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Sample records for osmoregulation heme biosynthesis

  1. Heme biosynthesis and its regulation: towards understanding and improvement of heme biosynthesis in filamentous fungi.

    PubMed

    Franken, Angelique C W; Lokman, B Christien; Ram, Arthur F J; Punt, Peter J; van den Hondel, Cees A M J J; de Weert, Sandra

    2011-08-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally produced in small amounts by basidiomycetes. Filamentous fungi like Aspergillus sp. are considered as suitable hosts for protein production due to their high capacity of protein secretion. For the purpose of peroxidase production, heme is considered a putative limiting factor. However, heme addition is not appropriate in large-scale production processes due to its high hydrophobicity and cost price. The preferred situation in order to overcome the limiting effect of heme would be to increase intracellular heme levels. This requires a thorough insight into the biosynthetic pathway and its regulation. In this review, the heme biosynthetic pathway is discussed with regards to synthesis, regulation, and transport. Although the heme biosynthetic pathway is a highly conserved and tightly regulated pathway, the mode of regulation does not appear to be conserved among eukaryotes. However, common factors like feedback inhibition and regulation by heme, iron, and oxygen appear to be involved in regulation of the heme biosynthesis pathway in most organisms. Therefore, they are the initial targets to be investigated in Aspergillus niger. PMID:21687966

  2. Sampangine inhibits heme biosynthesis in both yeast and human

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the p...

  3. Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

    PubMed

    Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E

    2015-07-14

    Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.

  4. Deconvoluting heme biosynthesis to target blood-stage malaria parasites

    PubMed Central

    Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E

    2015-01-01

    Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. DOI: http://dx.doi.org/10.7554/eLife.09143.001 PMID:26173178

  5. Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

    PubMed

    Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E

    2015-01-01

    Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. PMID:26173178

  6. Structure and function of enzymes in heme biosynthesis.

    PubMed

    Layer, Gunhild; Reichelt, Joachim; Jahn, Dieter; Heinz, Dirk W

    2010-06-01

    Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the "Radical SAM enzyme" coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.

  7. Regulation of heme biosynthesis and transport in metazoa.

    PubMed

    Sun, FengXiu; Cheng, YongJiao; Chen, CaiYong

    2015-08-01

    Heme is an iron-containing tetrapyrrole that plays a critical role in regulating a variety of biological processes including oxygen and electron transport, gas sensing, signal transduction, biological clock, and microRNA processing. Most metazoan cells synthesize heme via a conserved pathway comprised of eight enzyme-catalyzed reactions. Heme can also be acquired from food or extracellular environment. Cellular heme homeostasis is maintained through the coordinated regulation of synthesis, transport, and degradation. This review presents the current knowledge of the synthesis and transport of heme in metazoans and highlights recent advances in the regulation of these pathways.

  8. Impairment of heme biosynthesis induces short circadian period in body temperature rhythms in mice.

    PubMed

    Iwadate, Reiko; Satoh, Yoko; Watanabe, Yukino; Kawai, Hiroshi; Kudo, Naomi; Kawashima, Yoichi; Mashino, Tadahiko; Mitsumoto, Atsushi

    2012-07-01

    It has been demonstrated that the function of mammalian clock gene transcripts is controlled by the binding of heme in vitro. To examine the effects of heme on biological rhythms in vivo, we measured locomotor activity (LA) and core body temperature (T(b)) in a mouse model of porphyria with impaired heme biosynthesis by feeding mice a griseofulvin (GF)-containing diet. Mice fed with a 2.0% GF-containing diet (GF2.0) transiently exhibited phase advance or phase advance-like phenomenon by 1-3 h in terms of the biological rhythms of T(b) or LA, respectively (both, P < 0.05) while mice were kept under conditions of a light/dark cycle (12 h:12 h). We also observed a transient, ~0.3 h shortening of the period of circadian T(b) rhythms in mice kept under conditions of constant darkness (P < 0.01). Interestingly, the observed duration of abnormal circadian rhythms in GF2.0 mice lasted between 1 and 3 wk after the onset of GF ingestion; this finding correlated well with the extent of impairment of heme biosynthesis. When we examined the effects of therapeutic agents for acute porphyria, heme, and hypertonic glucose on the pathological status of GF2.0 mice, it was found that the intraperitoneal administration of heme (10 mg·kg(-1)·day(-1)) or glucose (9 g·kg(-1)·day(-1)) for 7 days partially reversed (50%) increases in urinary δ-aminolevulinic acids levels associated with acute porphyria. Treatment with heme, but not with glucose, suppressed the phase advance (-like phenomenon) in the diurnal rhythms (P < 0.05) and restored the decrease of heme (P < 0.01) in GF2.0 mice. These results suggest that impairments of heme biosynthesis, in particular a decrease in heme, may affect phase and period of circadian rhythms in animals.

  9. Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

    PubMed

    Zhao, Amy H; Tu, Lan N; Mukai, Chinatsu; Sirivelu, Madhu P; Pillai, Viju V; Morohaku, Kanako; Cohen, Roy; Selvaraj, Vimal

    2016-01-22

    Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

  10. A novel pathway for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions and experimental evidence.

    PubMed

    Storbeck, Sonja; Rolfes, Sarah; Raux-Deery, Evelyne; Warren, Martin J; Jahn, Dieter; Layer, Gunhild

    2010-12-13

    Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d(1) biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified.

  11. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli

    PubMed Central

    Zhang, Junli; Kang, Zhen; Chen, Jian; Du, Guocheng

    2015-01-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway. PMID:25716896

  12. Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

    PubMed Central

    Novotna, Jitka; Olsovska, Jana; Novak, Petr; Mojzes, Peter; Chaloupkova, Radka; Kamenik, Zdenek; Spizek, Jaroslav; Kutejova, Eva; Mareckova, Marketa; Tichy, Pavel; Damborsky, Jiri; Janata, Jiri

    2013-01-01

    The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis. PMID:24324587

  13. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    PubMed

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  14. Heme biosynthesis modulation via δ-aminolevulinic acid administration attenuates chronic hypoxia-induced pulmonary hypertension

    PubMed Central

    Alhawaj, Raed; Patel, Dhara; Kelly, Melissa R.; Sun, Dong

    2015-01-01

    This study examines how heme biosynthesis modulation with δ-aminolevulinic acid (ALA) potentially functions to prevent 21-day hypoxia (10% oxygen)-induced pulmonary hypertension in mice and the effects of 24-h organoid culture with bovine pulmonary arteries (BPA) with the hypoxia and pulmonary hypertension mediator endothelin-1 (ET-1), with a focus on changes in superoxide and regulation of micro-RNA 204 (miR204) expression by src kinase phosphorylation of signal transducer and activator of transcription-3 (STAT3). The treatment of mice with ALA attenuated pulmonary hypertension (assessed through echo Doppler flow of the pulmonary valve, and direct measurements of right ventricular systolic pressure and right ventricular hypertrophy), increases in pulmonary arterial superoxide (detected by lucigenin), and decreases in lung miR204 and mitochondrial superoxide dismutase (SOD2) expression. ALA treatment of BPA attenuated ET-1-induced increases in mitochondrial superoxide (detected by MitoSox), STAT3 phosphorylation, and decreases in miR204 and SOD2 expression. Because ALA increases BPA protoporphyrin IX (a stimulator of guanylate cyclase) and cGMP-mediated protein kinase G (PKG) activity, the effects of the PKG activator 8-bromo-cGMP were examined and found to also attenuate the ET-1-induced increase in superoxide. ET-1 increased superoxide production and the detection of protoporphyrin IX fluorescence, suggesting oxidant conditions might impair heme biosynthesis by ferrochelatase. However, chronic hypoxia actually increased ferrochelatase activity in mouse pulmonary arteries. Thus, a reversal of factors increasing mitochondrial superoxide and oxidant effects that potentially influence remodeling signaling related to miR204 expression and perhaps iron availability needed for the biosynthesis of heme by the ferrochelatase reaction could be factors in the beneficial actions of ALA in pulmonary hypertension. PMID:25659899

  15. Overexpression of NUDT7, a candidate quantitative trait locus for pork color, downregulates heme biosynthesis in L6 myoblasts.

    PubMed

    Taniguchi, Masaaki; Hayashi, Takeshi; Nii, Masahiro; Yamaguchi, Tomomi; Fujishima-Kanaya, Naoe; Awata, Takashi; Mikawa, Satoshi

    2010-11-01

    While testing a quantitative trait locus (QTL) for pork color in a cross population of pigs from the mating of Large White dams to a Japanese wild boar, our laboratory discovered a candidate gene (NUDT7) that might affect heme biosynthesis in porcine muscle. Therefore, this experiment was designed to test the effect of NUDT7 on heme biosynthesis in cultured myoblasts. Rat L6 myoblasts were transfected with a mammalian expression vector for pig NUDT7 immediately after the induction of cell differentiation, and samples were harvested at 2, 4, 6, and 8 days. Expression of exogenous NUDT7 mRNA was highest on day 4, when the heme content was substantially lower (P<0.01) than that of the control (14.2 vs. 63.9 pmol/10(5) cells). These results suggest that overexpression of pig NUDT7 may be associated with heme biosynthesis downregulation in skeletal muscle, which may partially explain differences in meat color among breeds of livestock.

  16. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli.

    PubMed

    Louis, P; Galinski, E A

    1997-04-01

    The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4.4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.

  17. The Heme Biosynthesis Pathway Is Essential for Plasmodium falciparum Development in Mosquito Stage but Not in Blood Stages*

    PubMed Central

    Ke, Hangjun; Sigala, Paul A.; Miura, Kazutoyo; Morrisey, Joanne M.; Mather, Michael W.; Crowley, Jan R.; Henderson, Jeffrey P.; Goldberg, Daniel E.; Long, Carole A.; Vaidya, Akhil B.

    2014-01-01

    Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[13C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs. PMID:25352601

  18. Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis

    SciTech Connect

    You,Z.; Omura, S.; Ikeda, H.; Cane, D.; Jogl, G.

    2007-01-01

    The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/{alpha}-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, a-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Angstroms resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and a-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal {alpha}-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.

  19. Crystal Structure of the Non-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis

    PubMed Central

    You, Zheng; Omura, Satoshi; Ikeda, Haruo; Cane, David E.; Jogl, Gerwald

    2010-01-01

    The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/α-ketoglutarate–dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the X-ray crystal structure of PtlH in several complexes with the cofactors iron, α-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Å resolution. The overall structure of PtlH forms a double-stranded barrel helix fold and the cofactor-binding site for iron and α-keto-glutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal α–helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate. PMID:17942405

  20. Cysteine Oxidation Reactions Catalyzed by a Mononuclear Non-heme Iron Enzyme (OvoA) in Ovothiol Biosynthesis

    PubMed Central

    2015-01-01

    OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine. PMID:24684381

  1. Effect of Hydrogen Peroxide on the Biosynthesis of Heme and Proteins: Potential Implications for the Partitioning of Glu-tRNAGlu between These Pathways

    PubMed Central

    Farah, Carolina; Levicán, Gloria; Ibba, Michael; Orellana, Omar

    2014-01-01

    Glutamyl-tRNA (Glu-tRNAGlu) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNAGlu can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNAGlu. Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNAGlu distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions. PMID:25514408

  2. Effect of hydrogen peroxide on the biosynthesis of heme and proteins: potential implications for the partitioning of Glu-tRNA(Glu) between these pathways.

    PubMed

    Farah, Carolina; Levicán, Gloria; Ibba, Michael; Orellana, Omar

    2014-12-11

    Glutamyl-tRNA (Glu-tRNA(Glu)) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNA(Glu) can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNA(Glu). Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNA(Glu) distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.

  3. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

    PubMed

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D; Wurtzel, Eleanore T

    2015-08-01

    Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

  4. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase

    PubMed Central

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P.; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H.; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D.; Wurtzel, Eleanore T.

    2015-01-01

    Plants synthesize carotenoids essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway leading to all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not certain whether isomerization was achieved by Z-ISO alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-to-trans isomerization of the 15–15′ C=C bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the following biosynthetic pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand-switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large, hydrophobic compound in a membrane is unprecedented. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially utilizes a novel chemical mechanism. PMID:26075523

  5. Heme content in developing chloroplasts

    SciTech Connect

    Thomas, J.; Weinstein, J.D. )

    1991-05-01

    Heme regulates tetrapyrrole biosynthesis by inhibition of {delta}-aminolevulinic acid synthesis, product inhibition of heme synthesis, and possibly other mechanisms. Determination of the physiological relevance of this inhibition requires a sensitive measurement which can distinguish regulatory free heme from heme which is an integral part of functional hemoproteins. A preliminary estimate was provided by reconstituting peroxidase activity from apo-peroxidase and the heme contained in broken plastids. However, subsequent experiments have suggested that this initial estimate was too large due to reconstitution of apo-peroxidase with heme from functional hemoproteins (i.e. heme stealing). The authors have now refined the measurement techniques to greatly reduce the extent of this heme stealing. Incubation of broken plastids with apo-peroxidase at 10C resolves the kinetics of reconstitution into two components. A fast component levels off after 100 min, and a slow component increases linearly for up to 6 hours. They believe that the heme which reconstitutes during the fast phase represents free heme, and the linear slow component represents heme stealing. In support of this theory, incubation at 15C increases the rate of both components. However, extrapolation to zero time of the slow components of the 10C and 15C time courses results in equivalent amounts of heme. Based on this kinetic differentiation between free heme and hemoprotein heme, chloroplasts isolated from cucumber cotyledons after 30 h of greening contain substantially greater amounts of free heme than etioplasts.

  6. Expression of genes involved in heme biosynthesis in the human retinoblastoma cell lines WERI-Rb-1 and Y79: implications for photodynamic therapy.

    PubMed

    Ruiz-Galindo, E; Arenas-Huertero, F; Ramón-Gallegos, E

    2007-06-01

    Photodynamic therapy (PDT), using protoporphyrin IX (PpIX) as a natural photosensitizer, may be a viable alternative therapy of retinoblastoma. In order to evaluate the potential value of PpIX, the expression profiles of genes involved in heme biosynthesis in human retinoblastoma WERI-Rb-1 and Y79 cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression levels were highest in protoporphyrinogen oxidase (PPOX), uroporphyrinogen synthase and aminolevulinic acid synthase. Ferrochelatase expression showed a reduction compared to PPOX. PpIX levels were 15- and 18-fold higher in WERI-Rb-1 and Y79 cells, respectively, following induction by delta-aminolevulinic acid. PDT may thus be a promising treatment in vitro, at least in these two retinoblastoma cell lines. PMID:17725098

  7. Effect of lead (Pb) exposure on the activity of superoxide dismutase and catalase in battery manufacturing workers (BMW) of Western Maharashtra (India) with reference to heme biosynthesis.

