günter; Linder, Robert E.; Bunnenberg, Edward; Djerassi, Carl

    1974-01-01

    The application of magnetic circular dichroism as an optical probe for simultaneous identification and determination of at least two microsomal cytochromes is demonstrated. The assignments of the bands in the spectra of microsomal suspensions are made from the spectra of soluble preparations of cytochrome P-450 obtained from Pseudomonas putida and of cytochrome b5 obtained from rat livers. PMID:4521811

  18. Biochemical responses in mussels Perna perna exposed to diesel B5.

    PubMed

    Nogueira, Lílian; Garcia, Danielly; Trevisan, Rafael; Sanches, Ana Letícia Madeira; da Silva Acosta, Daiane; Dafre, Alcir Luiz; Oliveira, Thiago Yukio Kikuchi; de Almeida, Eduardo Alves

    2015-09-01

    In Brazil B5 blend (5% biodiesel and 95% diesel oil) has been adopted as mandatory fuel since 2010 for automotive vehicles. Since little is known about the effects of B5 exposure can promote on antioxidant system of marine biota this study aimed to assess if B5 can generate modifications in antioxidant parameters of mussels Perna perna. To address this question mussels were exposed to two concentrations of B5 (0.01 mL L(-1) and 0.1 mL L(-1)) for 6h, 12h, 48 h and 168 h. Then the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were evaluated in gills and digestive gland as well as the contents of glutathione (GSH) and lipid peroxidation by measuring the malondialdehyde concentration (MDA). In the gills, GST activity decreased after 48 h and GR after 12h of exposure to B5. In digestive glands, the activities of SOD, GPx and GR were changed due to treatments. GSH concentration increased in digestive gland after 6h and 12h and in gills after 48 h for B5 0.1 mL L(-1) and after 168 h in the digestive gland for B5 0.01 mL L(-1) treatment. No lipid peroxidation was detected. The integrated biomarker response index (IBR) evidenced a B5 effect in the digestive gland after 168 h of exposure. Regarding the experimental conditions and species used in this study, long-term exposure to B5 is apparently more likely to affect the parameters tested in P. perna mussels. PMID:25950138

  19. Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

    PubMed Central

    Petersen, M; Seitz, H U

    1988-01-01

    Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase. PMID:3137929

  20. Dynamics of cytochrome P450 inducers in polluted sites of São Paulo city reservoirs.

    PubMed

    Rocha-e-Silva, Thomaz A A; Rantin, Francisco T; Matsumura-Tundisi, Jose E; Matsumura-Tundisi, Takako; Tundisi, Jose G; Degterev, Igor A

    2004-09-01

    The first analysis of water pollutants using biomarkers at the Guarapiranga Reservoir, which supplies water for one-third of the population of the São Paulo megalopolis (Brazil), is reported. Studies were performed before and after the start of water pumping to the Guarapiranga from the highly polluted Billings reservoir. Billings's water was purified by passing through the natural wetland located near Guarapiranga. Liver enzymes of Oreochromis niloticus (tilopias) obtained from both reservoirs served as biomarkers of pollution in a comparison with animals obtained from a reference site. Enhanced levels of total cytochromes P450 (3.4 times) and b5 (2.7 times) and activity of cytochrome c (P450) reductase (2.2 times) were observed in specimens collected near the water influx from the Billings before the pumping started. However, these parameters were significantly decreased 3 months later. This effect is probably due to dilution of pollutants because of the increased level of water in the Guarapiranga. PMID:15261731

  1. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  2. Technical description of Stack 296-B-5

    SciTech Connect

    Ridge, T.M.

    1994-11-15

    Of particular concern to facilities on the Hanford site is Title 40, Code of Federal Regulations, Chapter 40, Part 61, Subpart H, ``National emission Standards for Emissions of Radionuclides Other Than Radon From Department of Energy Facilities.`` Assessments of facility stacks and potential radionuclide emissions determined whether these stacks would be subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. Stack 296-B-5 exhausts 221-BB building which houses tanks containing B Plant steam condensate and B Plant process condensate from the operation of the low-level waste concentrator. The assessment of potential radionuclide emissions from the 296-B-5 stack resulted in an effective dose equivalent to the maximally exposed individual of less than 0.1 millirem per year. Therefore, the stack is not subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. However, the sampling and monitoring system must be in compliance with the Environmental Compliance Manual, WHC-CM-7-5. Currently, 296-B-5 is sampled continuously with a record sampler and continuous air monitor (CAM).