    PubMed

    Patil, Arun J; Bhagwat, Vinod R; Patil, Jyotsna A; Dongre, Nilima N; Ambekar, Jeevan G; Jailkhani, Rama; Das, Kusal K

    2006-12-01

    The aim of this study was to estimate the activity of superoxide dismutase (SOD) and catalase in erythrocytes and malondialdehyde (MDA) in plasma of battery manufacturing workers (BMW) of Western Maharashtra (India) who were occupationally exposed to lead (Pb) over a long period of time (about 15 years). This study was also aimed to determine the Pb intoxication resulted in a disturbance of heme biosynthesis in BMW group. The blood Pb level of BMW group (n = 28) was found to be in the range of 25.8 - 78.0 microg/dL (mean + SD, 53.63 + 16.98) whereas in Pb unexposed control group (n = 35) the range was 2.8 - 22.0 microg/dL (mean + SD, 12.52 + 4.08). The blood level (Pb-B) and urinary lead level (Pb-U) were significantly increased in BMW group as compared to unexposed control. Though activated d- aminolevulinic acid dehydratase (ALAD) activities in BMW group did not show any significant change when compared to control group but activated / non activated erythrocyte - ALAD activities in BMW group showed a significant increase. Erythrocyte- zinc protoporphyrin (ZPP), urinary daminolevulinic acid (ALA-U) and porphobilinogen (PBG-U) of BMW groups elevated significantly as compared to control. A positive correlation (r = 0.66, p < 0.001) between Pb-B and ALA-U were found in BMW group but no such significant correlation (r = 0.02, p> 1.0) were observed in control group. Hematological study revealed a significant decrease of hemoglobin concentration, packed cell volume (%) and other blood indices and a significant increase of total leucocytes count in BMW group in comparison to control group. The serum MDA content was significantly increased (p < 0.001) and the activities of antioxidant enzymes such as erythrocyte- SOD (p < 0.001) and erythrocytecatalase (p < 0.001) were significantly reduced in BMW group as compared to control group. A positive correlation (r = 0.45, p < 0.02) between Pb-B and serum MDA level was observed in BMW group (Pb-B range 25.8 - 78.0 microg / d

  8. Osmoregulation in marine mammals

    NASA Technical Reports Server (NTRS)

    Ortiz, R. M.

    2001-01-01

    Osmoregulation in marine mammals has been investigated for over a century; however, a review of recent advances in our understanding of water and electrolyte balance and of renal function in marine mammals is warranted. The following topics are discussed: (i) kidney structure and urine concentrating ability, (ii) sources of water, (iii) the effects of feeding, fasting and diving, (iv) the renal responses to infusions of varying salinity and (v) hormonal regulation. The kidneys of pinnipeds and cetaceans are reniculate in structure, unlike those of terrestrial mammals (except bears), but this difference does not confer any greater concentrating ability. Pinnipeds, cetaceans, manatees and sea otters can concentrate their urine above the concentration of sea water, but only pinnipeds and otters have been shown to produce urine concentrations of Na+ and Cl- that are similar to those in sea water. This could afford them the capacity to drink sea water and not lose fresh water. However, with few exceptions, drinking is not a common behavior in pinnipeds and cetaceans. Water balance is maintained in these animals via metabolic and dietary water, while incidental ingestion and dietary salt may help maintain electrolyte homeostasis. Unlike most other aquatic mammals, sea otters commonly drink sea water and manatees frequently drink fresh water. Among the various taxonomic groups of marine mammals, the sensitivity of the renin-angiotensin-aldosterone system appears to be influenced by the availability of Na+. The antidiuretic role of vasopressin remains inconclusive in marine mammals, while the natriuretic function of atrial natriuretic peptide has yet to be examined. Ideas on the direction of future studies are presented.

  9. Heme content and breakdown in developing chloroplasts

    SciTech Connect

    Thomas, J.; Weinstein, J.D. )

    1990-05-01

    Heme regulates tetrapyrrole biosynthesis in plants by inhibition of {delta}-aminolevulinic acid (ALA) synthesis, product inhibition of heme synthesis, and possibly other mechanisms. Plastid heme levels may be modulated by heme synthesis, breakdown and/or efflux. Heme breakdown may be catalyzed by a chloroplast localized heme oxygenase. Chloroplasts isolated from greening cucumber cotyledons were incubated in the presence or absence of various components thought to modulate heme breakdown. Following the incubations, the chloroplasts were broken (freeze-thaw) and then supplemented with horseradish peroxidase apoenzyme. The reconstituted peroxidase activity was used to determine the amount of free heme remaining (Thomas Weinstein (1989) Plant Physiol. 89S: 74). Chloroplasts, freshly isolated from seedlings greened for 16 hours, contained approximately 37 pmol heme/mg protein. When chloroplasts were incubated with 5 mM NADPH for 30 min, the endogenous heme dropped to unmeasurable levels. Exogenous heme was also broken down when NADPH was included in the incubation. Heme levels could be increased by the inclusion of 50 {mu}M ALA and/or p-hydroxymercuribenzoate. The increase due to exogenous ALA was blocked by levulinic acid, an inhibitor of ALA utilization. NADPH-dependent heme breakdown acid was inhibited by p-hydroxymercuribenzoate.

  10. Hepatic Heme Metabolism and Its Control

    PubMed Central

    Bonkowsky, Herbert L.; Sinclair, Peter R.; Sinclair, Jacqueline F.

    1979-01-01

    This review summarizes heme metabolism and focuses especially upon the control of hepatic heme biosynthesis. Activity of δ-aminolevulinic acid synthetase, the first enzyme of heme biosynthesis, is of primary importance in controlling the overall activity of this biosynthetic pathway. Δ-aminolevulinic acid synthetase is subject to inhibition and repression by heme, and numerous basic and clinical studies support the concept that there exists within hepatocytes a “regulatory” heme pool which controls activity of δ-aminolevulinic acid synthetase. In addition, activity of this enzyme is repressed by feeding, especially by ingestion of carbohydrates (the so-called “glucose effect”). Studies pertaining to the mechanisms underlying this effect are also reviewed. The “glucose effect” appears to be mediated by glucose or perhaps by glucose-6-phosphate or uridine diphosphate glucose, rather than by metabolites further removed from glucose itself. Unlike the situation in E. coli, the “glucose effect” in liver of higher organisms is not mediated by alterations in intracellular concentrations of cyclic AMP. Effects of heavy metals, especially iron, on hepatic heme metabolism are also considered. Iron has been found to inhibit formation and utilization of uroporphyrinogen III and to lead to decreased concentrations of microsomal heme and cytochrome P-450. Administration of large amounts of iron is also associated with an increase in activity of heme oxygenase, a property shared by several other metal ions, most notably cobalt. This effect of iron or cobalt administration is similar to the effect of heme administration in increasing heme oxygenase activity; however, we believe it is unlikely that iron, rather than heme itself, is a physiologic regulator of hepatic heme metabolism, although this hypothesis has lately been proposed. PMID:222077

  11. An integrated view on a eukaryotic osmoregulation system.

    PubMed

    Hohmann, Stefan

    2015-08-01

    Osmoregulation encompasses active homeostatic processes that ensure proper cell volume, shape and turgor as well as an intercellular milieu optimal for the diverse biochemical processes. Recent studies demonstrate that yeast cells operate within a tight window of cellular water concentrations that still allows rapid diffusion of biomolecules while already moderate cell compression following hyper-osmotic stress leads to macromolecular crowding and a slow-down of cellular processes. Yeast cells accumulate glycerol as compatible osmolyte under hyper-osmotic stress to regain cell volume and turgor and release glycerol following a hypo-osmotic shock. The high osmolarity glycerol (HOG) response pathway controls glycerol accumulation at various levels, where each mechanism contributes to the temporal and quantitative pattern of volume recovery: inhibition of glycerol efflux, direct activation of the first enzyme in glycerol biosynthesis, stimulation of glycolytic flux as well as upregulation of expression of genes encoding enzymes in glycerol biosynthesis and an active glycerol uptake system. The HOG mitogen-activated protein kinase (MAPK) pathway communicates with the other yeast MAPK pathways to control cell morphogenesis. Cross-talk between the MAPK pathways has recently been used to re-wire osmostress-controlled expression of glycerol biosynthesis genes from Hog1 to Kss1-Fus3. The results of this study further illustrate the key importance of glycerol accumulation under osmostress and allow studying Hog1-dependent and independent processes as well as redundancy and robustness of the MAPK system. PMID:25663258

  12. The role of sulfur in osmoregulation and salinity tolerance in cyanobacteria, algae, and plants

    NASA Technical Reports Server (NTRS)

    Yopp, J. H.

    1985-01-01

    Organosulfur compounds are involved in osmoregulation and salinity tolerance in some cyanobacteria and photosynthetic eukaryotes. Glycinebetaine, the osmolyte of the halotolerant cyanobacterium, Aphanothece halophytica, requires the sulfonium compound. S-adenosyl-methionine (SAM) for its synthesis. Glutamate is the nitrogen source, SAM is the methyl carbon and serine the carbon backbone source of this unique osmolyte. Inhibitor studies suggest that photorespiration interacts with sulfur metabolism to control betaine synthesis in cyanobacteria. The limiting factor for SAM synthesis is formate from photorespiration. SAM is, in turn, the methyl donor for betaine synthesis from serine. The nitrogen component of serine is from glutamate. Betaine synthesis is hypothesized to be regulated via potassium. The biosynthesis of dimethyl-B-propiothetin (DMPT, which is the same as beta-dimethyl sulfonioprpionate) and diacylsulfoquinovosylglycerol were elucidated as having their roles in osmoregulation and salinity tolerance. The relation between these sulfolipids and the sulfur cycle was discussed.

  13. Molecular Phylogeny of Heme Peroxidases

    NASA Astrophysics Data System (ADS)

    Zámocký, Marcel; Obinger, Christian

    All currently available gene sequences of heme peroxidases can be phylogenetically divided in two superfamilies and three families. In this chapter, the phylogenetics and genomic distribution of each group are presented. Within the peroxidase-cyclooxygenase superfamily, the main evolutionary direction developed peroxidatic heme proteins involved in the innate immune defense system and in biosynthesis of (iodinated) hormones. The peroxidase-catalase superfamily is widely spread mainly among bacteria, fungi, and plants, and particularly in Class I led to the evolution of bifunctional catalase-peroxidases. Its numerous fungal representatives of Class II are involved in carbon recycling via lignin degradation, whereas Class III secretory peroxidases from algae and plants are included in various forms of secondary metabolism. The family of di-heme peroxidases are predominantly bacteria-inducible enzymes; however, a few corresponding genes were also detected in archaeal genomes. Four subfamilies of dyp-type peroxidases capable of degradation of various xenobiotics are abundant mainly among bacteria and fungi. Heme-haloperoxidase genes are widely spread among sac and club fungi, but corresponding genes were recently found also among oomycetes. All described families herein represent heme peroxidases of broad diversity in structure and function. Our accumulating knowledge about the evolution of various enzymatic functions and physiological roles can be exploited in future directed evolution approaches for engineering peroxidase genes de novo for various demands.

  14. Osmoregulation in a parasitic nematode, Setaria cervi.

    PubMed

    Singh, K; Sharma, S

    1984-03-01

    Studies on osmotic and ionic regulation in Setaria cervi indicate that it can osmoregulate in hypertonic solutions but not in hypotonic solutions. The depression of the freezing point (delta degrees C) of host's peritoneal fluid was -0.71 degrees C. PMID:6721246

  15. Osmoregulation in a parasitic nematode, Setaria cervi.

    PubMed

    Singh, K; Sharma, S

    1984-03-01

    Studies on osmotic and ionic regulation in Setaria cervi indicate that it can osmoregulate in hypertonic solutions but not in hypotonic solutions. The depression of the freezing point (delta degrees C) of host's peritoneal fluid was -0.71 degrees C.

  16. Heme, iron, and the mitochondrial decay of ageing.

    PubMed

    Atamna, Hani

    2004-07-01

    Heme, the major functional form of iron, is synthesized in the mitochondria. Although disturbed heme metabolism causes mitochondrial decay, oxidative stress, and iron accumulation, all of which are hallmarks of ageing, heme has been little studied in nutritional deficiency, in ageing, or age-related disorders such as Alzheimer's disease (AD). Biosynthesis of heme requires Vitamin B(6), riboflavin, biotin, pantothenic acid, and lipoic acid and the minerals zinc, iron, and copper, micronutrients are essential for the production of succinyl-CoA, the precursor for porphyrins, by the TCA (Krebs) cycle. Only a small fraction of the porphyrins synthesized from succinyl-CoA are converted to heme, the rest are excreted out of the body together with the degradation products of heme (e.g. bilirubin). Therefore, the heme biosynthetic pathway causes a net loss of succinyl-CoA from the TCA cycle. The mitochondrial pool of succinyl-CoA may limit heme biosynthesis in deficiencies for micronutrients (e.g. iron or biotin deficiency). Ageing and AD are also associated with hypometabolism, increase in heme oxygenase-1, loss of complex IV, and iron accumulation. Heme is a common denominator for all these changes, suggesting that heme metabolism maybe altered in age-related disorders. Heme can also be a prooxidant: it converts less reactive oxidants to highly reactive free radicals. Free heme has high affinity for different cell structures (protein, membranes, and DNA), triggering site-directed oxidative damage. This review discusses heme metabolism as related to metabolic changes seen in ageing and age-related disorders and highlights the possible role in iron deficiency.

  17. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    PubMed Central

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  18. Osmoregulation or turgor regulation in chara?

    PubMed

    Bisson, M A; Bartholomew, D

    1984-02-01

    Chara corallina Klein ex Willd. wm. R.D.W. (= C. australis R. Br.), a fresh water alga, maintains a constant internal osmotic pressure when external osmotic pressure is increased. This results in a decrease in turgor pressure. Chara osmoregulates effectively in the presence of high CaCl(2) and raffinose, but is less efficient in response to increased NaCl. Decreasing external pH from 7 to 5 results in a decrease in turgor, but increasing it to values as high as 9 has no effect. Increasing the daily amount of light from 0.5 to 24 hours has no effect on turgor.

  19. Molecular hijacking of siroheme for the synthesis of heme and d1 heme

    PubMed Central

    Bali, Shilpa; Lawrence, Andrew D.; Lobo, Susana A.; Saraiva, Lígia M.; Golding, Bernard T.; Palmer, David J.; Howard, Mark J.; Ferguson, Stuart J.; Warren, Martin J.

    2011-01-01

    Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B12, coenzyme F430, and heme d1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d1 heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined. PMID:21969545

  20. Osmoregulation in anthozoan-dinoflagellate symbiosis.

    PubMed

    Mayfield, Anderson B; Gates, Ruth D

    2007-05-01

    Endosymbiosis creates a unique osmotic circumstance. Hosts are not only responsible for balancing their internal osmolarity with respect to the external environment, but they must also maintain a compatible osmotic environment for their endosymbionts, which may themselves contribute to the net osmolarity of the host cell through molecular fluxes and/or exchange. Cnidarian hosts that harbor intracellular dinoflagellates (zooxanthellae) are excellent examples of such a symbiosis. These associations are characterized by the exchange of osmotically active compounds, but they are temporally stable under normal environmental conditions indicating that these osmotically driven exchanges are effectively and rapidly regulated. Although we have some knowledge about how asymbiotic anthozoans and algae osmoregulate, our understanding of the physiological mechanisms involved in regulating an intact anthozoan-dinoflagellate symbiosis is poor. Large-scale expulsion of endosymbiotic zooxanthellae, or bleaching, is currently considered to be one of the greatest threats to coral reefs worldwide. To date, there has been little consideration of the osmotic scenarios that occur when these symbioses are exposed to the conditions that normally elicit bleaching, such as increased seawater temperatures and UV radiation. Here we review what is known about osmoregulation and osmotic stress in anthozoans and dinoflagellates and discuss the osmotic implications of exposure to environmental stress in these globally distributed and ecologically important symbioses.

  1. Caenorhabditis elegans ATAD-3 modulates mitochondrial iron and heme homeostasis.

    PubMed

    van den Ecker, Daniela; Hoffmann, Michael; Müting, Gesine; Maglioni, Silvia; Herebian, Diran; Mayatepek, Ertan; Ventura, Natascia; Distelmaier, Felix

    2015-11-13

    ATAD3 (ATPase family AAA domain-containing protein 3) is a mitochondrial protein, which is essential for cell viability and organismal development. ATAD3 has been implicated in several important cellular processes such as apoptosis regulation, respiratory chain function and steroid hormone biosynthesis. Moreover, altered expression of ATAD3 has been associated with several types of cancer. However, the exact mechanisms underlying ATAD3 effects on cellular metabolism remain largely unclear. Here, we demonstrate that Caenorhabditis elegans ATAD-3 is involved in mitochondrial iron and heme homeostasis. Knockdown of atad-3 caused mitochondrial iron- and heme accumulation. This was paralleled by changes in the expression levels of several iron- and heme-regulatory genes as well as an increased heme uptake. In conclusion, our data indicate a regulatory role of C. elegans ATAD-3 in mitochondrial iron and heme metabolism.

  2. Osmosensing and osmoregulation in unicellular eukaryotes.

    PubMed

    Suescún-Bolívar, Luis Parmenio; Thomé, Patricia Elena

    2015-03-01

    Eukaryotic microorganisms possess mechanisms to detect osmotic variations in their surroundings, from specialized receptors and membrane transporters, to sophisticated systems such as two-component histidine kinases. Osmotic stimuli are transduced through conserved phosphorylation cascades that result in a rapid response to mitigate stress. This response allows for the maintenance of an optimal biochemical environment for cell functioning, as well as a suitable recovery in suboptimal environments that would otherwise endanger cell survival. The molecular basis of these responses has been largely studied in yeasts and bacteria. However, fewer studies have been published concerning the molecular basis of osmoregulation in other eukaryotic microorganisms such as protozoans and microalgae. Here, we review the main osmosensors reported in unicellular eukaryotic microorganisms (yeasts, microalgae and protozoa) and the pathways that maintain homeostasis in cells encountering hyperosmotic challenges.

  3. The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake

    PubMed Central

    Hernández, Josefina; Barisón, María Julia; Pral, Elizabeth M. F.; Silber, Ariel M.; Cricco, Julia A.

    2016-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite’s replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE) with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport), which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi. PMID:26752206

  4. The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake.

    PubMed

    Merli, Marcelo L; Pagura, Lucas; Hernández, Josefina; Barisón, María Julia; Pral, Elizabeth M F; Silber, Ariel M; Cricco, Julia A

    2016-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite's replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE) with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport), which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi. PMID:26752206

  5. Measurement of heme concentration.

    PubMed

    Sinclair, P R; Gorman, N; Jacobs, J M

    2001-05-01

    Heme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P-450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectrophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed-phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.

  6. Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osmoregulated periplasmic glucans (OPGs) of foodborne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100 percent glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominan...

  7. TMEM14C is required for erythroid mitochondrial heme metabolism.

    PubMed

    Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J; Takahashi-Makise, Naoko; Gwynn, Babette; Bauer, Daniel E; Dass, Abhishek; Yi, Gloria; Li, Liangtao; Hildick-Smith, Gordon J; Cooney, Jeffrey D; Pierce, Eric L; Mohler, Kyla; Dailey, Tamara A; Miyata, Non; Kingsley, Paul D; Garone, Caterina; Hattangadi, Shilpa M; Huang, Hui; Chen, Wen; Keenan, Ellen M; Shah, Dhvanit I; Schlaeger, Thorsten M; DiMauro, Salvatore; Orkin, Stuart H; Cantor, Alan B; Palis, James; Koehler, Carla M; Lodish, Harvey F; Kaplan, Jerry; Ward, Diane M; Dailey, Harry A; Phillips, John D; Peters, Luanne L; Paw, Barry H

    2014-10-01

    The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

  8. TMEM14C is required for erythroid mitochondrial heme metabolism

    PubMed Central

    Yien, Yvette Y.; Robledo, Raymond F.; Schultz, Iman J.; Takahashi-Makise, Naoko; Gwynn, Babette; Bauer, Daniel E.; Dass, Abhishek; Yi, Gloria; Li, Liangtao; Hildick-Smith, Gordon J.; Cooney, Jeffrey D.; Pierce, Eric L.; Mohler, Kyla; Dailey, Tamara A.; Miyata, Non; Kingsley, Paul D.; Garone, Caterina; Hattangadi, Shilpa M.; Huang, Hui; Chen, Wen; Keenan, Ellen M.; Shah, Dhvanit I.; Schlaeger, Thorsten M.; DiMauro, Salvatore; Orkin, Stuart H.; Cantor, Alan B.; Palis, James; Koehler, Carla M.; Lodish, Harvey F.; Kaplan, Jerry; Ward, Diane M.; Dailey, Harry A.; Phillips, John D.; Peters, Luanne L.; Paw, Barry H.

    2014-01-01

    The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias. PMID:25157825

  9. Mitochondrial Atpif1 regulates heme synthesis in developing erythroblasts

    PubMed Central

    Shah, Dhvanit I.; Takahashi-Makise, Naoko; Cooney, Jeffrey D.; Li, Liangtao; Schultz, Iman J.; Pierce, Eric L.; Narla, Anupama; Seguin, Alexandra; Hattangadi, Shilpa M.; Medlock, Amy E.; Langer, Nathaniel B.; Dailey, Tamara A.; Hurst, Slater N.; Faccenda, Danilo; Wiwczar, Jessica M.; Heggers, Spencer K.; Vogin, Guillaume; Chen, Wen; Chen, Caiyong; Campagna, Dean R.; Brugnara, Carlo; Zhou, Yi; Ebert, Benjamin L.; Danial, Nika N.; Fleming, Mark D.; Ward, Diane M.; Campanella, Michelangelo; Dailey, Harry A.; Kaplan, Jerry; Paw, Barry H.

    2012-01-01

    SUMMARY Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies. PMID:23135403

  10. Heme, an Essential Nutrient from Dietary Proteins, Critically Impacts Diverse Physiological and Pathological Processes

    PubMed Central

    Hooda, Jagmohan; Shah, Ajit; Zhang, Li

    2014-01-01

    Heme constitutes 95% of functional iron in the human body, as well as two-thirds of the average person’s iron intake in developed countries. Hence, a wide range of epidemiological studies have focused on examining the association of dietary heme intake, mainly from red meat, with the risks of common diseases. High heme intake is associated with increased risk of several cancers, including colorectal cancer, pancreatic cancer and lung cancer. Likewise, the evidence for increased risks of type-2 diabetes and coronary heart disease associated with high heme intake is compelling. Furthermore, recent comparative metabolic and molecular studies of lung cancer cells showed that cancer cells require increased intracellular heme biosynthesis and uptake to meet the increased demand for oxygen-utilizing hemoproteins. Increased levels of hemoproteins in turn lead to intensified oxygen consumption and cellular energy generation, thereby fueling cancer cell progression. Together, both epidemiological and molecular studies support the idea that heme positively impacts cancer progression. However, it is also worth noting that heme deficiency can cause serious diseases in humans, such as anemia, porphyrias, and Alzheimer’s disease. This review attempts to summarize the latest literature in understanding the role of dietary heme intake and heme function in diverse diseases. PMID:24633395

  11. Ontogeny of osmoregulation in postembryonic fish: a review.

    PubMed

    Varsamos, Stamatis; Nebel, Catherine; Charmantier, Guy

    2005-08-01

    Salinity and its variations are among the key factors that affect survival, metabolism and distribution during the fish development. The successful establishment of a fish species in a given habitat depends on the ability of each developmental stage to cope with salinity through osmoregulation. It is well established that adult teleosts maintain their blood osmolality close to 300 mosM kg(-1) due to ion and water regulation effected at several sites: tegument, gut, branchial chambers, urinary organs. But fewer data are available in developing fish. We propose a review on the ontogeny of osmoregulation based on studies conducted in different species. Most teleost prelarvae are able to osmoregulate at hatch, and their ability increases in later stages. Before the occurrence of gills, the prelarval tegument where a high density of ionocytes (displaying high contents of Na+/K+-ATPase) is located appears temporarily as the main osmoregulatory site. Gills develop gradually during the prelarval stage along with the numerous ionocytes they support. The tegument and gill Na+/K+-ATPase activity varies ontogenetically. During the larval phase, the osmoregulatory function shifts from the skin to the gills, which become the main osmoregulatory site. The drinking rate normalized to body weight tends to decrease throughout development. The kidney and urinary bladder develop progressively during ontogeny and the capacity to produce hypotonic urine at low salinity increases accordingly. The development of the osmoregulatory functions is hormonally controlled. These events are inter-related and are correlated with changes in salinity tolerance, which often increases markedly at the metamorphic transition from larva to juvenile. In summary, the ability of ontogenetical stages of fish to tolerate salinity through osmoregulation relies on integumental ionocytes, then digestive tract development and drinking rate, developing branchial chambers and urinary organs. The physiological

  12. Ontogeny of osmoregulation in postembryonic fish: a review.

    PubMed

    Varsamos, Stamatis; Nebel, Catherine; Charmantier, Guy

    2005-08-01

    Salinity and its variations are among the key factors that affect survival, metabolism and distribution during the fish development. The successful establishment of a fish species in a given habitat depends on the ability of each developmental stage to cope with salinity through osmoregulation. It is well established that adult teleosts maintain their blood osmolality close to 300 mosM kg(-1) due to ion and water regulation effected at several sites: tegument, gut, branchial chambers, urinary organs. But fewer data are available in developing fish. We propose a review on the ontogeny of osmoregulation based on studies conducted in different species. Most teleost prelarvae are able to osmoregulate at hatch, and their ability increases in later stages. Before the occurrence of gills, the prelarval tegument where a high density of ionocytes (displaying high contents of Na+/K+-ATPase) is located appears temporarily as the main osmoregulatory site. Gills develop gradually during the prelarval stage along with the numerous ionocytes they support. The tegument and gill Na+/K+-ATPase activity varies ontogenetically. During the larval phase, the osmoregulatory function shifts from the skin to the gills, which become the main osmoregulatory site. The drinking rate normalized to body weight tends to decrease throughout development. The kidney and urinary bladder develop progressively during ontogeny and the capacity to produce hypotonic urine at low salinity increases accordingly. The development of the osmoregulatory functions is hormonally controlled. These events are inter-related and are correlated with changes in salinity tolerance, which often increases markedly at the metamorphic transition from larva to juvenile. In summary, the ability of ontogenetical stages of fish to tolerate salinity through osmoregulation relies on integumental ionocytes, then digestive tract development and drinking rate, developing branchial chambers and urinary organs. The physiological

  13. DDE feeding and plasma osmoregulation in ducks, guillemots, and puffins.

    PubMed

    Miller, D S; Kinter, W B; Peakall, D B; Risebrough, R W

    1976-08-01

    To assess the possibility that organochlorine pesticide disruption of osmoregulation is responsible for recent large kills of young seabirds, we have studied the effects of DDE feeding (10-250 ppm) on plasma osmoregulation and nasal gland function in the following species: mallared and white Pekin ducks (both Anas platyrhynchos), black guillemot (Cepphus grylle), and common (Fratercula arctica). Other investigators have recently reported that dietary DDE (10-1,000 ppm) inhibits nasal gland secretion in freshwater-maintained mallards; our initial experiments with white Pekins showed no such inhibition during either freshwater or seawater maintainance. Moreover, DDE had minimal effects on plasma electrolyte levels and total nasal gland Na-K-ATPase activities in all species studied. Liver DDE levels in experimental ducks and guillemots were comparable to those reported for seabirds found dead after kills; levels in starved experimental puffins were much higher. Thus DDE at environmental levels does not affect osmoregulation or nasal gland Na-K-ATPase either in ducks or in two species of oceanic birds. PMID:134641

  14. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    PubMed

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways.

  15. Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells.

    PubMed

    Hooda, Jagmohan; Cadinu, Daniela; Alam, Md Maksudul; Shah, Ajit; Cao, Thai M; Sullivan, Laura A; Brekken, Rolf; Zhang, Li

    2013-01-01

    Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer

  16. Rapid and sensitive quantitation of heme in hemoglobinized cells.

    PubMed

    Marcero, Jason R; Piel Iii, Robert B; Burch, Joseph S; Dailey, Harry A

    2016-01-01

    Rapid and accurate heme quantitation in the research lab has become more desirable as the crucial role that intracellular hemoproteins play in metabolism continues to emerge. Here, the time-honored approaches of pyridine hemochromogen and fluorescence heme assays are compared with direct absorbance-based technologies using the CLARiTY spectrophotometer. All samples tested with these methods were rich in hemoglobin-associated heme, including buffered hemoglobin standards, whole blood from mice, and murine erythroleukemia (MEL) and K562 cells. While the pyridine hemochromogen assay demonstrated the greatest linear range of heme detection, all 3 methods demonstrated similar analytical sensitivities and normalized limits of quantitation of ∼1 µM. Surprisingly, the fluorescence assay was only shown to be distinct in its ability to quantitate extremely small samples. Using the CLARiTY system in combination with pyridine hemochromogen and cell count data, a common hemoglobin extinction coefficient for blood and differentiating MEL and K562 cells of 0.46 µM-1 cm-1 was derived. This value was applied to supplemental experiments designed to measure MEL cell hemoglobinization in response to the addition or removal of factors previously shown to affect heme biosynthesis (e.g., L-glutamine, iron). PMID:27528073

  17. Non-heme iron enzymes: Contrasts to heme catalysis

    PubMed Central

    Solomon, Edward I.; Decker, Andrea; Lehnert, Nicolai

    2003-01-01

    Non-heme iron enzymes catalyze a wide range of O2 reactions, paralleling those of heme systems. Non-heme iron active sites are, however, much more difficult to study because they do not exhibit the intense spectral features characteristic of the porphyrin ligand. A spectroscopic methodology was developed that provides significant mechanistic insight into the reactivity of non-heme ferrous active sites. These studies reveal a general mechanistic strategy used by these enzymes and differences in substrate and cofactor interactions dependent on their requirement for activation by iron. Contributions to O2 activation have been elucidated for non-heme relative to heme ligand sets, and major differences in reactivity are defined with respect to the heterolytic and homolytic cleavage of O—O bonds. PMID:12598659

  18. Heme degradation and vascular injury.

    PubMed

    Belcher, John D; Beckman, Joan D; Balla, Gyorgy; Balla, Jozsef; Vercellotti, Gregory

    2010-02-01

    Heme is an essential molecule in aerobic organisms. Heme consists of protoporphyrin IX and a ferrous (Fe(2+)) iron atom, which has high affinity for oxygen (O(2)). Hemoglobin, the major oxygen-carrying protein in blood, is the most abundant heme-protein in animals and humans. Hemoglobin consists of four globin subunits (alpha(2)beta(2)), with each subunit carrying a heme group. Ferrous (Fe(2+)) hemoglobin is easily oxidized in circulation to ferric (Fe(3+)) hemoglobin, which readily releases free hemin. Hemin is hydrophobic and intercalates into cell membranes. Hydrogen peroxide can split the heme ring and release "free" redox-active iron, which catalytically amplifies the production of reactive oxygen species. These oxidants can oxidize lipids, proteins, and DNA; activate cell-signaling pathways and oxidant-sensitive, proinflammatory transcription factors; alter protein expression; perturb membrane channels; and induce apoptosis and cell death. Heme-derived oxidants induce recruitment of leukocytes, platelets, and red blood cells to the vessel wall; oxidize low-density lipoproteins; and consume nitric oxide. Heme metabolism, extracellular and intracellular defenses against heme, and cellular cytoprotective adaptations are emphasized. Sickle cell disease, an archetypal example of hemolysis, heme-induced oxidative stress, and cytoprotective adaptation, is reviewed. PMID:19697995

  19. [Hereditary porphyrias and heme related disorders].