  3. Lycopene protects against atrazine-induced hepatotoxicity through modifications of cytochrome P450 enzyme system in microsomes.

    PubMed

    Xia, Jun; Lin, Jia; Zhu, Shi-Yong; Du, Zheng-Hai; Guo, Jing-Ao; Han, Zi-Xuan; Li, Jin-Long; Zhang, Ying

    2016-04-01

    Atrazine (ATR) is primarily distributed in liver and hazardous to animal health. Cytochrome P450 enzyme system (CYP450s) is responsible for the biotransformation of toxic substances. Lycopene (LYC) prevents the herbicide-induced toxicity. However, it is unclear that LYC protects against ATR-induced hepatotoxicity via modifying CYP450s. To ascertain the chemoprevention of LYC on ATR-induced hepatotoxicity, male Kunming mice were treated with LYC (5mg/kg) and/or ATR (50mg/kg or 200mg/kg) by gavage administration for 21 days. These results showed that ATR induced the increase of total CYP450 and Cytochrome b5 (Cyt b5) contents and stimulated the activities of CYP450s enzymes (erythromycin N-demethylase (ERND), aminopyrin N-demethylase (APND), aniline-4-hydeoxylase (AH) and NADPH-cytochrome c reductase (NCR)) in hepatic microsomes. The mRNA expressions of six CYP450s genes (increase: CYP1a1, CYP2a4, CYP3a57 and decrease: CYP2f2, CYP3a11, CYP4a31) were significantly influenced by ATR. LYC modulated the contents and activities of CYP450s and normalized the expressions of four CYP450s genes (CYP1b1, CYP2a4, CYP2e1, and 4A14). These findings suggested that ATR induced hepatic CYP450s disturbance and influenced the gene expression of CYP450s. Lycopene protected against hepatic CYP450s disturbance induced by ATR via modifying the hepatic CYP450s activities and transcription in mice. PMID:26775023

  4. Expression, purification, physicochemical characterization and structural analysis of cytochrome c554 from Vibrio parahaemolyticus strain RIMD2210633.

    PubMed

    Akazaki, Hideharu; Kawai, Fumihiro; Chida, Hirotaka; Hirano, Takako; Hakamata, Wataru; Park, Sam-Yong; Nishio, Toshiyuki; Oku, Tadatake

    2010-01-01

    The function of cytochrome c(554) of Vibrio parahaemolyticus has not yet been determined. We have determined the physicochemical properties and crystal structure of cytochrome c(554) at 1.8 A in order to help elucidate its function. The physicochemical properties and the tertiary structure of cytochrome c(554) resemble those of dimeric cytochrome c(552) from Pseudomonas nautica, but the Vibrio genus contains no gene for nitrite reductase, cytochrome cd(1), in its genome DNA. These results raise the possibility that both cytochromes denote an electron to an electron carrier and accept an electron from same electron carrier. PMID:20460700

  5. Cytochrome P450 System Proteins Reside in Different Regions of the Endoplasmic Reticulum

    PubMed Central

    Park, Ji Won; Reed, James R.; Brignac-Huber, Lauren M.; Backes, Wayne L.

    2015-01-01

    Cytochrome P450 function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of this study was to determine if the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4, and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient, and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450s in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

  6. Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

    PubMed

    Park, Ji Won; Reed, James R; Brignac-Huber, Lauren M; Backes, Wayne L

    2014-12-01

    Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

  7. Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris

    USGS Publications Warehouse

    Lovley, D.R.; Widman, P.K.; Woodward, J.C.; Phillips, E.J.P.

    1993-01-01

    The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium- contaminated waters and waste streams.

  8. Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

    NASA Technical Reports Server (NTRS)

    Tsapin, A. I.; Vandenberghe, I.; Nealson, K. H.; Scott, J. H.; Meyer, T. E.; Cusanovich, M. A.; Harada, E.; Kaizu, T.; Akutsu, H.; Leys, D.; Van Beeumen, J. J.

    2001-01-01

    Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

  9. Identification of a Small Tetraheme Cytochrome c and a Flavocytochrome c as Two of the Principal Soluble Cytochromes c in Shewanella oneidensis Strain MR1

    PubMed Central

    Tsapin, A. I.; Vandenberghe, I.; Nealson, K. H.; Scott, J. H.; Meyer, T. E.; Cusanovich, M. A.; Harada, E.; Kaizu, T.; Akutsu, H.; Leys, D.; Van Beeumen, J. J.

    2001-01-01

    Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c3 but define a new folding motif for small multiheme cytochromes c. PMID:11425747

  10. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.