    PubMed

    Puy, Hervé; Gouya, Laurent; Deybach, Jean-charles

    2014-06-01

    Hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway, characterised by acute neurovisceral symptoms and/or skin lesions. Each porphyria is caused by abnormal functioning of a particular enzymatic step, resulting in specific accumulation of heme precursors. Seven porphyrias are due to a partial enzyme deficiency, while a gain-of-function mechanism has recently been identify in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation and confusion, and are sometimes complicated by seizures and severe neurological disorders, which may be life-threatening. Cutaneous porphyrias can also be present, with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still under-diagnosed, but recent advances in the pathogenesis and genetics of human porphyrias are leading to better care of these patients and their families.

  20. Osmoregulation in zebrafish: ion transport mechanisms and functional regulation

    PubMed Central

    Guh, Ying-Jey; Lin, Chia-Hao; Hwang, Pung-Pung

    2015-01-01

    Fish, like mammals, have to maintain their body fluid ionic and osmotic homeostasis through sophisticated iono-/osmoregulation mechanisms, which are conducted mainly by ionocytes of the gill (the skin in embryonic stages), instead of the renal tubular cells in mammals. Given the advantages in terms of genetic database availability and manipulation, zebrafish is an emerging model for research into regulatory and integrative physiology. At least five types of ionocytes, HR, NaR, NCC, SLC26, and KS cells, have been identified to carry out Na+ uptake/H+ secretion/NH4+ excretion, Ca2+ uptake, Na+/Cl- uptake, K+ secretion, and Cl- uptake/HCO3- secretion, respectively, through distinct sets of transporters. Several hormones, namely isotocin, prolactin, cortisol, stanniocalcin-1, calcitonin, endothelin-1, vitamin D, parathyorid hormone 1, catecholamines, and the renin-angiotensin-system, have been demonstrated to positively or negatively regulate ion transport through specific receptors at different ionocytes stages, at either the transcriptional/translational or posttranslational level. The knowledge obtained using zebrafish answered many long-term contentious or unknown issues in the field of fish iono-/osmoregulation. The homology of ion transport pathways and hormone systems also means that the zebrafish model informs studies on mammals or other animal species, thereby providing insights into related fields. PMID:26600749

  1. The Legend of Sally Hemings

    ERIC Educational Resources Information Center

    Belz, Herman

    2012-01-01

    The part played by Sally Hemings in the life of Thomas Jefferson has been regarded as provocatively dubious since political enemy James Callender claimed in 1802 that Jefferson was the father of several of Hemings's children. Historian Merrill Peterson, observing that paternity is hard to prove, wrote in 1960 that no concrete evidence was ever…

  2. Mechanism and Catalytic Diversity of Rieske Non-Heme Iron-Dependent Oxygenases

    PubMed Central

    Barry, Sarah M.; Challis, Gregory L.

    2013-01-01

    Rieske non-heme iron-dependent oxygenases are important enzymes that catalyze a wide variety of reactions in the biodegradation of xenobiotics and the biosynthesis of bioactive natural products. In this perspective article, we summarize recent efforts to elucidate the catalytic mechanisms of Rieske oxygenases and highlight the diverse range of reactions now known to be catalyzed by such enzymes. PMID:24244885

  3. Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Sigala, Paul A.; Crowley, Jan R.; Hsieh, Samantha; Henderson, Jeffrey P.; Goldberg, Daniel E.

    2012-01-01

    Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection. PMID:22992734

  4. Impact of Aeration and Heme-Activated Respiration on Lactococcus lactis Gene Expression: Identification of a Heme-Responsive Operon▿ †

    PubMed Central

    Pedersen, Martin Bastian; Garrigues, Christel; Tuphile, Karine; Brun, Célia; Vido, Karin; Bennedsen, Mads; Møllgaard, Henrik; Gaudu, Philippe; Gruss, Alexandra

    2008-01-01

    Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O2 and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis. PMID:18487342

  5. Taurine in the osmoregulation of the Brattleboro rat

    SciTech Connect

    Nieminen, M.J.; Tuomisto, L.; Solatunturi, E.; Eriksson, L.; Paasonen, M.K.

    1988-01-01

    The function of taurine in mammalian osmoregulation was studied in the Brattleboro rat with hereditary hypothalamic diabetes insipidus (DI). DI rats are chronically dehydrated because of their inability to synthesize vasopressin. One day of water deprivation did not affect the water balance in rats with normal vasopressin synthesis, whereas DI rats were markedly dehydrated and lost considerably body weight. Taurine content and /sup 3/H-taurine accumulation by platelets were significantly higher in DI rats, with a further increase after one day of water deprivation. In DI rats, water deprivation also evoked a clear taurine increase in skeletal muscle and in the brain. These findings indicate that taurine has an osmoregulatory function in mammals.

  6. Osmoregulation in wild and captive West Indian manatees (Trichechus manatus).

    PubMed

    Ortiz, R M; Worthy, G A; MacKenzie, D S

    1998-01-01

    The ability of West Indian manatees (Trichechus manatus latirostris and Trichechus manatus manatus) to inhabit both freshwater and marine habitats presents an interesting model to study osmoregulation in sirenians. Blood samples were analyzed from manatees held in fresh- and saltwater and from wild animals captured in fresh-, brackish, and saltwater for concentrations of aldosterone, arginine vasopressin, plasma renin activity, Na+, K+, Cl-, and osmolality. Two separate experiments were also conducted on captive animals to evaluate osmoregulatory responses to acute saltwater exposure and freshwater deprivation. Spurious differences were observed in plasma electrolyte and osmolality among the captive and wild groups. Wild brackish water animals exhibited the highest vasopressin concentrations, while wild freshwater manatees had the highest aldosterone levels. A significant correlation between mean vasopressin and osmolality was demonstrated for captive and wild animals. When freshwater animals were acutely exposed to saltwater, osmolality, Na+, and Cl- increased 5.5%, 8.0%, and 14%, respectively, while aldosterone decreased 82.6%. Saltwater animals deprived of freshwater exhibited an almost twofold increase in aldosterone during the deprivation period and a fourfold decrease when freshwater was again provided. Within this group, osmolality increased significantly by 3.4% over the course of the study; however, electrolytes did not change. The lack of consistent differences in electrolyte and osmolality among wild and captive groups suggests that manatees are good osmoregulators regardless of the environment. The high aldosterone levels in wild freshwater animals may indicate a need to conserve Na+, while the high vasopressin levels in wild brackish-water manatees suggest an antidiuretic state to conserve water. Vasopressin levels appear to be osmotically mediated in manatees as in other mammals.

  7. TRPV4 is associated with central rather than nephrogenic osmoregulation.

    PubMed

    Janas, Sylvie; Seghers, François; Schakman, Olivier; Alsady, Mohammad; Deen, Peter; Vriens, Joris; Tissir, Fadel; Nilius, Bernd; Loffing, Johannes; Gailly, Philippe; Devuyst, Olivier

    2016-09-01

    TRPV4 is a polymodal cation channel expressed in osmosensitive neurons of the hypothalamus and in the mammalian nephron. The segmental distribution and role(s) of TRPV4 in osmoregulation remain debated. We investigated the renal distribution pattern of TRPV4 and the functional consequences of its disruption in mouse models. Using qPCR on microdissected segments, immunohistochemistry, and a LacZ reporter mouse, we found that TRPV4 is abundantly expressed in the proximal tubule, the late distal convoluted tubule, and throughout the connecting tubule and collecting duct, including principal and intercalated cells. TRPV4 was undetectable in the glomeruli and thick ascending limb and weakly abundant in the early distal convoluted tubule. Metabolic studies in Trpv4 (+/+) and Trpv4 (-/-) littermates revealed that the lack of TRPV4 did not influence activity, food and water intake, renal function, and urinary concentration at baseline. The mice showed a similar response to furosemide, water loading and deprivation, acid loading, and dietary NaCl restriction. However, Trpv4 (-/-) mice showed a significantly lower vasopressin synthesis and release after water deprivation, with a loss of the positive correlation between plasma osmolality and plasma vasopressin levels, and a delayed water intake upon acute administration of hypertonic saline. Specific activation of TRPV4 in primary cultures of proximal tubule cells increased albumin uptake, whereas no effect of TRPV4 deletion could be observed at baseline. These data reveal that, despite its abundant expression in tubular segments, TRPV4 does not play a major role in the kidney or is efficiently compensated when deleted. Instead, TRPV4 is critical for the release of vasopressin, the sensation of thirst, and the central osmoregulation. PMID:27364478

  8. Effects of gravistimuli on osmoregulation in azuki bean epicotyls

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2013-02-01

    The effects of hypergravity on growth and osmoregulation were examined in dark-grown azuki bean epicotyls. Elongation growth of epicotyls was promptly suppressed by hypergravity at 300g. On the contrary, the increase in fresh weight of epicotyls during incubation was not suppressed by hypergravity at 300g at least up to 6 h. Also, the level of total osmotic solutes increased during epicotyl growth for 6 h, which was not affected by hypergravity. These results suggest that azuki bean epicotyls are capable of maintaining osmoregulation even under 300g conditions for a short period. On the other hand, the increase in fresh weight of epicotyls was suppressed, in addition to suppression of elongation growth, when seedlings were treated with 300g for 24 h. The increase in level of total osmotic solutes was also inhibited by 24 h hypergravity treatment, which was accounted by the reduced levels of organic solutes, such as sugars and amino acids. Furthermore, the dry weight of seeds decreased during incubation for 24 h, but the decrease was inhibited by hypergravity at 300g. Hypergravity treatment at 300g for 24 h also increased the pH value of apoplastic solution in epicotyls. Taken together, these results suggest that the translocation of organic solutes from the seed to epicotyls is inhibited by prolonged hypergravity treatment, which may underlie the suppression of epicotyl growth, and that the breakdown of H+ gradient across the plasma membrane in epicotyl cells may be at least partly involved in the reduction of organic solute accumulation under hypergravity conditions.

  9. Heme on innate immunity and inflammation

    PubMed Central

    Dutra, Fabianno F.; Bozza, Marcelo T.

    2014-01-01

    Heme is an essential molecule expressed ubiquitously all through our tissues. Heme plays major functions in cellular physiology and metabolism as the prosthetic group of diverse proteins. Once released from cells and from hemeproteins free heme causes oxidative damage and inflammation, thus acting as a prototypic damage-associated molecular pattern. In this context, free heme is a critical component of the pathological process of sterile and infectious hemolytic conditions including malaria, hemolytic anemias, ischemia-reperfusion, and hemorrhage. The plasma scavenger proteins hemopexin and albumin reduce heme toxicity and are responsible for transporting free heme to intracellular compartments where it is catabolized by heme-oxygenase enzymes. Upon hemolysis or severe cellular damage the serum capacity to scavenge heme may saturate and increase free heme to sufficient amounts to cause tissue damage in various organs. The mechanism by which heme causes reactive oxygen generation, activation of cells of the innate immune system and cell death are not fully understood. Although heme can directly promote lipid peroxidation by its iron atom, heme can also induce reactive oxygen species generation and production of inflammatory mediators through the activation of selective signaling pathways. Heme activates innate immune cells such as macrophages and neutrophils through activation of innate immune receptors. The importance of these events has been demonstrated in infectious and non-infectious diseases models. In this review, we will discuss the mechanisms behind heme-induced cytotoxicity and inflammation and the consequences of these events on different tissues and diseases. PMID:24904418

  10. Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

    PubMed Central

    Muraki, Norifumi; Kitatsuji, Chihiro; Ogura, Mariko; Uchida, Takeshi; Ishimori, Koichiro; Aono, Shigetoshi

    2016-01-01

    Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow. PMID:27240352

  11. Spectroscopic detection and quantification of heme and heme degradation products.

    PubMed

    Neugebauer, U; März, A; Henkel, T; Schmitt, M; Popp, J

    2012-12-01

    Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752 nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3 μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.

  12. Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus.

    PubMed

    Brücker, G; Zeidler, M; Kohchi, T; Hartmann, E; Lamparter, T

    2000-03-01

    In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one. PMID:10787045

  13. Hydroxylamine and hydrazine bind directly to the heme iron of the heme-heme oxygenase-1 complex.

    PubMed

    Sakamoto, Hiroshi; Higashimoto, Yuichiro; Hayashi, Shunsuke; Sugishima, Masakazu; Fukuyama, Keiichi; Palmer, Graham; Noguchi, Masato

    2004-07-01

    We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron. With a concentrated solution of the heme-enzyme complex, however, another species of HA binding appeared, in which the oxygen atom of HA is coordinated to the iron. This species showed an unusual low-spin signal which is similar to that of the ferric hydroperoxide species in the heme oxygenase reaction.

  14. Non-heme iron as ferrous sulfate does not interact with heme iron absorption in humans.

    PubMed

    Gaitán, Diego; Olivares, Manuel; Lönnerdal, Bo; Brito, Alex; Pizarro, Fernando

    2012-12-01

    The absorption of heme iron has been described as distinctly different from that of non-heme iron. Moreover, whether heme and non-heme iron compete for absorption has not been well established. Our objective was to investigate the potential competition between heme and non-heme iron as ferrous sulfate for absorption, when both iron forms are ingested on an empty stomach. Twenty-six healthy nonpregnant women were selected to participate in two iron absorption studies using iron radioactive tracers. We obtained the dose-response curve for absorption of 0.5, 10, 20, and 50 mg heme iron doses, as concentrated red blood cells. Then, we evaluated the absorption of the same doses, but additionally we added non-heme iron, as ferrous sulfate, at constant heme/non-heme iron molar ratio (1:1). Finally, we compare the two curves by a two-way ANOVA. Iron sources were administered on an empty stomach. One factor analysis showed that heme iron absorption was diminished just by increasing total heme iron (P < 0.0001). The addition of non-heme iron as ferrous sulfate did not have any effect on heme iron absorption (P = NS). We reported evidence that heme and non-heme iron as ferrous sulfate does not compete for absorption. The mechanism behind the absorption of these iron sources is not clear. PMID:22935997

  15. Measurement of heme efflux and heme content in isolated developing chloroplasts. [Cucumis sativus, cv. Sumter

    SciTech Connect

    Thomas, J.; Weinstein, J.D. )

    1990-11-01

    Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, {delta}-aminolevulinic acid. Cold acetone extraction removed 47 {plus minus} 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 {plus minus} 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 {plus minus} 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a free heme pool which exists in the chloroplast.

  16. Digestive tract ontogeny of Dicentrarchus labrax: implication in osmoregulation.

    PubMed

    Giffard-Mena, Ivone; Charmantier, Guy; Grousset, Evelyse; Aujoulat, Fabien; Castille, René

    2006-04-01

    The ontogeny of the digestive tract (DT) and of Na(+)/K(+)-ATPase localization was investigated during the early postembryonic development (from yolk sac larva to juvenile) of the euryhaline teleost Dicentrarchus labrax reared at two salinities: seawater and diluted seawater. Histology, electron microscopy and immunocytochemistry were used to determine the presence and differentiation of ion transporting cells. At hatching, the DT is an undifferentiated straight tube over the yolk sac. At the mouth opening (day 5), it comprises six segments: buccopharynx, esophagus, stomach, anterior intestine, posterior intestine and rectum, well differentiated at the juvenile stage (day 72). The enterocytes displayed ultrastructural features similar to those of mitochondria-rich cells known to be involved in active ion transport. At hatching, ion transporting cells lining the intestine and the rectum exhibited a Na(+)/K(+)-ATPase activity which increased mainly after the larva/juvenile (20 mm) metamorphic transition. The immunofluorescence intensity was dependent upon the stage of development of the gut as well as on the histological configuration of the analyzed segment. The appearance and distribution of enteric ionocytes and the implication of the DT in osmoregulation are discussed.

  17. Identification of an intestinal heme transporter.

    PubMed

    Shayeghi, Majid; Latunde-Dada, Gladys O; Oakhill, Jonathan S; Laftah, Abas H; Takeuchi, Ken; Halliday, Neil; Khan, Yasmin; Warley, Alice; McCann, Fiona E; Hider, Robert C; Frazer, David M; Anderson, Gregory J; Vulpe, Christopher D; Simpson, Robert J; McKie, Andrew T

    2005-09-01

    Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.

  18. Auxin Biosynthesis

    PubMed Central

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development. PMID:24955076

  19. An intercellular heme trafficking protein delivers maternal heme to the embryo during development in C. elegans

    PubMed Central

    Chen, Caiyong; Samuel, Tamika K.; Sinclair, Jason; Dailey, Harry A.; Hamza, Iqbal

    2011-01-01

    Summary Heme is a cytotoxic, hydrophobic tetrapyrrole that crosses multiple biological membranes for incorporation into proteins critical for numerous biological processes. Thus, a prima facie argument can be made that heme trafficking within the aqueous cellular milieu must be mediated by specific intra- and intercellular pathways. Embryonic development in Caenorhabditis elegans, a heme auxotroph, is inextricably dependent on maternal heme acquisition. Here we show that HRG-3 is required to deliver maternal heme to oocytes for zygotic development. HRG-3 binds heme and is exclusively secreted by the intestine during heme insufficiency into the interstitial fluid for transport of maternal heme to extra-intestinal cells. HRG-3 deficiency results either in death during embryogenesis or in developmental arrest immediately post hatching – phenotypes that are fully suppressed by maternal but not zygotic hrg-3 expression. Our results establish an unprecedented role for HRG-3 as an intercellular heme chaperone in zygotic development and maternal-embryonic nutrition in C. elegans. PMID:21620137

  20. Ontogenetic taurine biosynthesis ability in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Wang, Xuan; He, Gen; Mai, Kangsen; Xu, Wei; Zhou, Huihui

    2015-07-01

    Taurine (2-aminoethane sulfonic acid) plays important roles in multiple physiological processes including osmoregulation, bile salt conjugation and membrane protection. It is known that taurine biosynthesis varies in different fish species. However, its ontogenetic regulation has not been clear. In the present study, we found that the hepatic concentrations of taurine increased marginally with rainbow trout growth. The mRNA expression, protein levels and enzyme activities of key enzymes involved in taurine biosynthesis, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD), were analyzed. Our results showed that the mRNA levels and protein abundances of CSD increased dramatically with the development of rainbow trout stages while the enzyme activities showed a slight improvement. However, the expression and activities of CDO decreased with rainbow trout growth. These results provide valuable information on defining the exact supplementation of taurine in diets for different stages of rainbow trout and give new insights into elucidating the regulation of taurine metabolism in rainbow trout.

  1. Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake

    PubMed Central

    Owens, Cedric P.; Du, Jing; Dawson, John H.; Goulding, Celia W.

    2012-01-01

    The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work we use spectroscopic (absorption, electron paramagnetic resonance and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low spin in both ferric and ferrous oxidation states. Rv0203-notag is high spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen ligated heme proteins. Mutagenesis experiments identified that residues Tyr59, His63 and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63 and His89 are required for heme binding to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggest the heme-ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for heme binding to Rv0203-His and Rv0203-notag are similar, 115 (μM s)-1 and 133 (μM s)-1, respectively. However, the heme off-rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 s-1 and 0.0002 s-1, respectively, and Rv0203-notag has a single off-rate of 0.082 s-1. The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His-tag interferes with heme binding. Furthermore, these results imply that the His-tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme

  2. Heme and the endothelium. Effects of nitric oxide on catalytic iron and heme degradation by heme oxygenase.

    PubMed

    Juckett, M; Zheng, Y; Yuan, H; Pastor, T; Antholine, W; Weber, M; Vercellotti, G

    1998-09-01

    We studied the effects of nitric oxide (NO) on the control of excess cellular heme and release of catalytically active iron. Endothelial cells (ECs) exposed to hemin followed by a NO donor have a ferritin content that is 16% that of cells exposed to hemin alone. Hemin-treated ECs experience a 3.5-fold rise in non-heme, catalytic iron 2 h later, but a hemin rechallenge 20 h later results in only a 24% increase. The addition of a NO donor after the first hemin exposure prevents this adaptive response, presumably due to effects on ferritin synthesis. NO donors were found to reduce iron release from hemin, while hemin accumulated in cells. A NO donor, in a dose-dependent fashion, inhibited heme oxygenase activity, measured by bilirubin production. Using low temperature EPR spectroscopy, heme oxygenase inhibition correlated with nitrosylation of free heme in microsomes. Nitrosylation of cellular heme prevented iron release, for while there was heme oxygenase-dependent release of iron in cells incubated with hemin for 24 h, the addition of a NO donor blocked iron release. This indicates that NO readily nitrosylates intracellular free heme and prevents its degradation by heme oxygenase. Nitrosylation of heme was found to reduce sensitization of cells to oxidative injury. PMID:9722574

  3. Identification and characterization of VPO1, a new animal heme-containing peroxidase.

    PubMed

    Cheng, Guangjie; Salerno, John C; Cao, Zehong; Pagano, Patrick J; Lambeth, J David

    2008-12-15

    Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K(m) for H(2)O(2) of 1.5 mM. When co-expressed in cells, VPO1 can use H(2)O(2) produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.

  4. Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ

    PubMed Central

    2016-01-01

    Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of −205 ± 3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 104 M–1 s–1). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria. PMID:27599156

  5. Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ.

    PubMed

    Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović-Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G; Oostenbrink, Chris; Obinger, Christian

    2016-09-27

    Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of -205 ± 3 mV for LmHemQ and -207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 10(4) M(-1) s(-1)). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria. PMID:27599156

  6. Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ.

    PubMed

    Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović-Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G; Oostenbrink, Chris; Obinger, Christian

    2016-09-27

    Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of -205 ± 3 mV for LmHemQ and -207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 10(4) M(-1) s(-1)). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.

  7. The integration of digestion and osmoregulation in the avian gut.

    PubMed

    McWhorter, Todd J; Caviedes-Vidal, Enrique; Karasov, William H

    2009-11-01

    We review digestion and osmoregulation in the avian gut, with an emphasis on the ways these different functions might interact to support or constrain each other and the ways they support the functioning of the whole animal in its natural environment. Differences between birds and other vertebrates are highlighted because these differences may make birds excellent models for study and may suggest interesting directions for future research. At a given body size birds, compared with mammals, tend to eat more food but have less small intestine and retain food in their gastrointestinal tract (GIT) for shorter periods of time, despite generally higher mass-specific energy demands. On most foods, however, they are not less efficient at digestion, which begs the question how they compensate. Intestinal tissue-specific rates of enzymatic breakdown of substrates and rates of active transport do not appear higher in birds than in mammals, nor is there a demonstrated difference in the extent to which those rates can be modulated during acclimation to different feeding regimes (e.g. diet, relative intake level). One compensation appears to be more extensive reliance on passive nutrient absorption by the paracellular pathway, because the avian species studied so far exceed the mammalian species by a factor of at least two- to threefold in this regard. Undigested residues reach the hindgut, but there is little evidence that most wild birds recover microbial metabolites of nutritional significance (essential amino acids and vitamins) by re-ingestion of faeces, in contrast to many hindgut fermenting mammals and possibly poultry. In birds, there is some evidence for hindgut capacity to breakdown either microbial protein or protein that escapes the small intestine intact, freeing up essential amino acids, and there is considerable evidence for an amino acid absorptive capacity in the hindgut of both avian and mammalian hindgut fermenters. Birds, unlike mammals, do not excrete

  8. Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100% glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structu...

  9. Swarm and swim motilities of Salmonella enterica serovar Typhimurium and role of osmoregulated periplasmic glucans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Salmonella enterica serovar Typhimurium strains synthesize osmoregulated periplasmic glucans (OPGs) under low osmolarity conditions (< 70 mos mol l-1). OPG synthesis is not observed when cells are grown in iso- or hyper-osmotic media (> 400 mos mol l-1). Mutation in OPG structural gene...

  10. [Heme-iron in the human body].

    PubMed

    Balla, József; Balla, György; Lakatos, Béla; Jeney, Viktória; Szentmihályi, Klára

    2007-09-01

    Iron is essential for all living organism, although in excess amount it is dangerous via catalyzing the formation of reactive oxygen species. Absorption of iron is strictly controlled resulting in a fine balance of iron-loss and iron-uptake. In countries where the ingestion of heme-iron is significant by meal, great part of iron content in the body originates from heme. Heme derived from food is absorbed by a receptor-mediated manner by enterocytes of small intestine then it is degraded in a reaction catalyzed by heme oxygenase. Iron released from the porphyrin ring leaves enterocytes as transferrin associated iron. Prosthetic group of several proteins contains heme, therefore, it is synthesized by all cells. One of the most significant heme proteins is hemoglobin which transports oxygen in the erythrocytes. Hemoglobin released from erythrocyte during intravascular hemolysis binds to haptoglobin and is taken up by cells of the monocyte-macrophage lineage. Oxidation of hemoglobin (ferro) to methemoglobin (ferri) is inhibited by the structure of hemoglobin although it is not hindered. Superoxide anion is also formed in the reaction that initiates further free radical reactions. In contrast to ferrohemoglobin, methemoglobin readily releases heme, therefore, oxidation of hemoglobin drives the formation of free heme in plasma. Heme binds to a plasma protein, hemopexin, and is internalized by cells of monocyte-macrophage lineage in a receptor-mediated manner, then degraded in reaction catalysed by heme oxygenase. Heme is also taken up by plasma lipoproteins and endothelial cells leading to oxidation of LDL and subsequent endothelial cell damage. The purpose of this work was to summarize the processes related to heme.

  11. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

    PubMed

    Hanna, David A; Harvey, Raven M; Martinez-Guzman, Osiris; Yuan, Xiaojing; Chandrasekharan, Bindu; Raju, Gheevarghese; Outten, F Wayne; Hamza, Iqbal; Reddi, Amit R

    2016-07-01

    Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines. PMID:27247412

  12. Adaptive evolution of the osmoregulation-related genes in cetaceans during secondary aquatic adaptation

    PubMed Central

    2013-01-01

    Background Osmoregulation was a primary challenge for cetaceans during the evolutionary transition from a terrestrial to a mainly hyperosmotic environment. Several physiological mechanisms have been suggested to maintain the water and salt balance in cetaceans, but their genetic and evolutionary bases remain poorly explored. The current study investigated the genes involved in osmoregulation in cetaceans and compared them with their counterparts in terrestrial mammals to test whether adaptive evolution occurred during secondary aquatic adaptation. Results The present study analyzed the molecular evolution of 11 osmoregulation-related genes in 11 cetacean species, which represented all of the major cetacean clades. The results demonstrated positive selection acting on angiotensin converting enzyme (ACE), angiotensinogen (AGT), SLC14A2, and aquaporin 2 (AQP2). This evidence for the positive selection of AQP2 and SLC14A2 suggests that the adaptive evolution of these genes has helped to enhance the capacity for water and urea transport, thereby leading to the concentration of urine, which is an efficient mechanism for maintaining the water balance. By contrast, a series of positively selected amino acid residues identified in the ACE and AGT (two key members of the renin-angiotensin-aldosterone system, RAAS) proteins of cetaceans suggests that RAAS might have been adapted to maintain the water and salt balance in response to a hyperosmotic environment. Radical amino acid changes in positively selected sites were distributed among most internal and terminal branches of the cetacean phylogeny, which suggests the pervasively adaptive evolution of osmoregulation since the origin of cetaceans and their subsequent diversification. Conclusions This is the first comprehensive analysis of the molecular evolution of osmoregulation-related genes in cetaceans in response to selection pressure from a generally hyperosmotic environment. Four genes, i.e., AQP2, SLC14A2, ACE, and AGT

  13. Mechanisms of Heme Utilization by Francisella tularensis

    PubMed Central

    Lindgren, Helena; Lindgren, Lena; Golovliov, Igor; Sjöstedt, Anders

    2015-01-01

    Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel. PMID:25756756

  14. A Twist on Heme Signaling

    PubMed Central

    Spiro, Thomas G.

    2009-01-01

    Proteins in the H-NOX family act as sensors of NO or O2. This family includes soluble guanylate cyclase, sGC, the NO sensor that is responsible for vasodilation and neurotransmission in mammals. The crystal structures of bacterial H-NOX domains have revealed a highly distorted heme cofactor. This distortion has now been shown, via replacement of a single sterically critical residue, to be associated with a concerted displacement of the entire N-terminal half of the protein. This displacement likely provides the mechanism for transducing the ligand binding event into signaling. PMID:19032089

  15. Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

    PubMed

    Fuse, Yuji; Nakajima, Hitomi; Nakajima-Takagi, Yaeko; Nakajima, Osamu; Kobayashi, Makoto

    2015-07-01

    The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.

  16. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

    PubMed

    Pang, Wei; Coghill, George M

    2015-05-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed.

  17. Glucocorticoids accelerate maturation of the heme pathway in fetal liver through effects on transcription and DNA methylation

    PubMed Central

    Khulan, Batbayar; Liu, Lincoln; Rose, Catherine M.; Boyle, Ashley K.; Manning, Jonathan R.; Drake, Amanda J.

    2016-01-01

    ABSTRACT Glucocorticoids are widely used in threatened preterm labor to promote maturation in many organ systems in preterm babies and have significant beneficial effects on morbidity and mortality. We performed transcriptional profiling in fetal liver in a rat model of prenatal glucocorticoid exposure and identified marked gene expression changes in heme biosynthesis, utilization, and degradation pathways in late gestation. These changes in gene expression associated with alterations in DNA methylation and with a reduction in hepatic heme concentration. There were no persistent differences in gene expression, DNA methylation, or heme concentrations at 4 weeks of age, suggesting that these are transient effects. Our findings are consistent with glucocorticoid-induced accelerated maturation of the haematopoietic system and support the hypothesis that glucocorticoids can drive changes in gene expression in association with alterations in DNA methylation. PMID:26889791

  18. Structural biology of heme monooxygenases

    SciTech Connect

    Poulos, Thomas L. . E-mail: poulos@uci.edu

    2005-12-09

    Over the past few years the number of crystal structures available for heme monooxygenases has substantially increased. Those most closely related to one another are cytochrome P450, nitric oxide synthase, and heme oxygenase. The present mini-review provides a summary of some recently published work on how crystallography and solution studies have provided new insights on function and especially the oxygen activation process. It now appears that in all three monooxygenases highly ordered solvent in the active site serves as direct proton donors to the iron-linked dioxygen; a requirement for splitting the O-O bond. This is in sharp contrast to the related peroxidase family of enzymes where strategically positioned amino acid side chains serve the function of shuttling protons. The P450cam-oxy-complex as well as various mutants in a complex with either oxygen or carbon monoxide have enabled a fairly detailed picture to be developed on the role of specific amino acids and conformational changes in both electron transfer and oxygen activation.

  19. Heme Oxygenase-1 in Tumors

    PubMed Central

    JOZKOWICZ, ALICJA; WAS, HALINA; DULAK, JOZEF

    2007-01-01

    Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to biologically active products: carbon monoxide (CO), biliverdin, and ferrous iron. It participates in maintaining cellular homeostasis and plays an important protective role in the tissues by reducing oxidative injury, attenuating the inflammatory response, inhibiting cell apoptosis, and regulating cell proliferation. HO-1 is also an important proangiogenic mediator. Most studies have focused on the role of HO-1 in cardiovascular diseases, in which its significant, beneficial activity is well recognized. A growing body of evidence indicates, however, that HO-1 activation may play a role in carcinogenesis and can potently influence the growth and metastasis of tumors. HO-1 is very often upregulated in tumor tissues, and its expression is further increased in response to therapies. Although the exact effect can be tissue specific, HO-1 can be regarded as an enzyme facilitating tumor progression. Accordingly, inhibition of HO-1 can be suggested as a potential therapeutic approach sensitizing tumors to radiation, chemotherapy, or photodynamic therapy. PMID:17822372

  20. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    PubMed

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

  1. Heme Oxygenase-1: A Metabolic Nike

    PubMed Central

    Nemeth, Zsuzsanna; Correa-Costa, Matheus; Bulmer, Andrew C.; Otterbein, Leo E.

    2014-01-01

    Abstract Significance: Heme degradation, which was described more than 30 years ago, is still very actively explored with many novel discoveries on its role in various disease models every year. Recent Advances: The heme oxygenases (HO) are metabolic enzymes that utilize NADPH and oxygen to break apart the heme moiety liberating biliverdin (BV), carbon monoxide (CO), and iron. Heme that is derived from hemoproteins can be toxic to the cells and if not removed immediately, it causes cell apoptosis and local inflammation. Elimination of heme from the milieu enables generation of three products that influences numerous metabolic changes in the cell. Critical Issues: CO has profound effects on mitochondria and cellular respiration and other hemoproteins to which it can bind and affect their function, while BV and bilirubin (BR), the substrate and product of BV, reductase, respectively, are potent antioxidants. Sequestration of iron into ferritin and its recycling in the tissues is a part of the homeodynamic processes that control oxidation-reduction in cellular metabolism. Further, heme is an important component of a number of metabolic enzymes, and, therefore, HO-1 plays an important role in the modulation of cellular bioenergetics. Future Directions: In this review, we describe the cross-talk between heme oxygenase-1 (HO-1) and its products with other metabolic pathways. HO-1, which we have labeled Nike, the goddess who personified victory, dictates triumph over pathophysiologic conditions, including diabetes, ischemia, and cancer. Antioxid. Redox Signal. 20, 1709–1722. PMID:24180257

  2. Physiological significance of heme oxygenase in hypertension

    PubMed Central

    Cao, Jian; Inoue, Kazuyoshi; Li, Xiaoying; Drummond, George; Abraham, Nader G.

    2009-01-01

    The last decade has witnessed an explosion in the elucidation of the role that the heme oxygenase system plays in human physiology. This system encompasses not only the heme degradative pathway, including heme oxygenase and biliverdin reductase, but also the products of heme degradation, carbon monoxide, iron, and biliverdin/bilirubin. Their role in diabetes, inflammation, heart disease, hypertension, transplantation, and pulmonary disease are areas of burgeoning research. The research has focused not only on heme itself but also on its metabolic products as well as endogenous compounds involved in a vast number of genetic and metabolic processes that are affected when heme metabolism is perturbed. It should be noted, however, that although the use of carbon monoxide and biliverdin/bilirubin as therapeutic agents has been successful, these agents can be toxic at high levels in tissue, e.g., kernicterus. Care must be used to ensure that when these compounds are used as therapeutic agents their deleterious effects are minimized or avoided. On balance, however, the strategies to target heme oxygenase-1 as described in this review offer promising therapeutic approaches to clinicians for the effective management of hypertension and renal function. The approaches detailed may prove to be seminal in the development of a new therapeutic strategy to treat hypertension. PMID:19027871

  3. Porphyrin and heme metabolism and the porphyrias.

    PubMed

    Bonkovsky, Herbert L; Guo, Jun-Tao; Hou, Weihong; Li, Ting; Narang, Tarun; Thapar, Manish

    2013-01-01

    Porphyrins and metalloporphyrins are the key pigments of life on earth as we know it, because they include chlorophyll (a magnesium-containing metalloporphyrin) and heme (iron protoporphyrin). In eukaryotes, porphyrins and heme are synthesized by a multistep pathway that involves eight enzymes. The first and rate-controlling step is the formation of delta-aminolevulinic acid (ALA) from glycine plus succinyl CoA, catalyzed by ALA synthase. Intermediate steps occur in the cytoplasm, with formation of the monopyrrole porphobilinogen and the tetrapyrroles hydroxymethylbilane and a series of porphyrinogens, which are serially decarboxylated. Heme is utilized chiefly for the formation of hemoglobin in erythrocytes, myoglobin in muscle cells, cytochromes P-450 and mitochondrial cytochromes, and other hemoproteins in hepatocytes. The rate-controlling step of heme breakdown is catalyzed by heme oxygenase (HMOX), of which there are two isoforms, called HMOX1 and HMOX2. HMOX breaks down heme to form biliverdin, carbon monoxide, and iron. The porphyrias are a group of disorders, mainly inherited, in which there are defects in normal porphyrin and heme synthesis. The cardinal clinical features are cutaneous (due to the skin-damaging effects of excess deposited porphyrins) or neurovisceral attacks of pain, sometimes with weakness, delirium, seizures, and the like (probably due mainly to neurotoxic effects of ALA). The treatment of choice for the acute hepatic porphyrias is intravenous heme therapy, which repletes a critical regulatory heme pool in hepatocytes and leads to downregulation of hepatic ALA synthase, which is a biochemical hallmark of all forms of acute porphyria in relapse.

  4. Free heme and sickle hemoglobin polymerization

    NASA Astrophysics Data System (ADS)

    Uzunova, Veselina V.

    This work investigates further the mechanism of one of the most interesting of the protein self-assembly systems---the polymerization of sickle hemoglobin and the role of free heme in it. Polymerization of sickle hemoglobin is the primary event in the pathology of a chronic hemolytic condition called sickle cell anemia with complex pathogenesis, unexplained variability and symptomatic treatment. Auto-oxidation develops in hemoglobin solutions exposed to room temperature and causes release of ferriheme. The composition of such solutions is investigated by mass spectrometry. Heme dimers whose amount corresponds to the initial amounts of heme released from the protein are followed. Differences in the dimer peak height are established for hemoglobin variants A, S and C and depending on the exposure duration. The effects of free heme on polymerization kinetics are studied. Growth rates and two characteristic parameters of nucleation are measured for stored Hb S. After dialysis of polymerizing solutions, no spherulites are detected at moderately high supersaturation and prolonged exposure times. The addition of 0.16-0.26 mM amounts of heme to dialyzed solutions leads to restoration of polymerization. The measured kinetic parameters have higher values compared to the ones before dialysis. The amount of heme in non-dialyzed aged solution is characterized using spectrophotometry. Three methods are used: difference in absorbance of dialyzed and non-dialyzed solutions, characteristic absorbance of heme-albumin complex and absorbance of non-dialyzed solutions with added potassium cyanide. The various approaches suggest the presence of 0.12 to 0.18 mM of free ferriheme in such solutions. Open questions are whether the same amounts of free heme are present in vivo and whether the same mechanism operates intracellulary. If the answer to those questions is positive, then removal of free heme from erythrocytes can influence their readiness to sickle.

  5. Shigella dysenteriae ShuS promotes utilization of heme as an iron source and protects against heme toxicity.

    PubMed

    Wyckoff, Elizabeth E; Lopreato, Gregory F; Tipton, Kimberly A; Payne, Shelley M

    2005-08-01

    Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.

  6. Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids.

    PubMed

    Oohora, K; Hayashi, T

    2016-01-01

    An important strategy used in engineering of hemoproteins to generate artificial enzymes involves replacement of heme with an artificial cofactor after removal of the native heme cofactor under acidic conditions. Replacement of heme in an enzyme with a nonnatural metalloporphyrinoid can significantly alter the reactivity of the enzyme. This chapter describes the design and synthesis of three types of artificial metalloporphyrinoid cofactors consisting of mono-, di-, and tri-anionic ligands (tetradehydrocorrin, porphycene, and corrole, respectively). In addition, practical procedures for the preparation of apo-hemoproteins, incorporation of artificial cofactors, and characterization techniques are presented. Furthermore, the representative catalytic activities of artificial enzymes generated by reconstitution of hemoproteins are summarized. PMID:27586344

  7. Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine▿ †

    PubMed Central

    Agarwal, Ameeta K.; Xu, Tao; Jacob, Melissa R.; Feng, Qin; Lorenz, Michael C.; Walker, Larry A.; Clark, Alice M.

    2008-01-01

    Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1Δ mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme. PMID:18156292

  8. Structural mechanisms of nonplanar hemes in proteins

    SciTech Connect

    Shelnutt, J.A.

    1997-05-01

    The objective is to assess the occurrence of nonplanar distortions of hemes and other tetrapyrroles in proteins and to determine the biological function of these distortions. Recently, these distortions were found by us to be conserved among proteins belonging to a functional class. Conservation of the conformation of the heme indicates a possible functional role. Researchers have suggested possible mechanisms by which heme distortions might influence biological properties; however, no heme distortion has yet been shown conclusively to participate in a structural mechanism of hemoprotein function. The specific aims of the proposed work are: (1) to characterize and quantify the distortions of the hemes in all of the more than 300 hemoprotein X-ray crystal structures in terms of displacements along the lowest-frequency normal coordinates, (2) to determine the structural features of the protein component that generate and control these nonplanar distortions by using spectroscopic studies and molecular-mechanics calculations for the native proteins, their mutants and heme-peptide fragments, and model porphyrins, (3) to determine spectroscopic markers for the various types of distortion, and, finally, (4) to discover the functional significance of the nonplanar distortions by correlating function with porphyrin conformation for proteins and model porphyrins.

  9. Heme Oxygenases in Cardiovascular Health and Disease.

    PubMed

    Ayer, Anita; Zarjou, Abolfazl; Agarwal, Anupam; Stocker, Roland

    2016-10-01

    Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies. PMID:27604527

  10. Planetary change and biochemical adaptation: molecular evolution of corrinoid and heme biosyntheses.

    PubMed

    Lazcano, Antonio

    2012-04-01

    This review examines the evidence suggesting that the anaerobic biosynthesis of cobalamin (vitamin B12) evolved during early stages of cell evolution and was quickly recruited in the pathway leading to deoxyribonucleotides, the building blocks of DNA genomes. Biochemical evolution preceding the synthesis of the heme group and related molecules is discussed within the framework of geological evolution in which the appearance and accumulation of an oxygen-rich atmosphere stands as one of the major events in the evolution of the planet and the biosphere.

  11. Heme acquisition in the parasitic filarial nematode Brugia malayi

    PubMed Central

    Luck, Ashley N.; Yuan, Xiaojing; Voronin, Denis; Slatko, Barton E.; Hamza, Iqbal; Foster, Jeremy M.

    2016-01-01

    Nematodes lack a heme biosynthetic pathway and must acquire heme from exogenous sources. Given the indispensable role of heme, this auxotrophy may be exploited to develop drugs that interfere with heme uptake in parasites. Although multiple heme-responsive genes (HRGs) have been characterized within the free-living nematode Caenorhabditis elegans, we have undertaken the first study of heme transport in Brugia malayi, a causative agent of lymphatic filariasis. Through functional assays in yeast, as well as heme analog, RNAi, and transcriptomic experiments, we have shown that the heme transporter B. malayi HRG-1 (BmHRG-1) is indeed functional in B. malayi. In addition, BmHRG-1 localizes both to the endocytic compartments and cell membrane when expressed in yeast cells. Transcriptomic sequencing revealed that BmHRG-1, BmHRG-2, and BmMRP-5 (all orthologs of HRGs in C. elegans) are down-regulated in heme-treated B. malayi, as compared to non–heme-treated control worms. Likely because of short gene lengths, multiple exons, other HRGs in B. malayi (BmHRG-3–6) remain unidentified. Although the precise mechanisms of heme homeostasis in a nematode with the ability to acquire heme remains unknown, this study clearly demonstrates that the filarial nematode B. malayi is capable of transporting exogenous heme.—Luck, A. N., Yuan, X., Voronin, D., Slatko, B. E., Hamza, I., Foster, J. M. Heme acquisition in the parasitic filarial nematode Brugia malayi. PMID:27363426

  12. Function and distribution of bilin biosynthesis enzymes in photosynthetic organisms.

    PubMed

    Dammeyer, Thorben; Frankenberg-Dinkel, Nicole

    2008-10-01

    Bilins are open-chain tetrapyrrole molecules essential for light-harvesting and/or sensing in many photosynthetic organisms. While they serve as chromophores in phytochrome-mediated light-sensing in plants, they additionally function in light-harvesting in cyanobacteria, red algae and cryptomonads. Associated to phycobiliproteins a variety of bile pigments is responsible for the specific light-absorbance properties of the organisms enabling efficient photosynthesis under different light conditions. The initial step of bilin biosynthesis is the cleavage of heme by heme oxygenases (HO) to afford the first linear molecule biliverdin. This reaction is ubiquitously found also in non-photosynthetic organisms. Biliverdin is then further reduced by site specific reductases most of them belonging to the interesting family of ferredoxin-dependent bilin reductases (FDBRs)-a new family of radical oxidoreductases. In recent years much progress has been made in the field of heme oxygenases but even more in the widespread family of FDBRs, revealing novel biochemical FDBR activities, new crystal structures and new ecological aspects, including the discovery of bilin biosynthesis genes in wild marine phage populations. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and remaining questions.

  13. Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

    PubMed

    Kassa, Tigist; Jana, Sirsendu; Meng, Fantao; Alayash, Abdu I

    2016-09-01

    Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage-associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states-ferrous (Fe(2+)), ferric (Fe(3+)), and ferryl (Fe(4+))-using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe(3+)) loses heme at rates substantially higher than that of ferryl Hb (Fe(4+)). This was also supported by a higher expression of heme oxygenase-1 (HO-1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen-carrying therapeutic. PMID:27642551

  14. A Brief History of the Study of Fish Osmoregulation: The Central Role of the Mt. Desert Island Biological Laboratory

    PubMed Central

    Evans, David H.

    2010-01-01

    The Mt. Desert Island Biological Laboratory (MDIBL) has played a central role in the study of fish osmoregulation for the past 80 years. In particular, scientists at the MDIBL have made significant discoveries in the basic pattern of fish osmoregulation, the function of aglomerular kidneys and proximal tubular secretion, the roles of NaCl cotransporters in intestinal uptake and gill and rectal gland secretion, the role of the shark rectal gland in osmoregulation, the mechanisms of salt secretion by the teleost fish gill epithelium, and the evolution of the ionic uptake mechanisms in fish gills. This short review presents the history of these discoveries and their relationships to the study of epithelial transport in general. PMID:21423356

  15. Heme-containing dioxygenases involved in tryptophan oxidation.

    PubMed

    Millett, Elizabeth S; Efimov, Igor; Basran, Jaswir; Handa, Sandeep; Mowat, Christopher G; Raven, Emma Lloyd

    2012-04-01

    Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.

  16. Heme/copper terminal oxidases

    SciTech Connect

    Ferguson-Miller, S.; Babcock, G.T.

    1996-11-01

    Spatially well-organized electron-transfer reactions in a series of membrane-bound redox proteins form the basis for energy conservation in both photosynthesis and respiration. The membrane-bound nature of the electron-transfer processes is critical, as the free energy made available in exergonic redox chemistry is used to generate transmembrane proton concentration and electrostatic potential gradients. These gradients are subsequently used to drive ATP formation, which provides the immediate energy source for constructive cellular processes. The terminal heme/copper oxidases in respiratory electron-transfer chains illustrate a number of the thermodynamic and structural principles that have driven the development of respiration. This class of enzyme reduces dioxygen to water, thus clearing the respiratory system of low-energy electrons so that sustained electron transfer and free-energy transduction can occur. By using dioxygen as the oxidizing substrate, free-energy production per electron through the chain is substantial, owing to the high reduction potential of O{sub 2} (0.815 V at pH 7). 122 refs.

  17. Distance metrics for heme protein electron tunneling.

    PubMed

    Moser, Christopher C; Chobot, Sarah E; Page, Christopher C; Dutton, P Leslie

    2008-01-01

    There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a approximately 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.

  18. ApoHRP-based assay to measure intracellular regulatory heme.

    PubMed

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A; Dhahbi, Joseph M

    2015-02-01

    The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells. PMID:25525887

  19. ApoHRP-based Assay to Measure Intracellular Regulatory Heme

    PubMed Central

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A.; Dhahbi, Joseph M.

    2015-01-01

    The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells. PMID:25525887

  20. Single or functionalized fullerenes interacting with heme group

    NASA Astrophysics Data System (ADS)

    Costa, Wallison Chaves; Diniz, Eduardo Moraes

    2014-09-01

    The heme group is responsible for iron transportation through the bloodstream, where iron participates in redox reactions, electron transfer, gases detection etc. The efficiency of such processes can be reduced if the whole heme molecule or even the iron is somehow altered from its original oxidation state, which can be caused by interactions with nanoparticles as fullerenes. To verify how such particles alter the geometry and electronic structure of heme molecule, here we report first principles calculations based on density functional theory of heme group interacting with single C60 fullerene or with C60 functionalized with small functional groups (-CH3, -COOH, -NH2, -OH). The calculations shown that the system heme + nanoparticle has a different spin state in comparison with heme group if the fullerene is functionalized. Also a functional group can provide a stronger binding between nanoparticle and heme molecule or inhibit the chemical bonding in comparison with single fullerene results. In addition heme molecule loses electrons to the nanoparticles and some systems exhibited a geometry distortion in heme group, depending on the binding energy. Furthermore, one find that such nanoparticles induce a formation of spin up states in heme group. Moreover, there exist modifications in density of states near the Fermi energy. Although of such changes in heme electronic structure and geometry, the iron atom remains in the heme group with the same oxidation state, so that processes that involve the iron might not be affected, only those that depend on the whole heme molecule.

  1. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  2. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  3. Radical S-Adenosylmethionine (SAM) Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions*

    PubMed Central

    Mehta, Angad P.; Abdelwahed, Sameh H.; Mahanta, Nilkamal; Fedoseyenko, Dmytro; Philmus, Benjamin; Cooper, Lisa E.; Liu, Yiquan; Jhulki, Isita; Ealick, Steven E.; Begley, Tadhg P.

    2015-01-01

    In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry. PMID:25477515

  4. Independent evolution of four heme peroxidase superfamilies.

    PubMed

    Zámocký, Marcel; Hofbauer, Stefan; Schaffner, Irene; Gasselhuber, Bernhard; Nicolussi, Andrea; Soudi, Monika; Pirker, Katharina F; Furtmüller, Paul G; Obinger, Christian

    2015-05-15

    Four heme peroxidase superfamilies (peroxidase-catalase, peroxidase-cyclooxygenase, peroxidase-chlorite dismutase and peroxidase-peroxygenase superfamily) arose independently during evolution, which differ in overall fold, active site architecture and enzymatic activities. The redox cofactor is heme b or posttranslationally modified heme that is ligated by either histidine or cysteine. Heme peroxidases are found in all kingdoms of life and typically catalyze the one- and two-electron oxidation of a myriad of organic and inorganic substrates. In addition to this peroxidatic activity distinct (sub)families show pronounced catalase, cyclooxygenase, chlorite dismutase or peroxygenase activities. Here we describe the phylogeny of these four superfamilies and present the most important sequence signatures and active site architectures. The classification of families is described as well as important turning points in evolution. We show that at least three heme peroxidase superfamilies have ancient prokaryotic roots with several alternative ways of divergent evolution. In later evolutionary steps, they almost always produced highly evolved and specialized clades of peroxidases in eukaryotic kingdoms with a significant portion of such genes involved in coding various fusion proteins with novel physiological functions.

  5. Horseradish Peroxidase Inactivation: Heme Destruction and Influence of Polyethylene Glycol

    PubMed Central

    Mao, Liang; Luo, Siqiang; Huang, Qingguo; Lu, Junhe

    2013-01-01

    Horseradish peroxidase (HRP) mediates efficient conversion of many phenolic contaminants and thus has potential applications for pollution control. Such potentially important applications suffer however from the fact that the enzyme becomes quickly inactivated during phenol oxidation and polymerization. The work here provides the first experimental data of heme consumption and iron releases to support the hypothesis that HRP is inactivated by heme destruction. Product of heme destruction is identified using liquid chromatography with mass spectrometry. The heme macrocycle destruction involving deprivation of the heme iron and oxidation of the 4-vinyl group in heme occurs as a result of the reaction. We also demonstrated that heme consumption and iron releases resulting from HRP destruction are largely reduced in the presence of polyethylene glycol (PEG), providing the first evidence to indicate that heme destruction is effectively suppressed by co-dissolved PEG. These findings advance a better understanding of the mechanisms of HRP inactivation. PMID:24185130

  6. Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish.

    PubMed

    Whittamore, Jonathan M

    2012-01-01

    For teleost fish living in seawater, drinking the surrounding medium is necessary to avoid dehydration. This is a key component of their osmoregulatory strategy presenting the challenge of excreting excess salts while achieving a net retention of water. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid is crucial to compensating for water losses to the hyperosmotic environment. Despite this, the potential for the teleost intestine to serve as a comparative model for detailed, integrative experimental studies on epithelial water transport has so far gone largely untapped. The following review aims to present an assessment of the teleost intestine as a fluid-transporting epithelium. Beginning with a brief overview of marine teleost osmoregulation, emphasis shifts to the processing of ingested seawater by the gastrointestinal tract and the characteristics of intestinal ion and fluid transport. Particular attention is given to acid-base transfers by the intestine, specifically bicarbonate secretion, which creates the distinctly alkaline gut fluids responsible for the formation of solid calcium carbonate precipitates. The respective contributions of these unique features to intestinal fluid absorption, alongside other recognised ion transport processes, are then subsequently considered within the wider context of the classic physiological problem of epithelial water transport. PMID:21735220

  7. Cloning and in vitro characterization of a Schistosoma japonicum aquaglyceroporin that functions in osmoregulation

    PubMed Central

    Huang, Yuzheng; Li, Wei; Lu, Wuguang; Xiong, Chunrong; Yang, Yang; Yan, Huaijiang; Liu, Kun Connie; Cao, Peng

    2016-01-01

    As one of the three major human pathogens that cause schistosomiasis, Schistosoma japonicum is the only one that is endemic in China. Despite great progress on schistosomiasis control over the past 50 years in China, S. japonicum transmission still occurs in certain endemic regions, which causes significant public health problems and enormous economic losses. During different life stages, parasites are able to survive dramatic osmolality changes between its vector, fresh water, and mammal host. However, the molecular mechanism of parasite osmoregulation remains unknown. To address this challenging question, we report the first cloning of an S. japonicum aquaglyceroporin (SjAQP) from an isolate from Jiangsu province, China. Expressing SjAQP in Xenopus oocytes facilitated the permeation of water, glycerol, and urea. The water permeability of SjAQP was inhibited by 1 mM HgCl2, 3 mM tetraethylammonium, 1 mM ZnCl2, and 1 mM CuSO4. SjAQP was constitutively expressed throughout the S. japonicum life cycle, including in the egg, miracidia, cercaria, and adult stages. The highest expression was detected during the infective cercaria stage. Our results suggest that SjAQP plays a role in osmoregulation throughout the S. japonicum life cycle, especially during cercariae transformation, which enables parasites to survive osmotic challenges. PMID:27733755

  8. A comparative study of osmoregulation in four fiddler crabs (Ocypodidae: Uca).

    PubMed

    Lin, Hui-Chen; Su, Yong-Chao; Su, Shan-Hui

    2002-06-01

    This study aims to give an integrative description of the correlation of physiological parameters of osmoregulation and the habitats of the four common Uca species in Taiwan. Uca arcuata inhabits areas close to fresh water in the upper beach. Uca formosensis is only found in the areas near the mean high water of spring tide where there is a clear dry-wet transition within a single semilunar cycle. Uca vocans is found in the lower intertidal zone. Uca lactea, the most widely distributed species, can easily be found on most muddy sand shores. The number of gills was observed and histological sectioning performed on each species. The range of salinity in which the fiddler crabs maintained their hemolymph osmolality without any significant change (i.e. osmoregulatory homeostasis) and the gill Na(+), K(+)-ATPase activity were determined by transferring individuals to different salinity tanks. The results suggest that U. formosensis and U. lactea can sustain a wider range of salinity change through both modification in gill morphology and Na(+), K(+)-ATPase activity. Uca arcuata can regulate in a hypo-osmotic condition and U. vocans tends to be a weak-osmoregulator.

  9. Relaxed selection causes microevolution of seawater osmoregulation and gene expression in landlocked Alewives

    USGS Publications Warehouse

    Velotta, Jonathan P.; McCormick, Stephen D.; O'Neill, Rachel J.; Schultz, Eric T.

    2014-01-01

    Ecological transitions from marine to freshwater environments have been important in the creation of diversity among fishes. Evolutionary changes associated with these transitions likely involve modifications of osmoregulatory function. In particular, relaxed selection on hypo-osmoregulation should strongly affect animals that transition into novel freshwater environments. We used populations of the Alewife (Alosa pseudoharengus) to study evolutionary shifts in hypo-osmoregulatory capacity and ion regulation associated with freshwater transitions. Alewives are ancestrally anadromous, but multiple populations in Connecticut have been independently restricted to freshwater lakes; these landlocked populations complete their entire life cycle in freshwater. Juvenile landlocked and anadromous Alewives were exposed to three salinities (1, 20 and 30 ppt) in small enclosures within the lake. We detected strong differentiation between life history forms: landlocked Alewives exhibited reduced seawater tolerance and hypo-osmoregulatory performance compared to anadromous Alewives. Furthermore, gill Na+/K+-ATPase activity and transcription of genes for seawater osmoregulation (NKCC—Na+/K+/2Cl− cotransporter and CFTR—cystic fibrosis transmembrane conductance regulator) exhibited reduced responsiveness to seawater challenge. Our study demonstrates that adaptations of marine-derived species to completely freshwater life cycles involve partial loss of seawater osmoregulatory performance mediated through changes to ion regulation in the gill.

  10. Osmoregulation in the Avena coleoptile in relation to auxin and growth

    SciTech Connect

    Stevenson, T.T.; Cleland, R.E.

    1981-04-01

    A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e., to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin-induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2-deoxy-O-glucose or 3-O-methyl-O-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells.

  11. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole.

  12. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole. PMID:27311484

  13. Ontogeny of osmoregulation and salinity tolerance in a mangrove crab, Sesarma curacaoense (Decapoda: Grapsidae).

    PubMed

    Anger; Charmantier

    2000-08-30

    The grapsid crab Sesarma curacaoense is believed to represent the closest saltwater relative to the ancestor which gave rise to an adaptive radiation of endemic freshwater and terrestrial species on the island of Jamaica. Living in mangrove swamps with variable salinity conditions and showing semiterrestrial behaviour, S. curacaoense exhibits ecological adaptations to non-marine conditions. In laboratory experiments, we studied the salinity tolerance during development from hatching to the end of the first juvenile stage. Successful development through metamorphosis occurred in the full salinity range tested (15-32 per thousand), although mortality was significantly enhanced and development delayed at 15 per thousand. In another series of experiments, we studied the ontogeny of the capability for osmoregulation, which is considered as the physiological basis of osmotic stress tolerance. Our results show that S. curacaoense is from hatching a fairly strong hyperosmoregulator in dilute media. This capability increased gradually from hatching throughout the larval and juvenile development. In seawater (32 per thousand) and at an enhanced salt concentration (44 per thousand), the zoeal stages remained hyperosmoconformers. The capability for hypoosmoregulation in concentrated media appeared first in the megalopa stage and increased thereafter. Adult crabs were observed to be strong hyper-hypo-osmoregulators in a salinity range from at least 1 per thousand to 44 per thousand. The unusually early appearance of strong regulatory capabilities, particularly in dilute media, is interpreted as a physiological preadaptation that should have facilitated the evolutionary process of adaptive radiation in non-marine environments on Jamaica. PMID:10960618

  14. Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes.

    PubMed

    Graves, Amanda B; Horak, Erik H; Liptak, Matthew D

    2016-06-14

    Recent work by several groups has established that MhuD, IsdG, and IsdI are non-canonical heme oxygenases that induce significant out-of-plane ruffling distortions of their heme substrates enroute to mycobilin or staphylobilin formation. However, clear explanations for the observations of "nested" S = ½ VTVH MCD saturation magnetization curves at cryogenic temperatures, and exchange broadened (1)H NMR resonances at physiologically-relevant temperatures have remained elusive. Here, MCD and NMR data have been acquired for F23A and F23W MhuD-heme-CN, in addition to MCD data for IsdI-heme-CN, in order to complete assembly of a library of spectroscopic data for cyanide-inhibited ferric heme with a wide range of ruffling deformations. The spectroscopic data were used to evaluate a number of computational models for cyanide-inhibited ferric heme, which ultimately led to the development of an accurate NEVPT2/CASSCF model. The resulting model has a shallow, double-well potential along the porphyrin ruffling coordinate, which provides clear explanations for the unusual MCD and NMR data. The shallow, double-well potential also implies that MhuD-, IsdG-, and IsdI-bound heme is dynamic, and the functional implications of these dynamics are discussed. PMID:27273757

  15. Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

    PubMed Central

    Waheed, Syed Mohsin; Ghosh, Arnab; Chakravarti, Ritu; Biswas, Ashis; Haque, Mohammad Mahfuzul; Panda, Koustubh; Stuehr, Dennis J.

    2010-01-01

    Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time period, and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structure, heme ligation, and function (three NO synthases, two cytochrome P450’s, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme level, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process. PMID:20211245

  16. Heme activation by DNA: isoguanine pentaplexes, but not quadruplexes, bind heme and enhance its oxidative activity

    PubMed Central

    Shumayrikh, Nisreen; Huang, Yu Chuan; Sen, Dipankar

    2015-01-01

    Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na+ or NH4+ was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme. PMID:25824944

  17. Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism.

    PubMed

    Sheftel, Alex D; Kim, Sangwon F; Ponka, Prem

    2007-04-01

    The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

  18. Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

    PubMed

    Draganova, Elizabeth B; Akbas, Neval; Adrian, Seth A; Lukat-Rodgers, Gudrun S; Collins, Daniel P; Dawson, John H; Allen, Courtni E; Schmitt, Michael P; Rodgers, Kenton R; Dixon, Dabney W

    2015-11-01

    The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT. PMID:26478504

  19. Heme compounds in dinosaur trabecular bone.

    PubMed

    Schweitzer, M H; Marshall, M; Carron, K; Bohle, D S; Busse, S C; Arnold, E V; Barnard, D; Horner, J R; Starkey, J R

    1997-06-10

    Six independent lines of evidence point to the existence of heme-containing compounds and/or hemoglobin breakdown products in extracts of trabecular tissues of the large theropod dinosaur Tyrannosaurus rex. These include signatures from nuclear magnetic resonance and electron spin resonance that indicate the presence of a paramagnetic compound consistent with heme. In addition, UV/visible spectroscopy and high performance liquid chromatography data are consistent with the Soret absorbance characteristic of this molecule. Resonance Raman profiles are also consistent with a modified heme structure. Finally, when dinosaurian tissues were extracted for protein fragments and were used to immunize rats, the resulting antisera reacted positively with purified avian and mammalian hemoglobins. The most parsimonious explanation of this evidence is the presence of blood-derived hemoglobin compounds preserved in the dinosaurian tissues.

  20. Molecular Simulations of Porphyrins and Heme Proteins

    SciTech Connect

    SHELNUTT,JOHN A.

    2000-01-18

    An overview of the use of classical mechanical molecular simulations of porphyrins, hydroporphyrins, and heme proteins is given. The topics cover molecular mechanics calculations of structures and conformer energies of porphyrins, energies of barriers for interconversion between stable conformers, molecular dynamics of porphyrins and heme proteins, and normal-coordinate structural analysis of experimental and calculated porphyrin structures. Molecular mechanics and dynamics are currently a fertile area of research on porphyrins. In the future, other computational methods such as Monte Carlo simulations, which have yet to be applied to porphyrins, will come into use and open new avenues of research into molecular simulations of porphyrins.

  1. Osmoregulated Periplasmic Glucans (OPGs) of Salmonella enterica serovars Typhimurium are needed for optimal growth under nutrient limiting- hypoosmotic conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of gram negative bacteria. Synthesis of OPGs is regulated by the osmolarity of the growth medium. The role of OPGs has been postulated in plant-symbiotic as well as pathogenic microorganisms. On the other hand, opg mutant...

  2. Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes.

    PubMed

    Boudour-Boucheker, Nesrine; Boulo, Viviane; Charmantier-Daures, Mireille; Anger, Klaus; Charmantier, Guy; Lorin-Nebel, Catherine

    2016-05-01

    In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

  3. Shu1 is a cell-surface protein involved in iron acquisition from heme in Schizosaccharomyces pombe.

    PubMed

    Mourer, Thierry; Jacques, Jean-François; Brault, Ariane; Bisaillon, Martin; Labbé, Simon

    2015-04-17

    Iron is an essential metal cofactor that is required for many biological processes. Eukaryotic cells have consequently developed different strategies for its acquisition. Until now, Schizosaccharomyces pombe was known to use reductive iron uptake and siderophore-bound iron transport to scavenge iron from the environment. Here, we report the identification of a gene designated shu1(+) that encodes a protein that enables S. pombe to take up extracellular heme for cell growth. When iron levels are low, the transcription of shu1(+) is induced, although its expression is repressed when iron levels rise. The iron-dependent down-regulation of shu1(+) requires the GATA-type transcriptional repressor Fep1, which strongly associates with a proximal promoter region of shu1(+) in vivo in response to iron repletion. HA4-tagged Shu1 localizes to the plasma membrane in cells expressing a functional shu1(+)-HA4 allele. When heme biosynthesis is selectively blocked in mutated S. pombe cells, their ability to acquire exogenous hemin or the fluorescent heme analog zinc mesoporphyrin IX is dependent on the expression of Shu1. Further analysis by absorbance spectroscopy and hemin-agarose pulldown assays showed that Shu1 interacts with hemin, with a KD of ∼2.2 μm. Taken together, results reported here revealed that S. pombe possesses an unexpected pathway for heme assimilation, which may also serve as a source of iron for cell growth.

  4. Heme enzymes. Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.

    PubMed

    Casadei, Cecilia M; Gumiero, Andrea; Metcalfe, Clive L; Murphy, Emma J; Basran, Jaswir; Concilio, Maria Grazia; Teixeira, Susana C M; Schrader, Tobias E; Fielding, Alistair J; Ostermann, Andreas; Blakeley, Matthew P; Raven, Emma L; Moody, Peter C E

    2014-07-11

    Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

  5. Combinational biosynthesis of phycocyanobilin using genetically-engineered Escherichia coli.

    PubMed

    Ge, Baosheng; Li, Yan; Sun, Haixiang; Zhang, Shuai; Hu, Peijie; Qin, Song; Huang, Fang

    2013-05-01

    Genes of the key enzymes for phycocyanobilin (PCB) biosynthesis were cloned into E. coli and combinationally expressed to produce phycocyanobilin, with autologous heme as substrate. Culture conditions were optimized to achieve ~3 mg PCB/l. A protocol for the purification of recombinant phycocyanobilin was established using solvent extraction combined with chromatography, which resulted in a final yield of ~0.3 mg PCB/l with a purity >95 %. Recombinant phycocyanobilin could scavenge hydroxyl radicals with an EC50 of 0.1 μM. PMID:23307652

  6. Thirty years of heme catalases structural biology.

    PubMed

    Díaz, Adelaida; Loewen, Peter C; Fita, Ignacio; Carpena, Xavi

    2012-09-15

    About thirty years ago the crystal structures of the heme catalases from Penicillium vitale (PVC) and, a few months later, from bovine liver (BLC) were published. Both enzymes were compact tetrameric molecules with subunits that, despite their size differences and the large phylogenetic separation between the two organisms, presented a striking structural similarity for about 460 residues. The high conservation, confirmed in all the subsequent structures determined, suggested a strong pressure to preserve a functional catalase fold, which is almost exclusively found in these mono-functional heme catalases. However, even in the absence of the catalase fold an efficient catalase activity is also found in the heme containing catalase-peroxidase proteins. The structure of these broad substrate range enzymes, reported for the first time less than ten years ago from the halophilic archaebacterium Haloarcula marismortui (HmCPx) and from the bacterium Burkholderia pseudomallei (BpKatG), showed a heme pocket closely related to that of plant peroxidases, though with a number of unique modifications that enable the catalase reaction. Despite the wealth of structural information already available, for both monofunctional catalases and catalase-peroxidases, a number of unanswered major questions require continuing structural research with truly innovative approaches. PMID:22209752

  7. Activation of lactoperoxidase by heme-linked protonation and heme-independent iodide binding.

    PubMed

    Toyama, Akira; Tominaga, Aya; Inoue, Tatsuo; Takeuchi, Hideo

    2010-01-01

    Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

  8. GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

    PubMed Central

    Chakravarti, Ritu; Aulak, Kulwant S.; Fox, Paul L.; Stuehr, Dennis J.

    2010-01-01

    Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target. Surprisingly, we found that GAPDH plays a key role. GAPDH was associated with iNOS in cells. Pure GAPDH bound tightly to heme or to iNOS in an NO-sensitive manner. GAPDH knockdown inhibited heme insertion into iNOS and a GAPDH mutant with defective heme binding acted as a dominant negative inhibitor of iNOS heme insertion. Exposing cells to NO either from a chemical donor or by iNOS induction caused GAPDH to become S-nitrosylated at Cys152. Expressing a GAPDH C152S mutant in cells or providing a drug to selectively block GAPDH S-nitrosylation both made heme insertion into iNOS resistant to the NO inhibition. We propose that GAPDH delivers heme to iNOS through a process that is regulated by its S-nitrosylation. Our findings may uncover a fundamental step in intracellular heme trafficking, and reveal a mechanism whereby NO can govern the process. PMID:20921417

  9. Heme and erythropoieis: more than a structural role

    PubMed Central

    Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own synthesis and regulates the expression of several erythroid-specific genes. Heme is synthesized in developing erythroid progenitors by the stage of proerythroblast, through a series of eight enzymatic reactions divided between mitochondria and cytosol. Defects of heme synthesis in the erythroid lineage result in sideroblastic anemias, characterized by microcytic anemia associated to mitochondrial iron overload, or in erythropoietic porphyrias, characterized by porphyrin deposition in erythroid cells. Here, we focus on the heme biosynthetic pathway and on human erythroid disorders due to defective heme synthesis. The regulatory role of heme during erythroid differentiation is discussed as well as the heme-mediated regulatory mechanisms that allow the orchestration of the adaptive cell response to heme deficiency. PMID:24881043

  10. The crimson conundrum: heme toxicity and tolerance in GAS.

    PubMed

    Sachla, Ankita J; Le Breton, Yoann; Akhter, Fahmina; McIver, Kevin S; Eichenbaum, Zehava

    2014-01-01

    The massive erythrocyte lysis caused by the Group A Streptococcus (GAS) suggests that the β-hemolytic pathogen is likely to encounter free heme during the course of infection. In this study, we investigated GAS mechanisms for heme sensing and tolerance. We compared the minimal inhibitory concentration of heme among several isolates and established that excess heme is bacteriostatic and exposure to sub-lethal concentrations of heme resulted in noticeable damage to membrane lipids and proteins. Pre-exposure of the bacteria to 0.1 μM heme shortened the extended lag period that is otherwise observed when naive cells are inoculated into heme-containing medium, implying that GAS is able to adapt. The global response to heme exposure was determined using microarray analysis revealing a significant transcriptome shift that included 79 up regulated and 84 down regulated genes. Among other changes, the induction of stress-related chaperones and proteases, including groEL/ES (8x), the stress regulators spxA2 (5x) and ctsR (3x), as well as redox active enzymes were prominent. The heme stimulon also encompassed a number of regulatory proteins and two-component systems that are important for virulence. A three-gene cluster that is homologous to the pefRCD system of the Group B Streptococcus was also induced by heme. PefR, a MarR-like regulator, specifically binds heme with stoichiometry of 1:2 and protoporphyrin IX (PPIX) with stoichiometry of 1:1, implicating it is one of the GAS mediators to heme response. In summary, here we provide evidence that heme induces a broad stress response in GAS, and that its success as a pathogen relies on mechanisms for heme sensing, detoxification, and repair.

  11. The crimson conundrum: heme toxicity and tolerance in GAS

    PubMed Central

    Sachla, Ankita J.; Le Breton, Yoann; Akhter, Fahmina; McIver, Kevin S.; Eichenbaum, Zehava

    2014-01-01

    The massive erythrocyte lysis caused by the Group A Streptococcus (GAS) suggests that the β-hemolytic pathogen is likely to encounter free heme during the course of infection. In this study, we investigated GAS mechanisms for heme sensing and tolerance. We compared the minimal inhibitory concentration of heme among several isolates and established that excess heme is bacteriostatic and exposure to sub-lethal concentrations of heme resulted in noticeable damage to membrane lipids and proteins. Pre-exposure of the bacteria to 0.1 μM heme shortened the extended lag period that is otherwise observed when naive cells are inoculated into heme-containing medium, implying that GAS is able to adapt. The global response to heme exposure was determined using microarray analysis revealing a significant transcriptome shift that included 79 up regulated and 84 down regulated genes. Among other changes, the induction of stress-related chaperones and proteases, including groEL/ES (8x), the stress regulators spxA2 (5x) and ctsR (3x), as well as redox active enzymes were prominent. The heme stimulon also encompassed a number of regulatory proteins and two-component systems that are important for virulence. A three-gene cluster that is homologous to the pefRCD system of the Group B Streptococcus was also induced by heme. PefR, a MarR-like regulator, specifically binds heme with stoichiometry of 1:2 and protoporphyrin IX (PPIX) with stoichiometry of 1:1, implicating it is one of the GAS mediators to heme response. In summary, here we provide evidence that heme induces a broad stress response in GAS, and that its success as a pathogen relies on mechanisms for heme sensing, detoxification, and repair. PMID:25414836

  12. New Insights into the Biological Role of the Osmoregulated Periplasmic Glucans in Pathogenic and Symbiotic Bacteria

    PubMed Central

    Bontemps-Gallo, Sébastien; Lacroix, Jean-Marie

    2015-01-01

    Summary This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). OPGs occur in almost all α, β and γ Proteobacteria. This polymer of glucose is required for full virulence. The roles of the OPGs are complex and vary depending on the species. Here, we outline the four major roles of the OPGs through four different pathogenic and one symbiotic bacterial models (Dickeya dadantii, Salmonella enterica, Pseudomonas aeruginosa, Brucella abortus and Sinorhizobium meliloti). When periplasmic, the OPGs are a part of the signal transduction pathway and indirectly regulate genes involved in virulence. The OPGs can also be secreted. When outside of the cell, they interact directly with antibiotics to protect the bacterial cell or interact with the host cell to facilitate the invasion process. When OPGs are not found, as in the ε Proteobacteria, OPG-like oligosaccharides are present. Their presence strengthens the evidence that OPGs play an important role in virulence. PMID:26265506

  13. Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin-probe techniques

    PubMed Central

    Blumwald, Eduardo; Mehlhorn, Rolf J.; Packer, Lester

    1983-01-01

    Sucrose is accumulated in response to NaCl-induced stress in the cyanobacterium Synechococcus 6311. Internal cell volume was measured by ESR spectra with 2,2,6,6-tetramethyl-4-oxopiperidinoxy free radical (TEMPONE) as a spin probe in order to calculate sucrose concentrations inside the cell. This method is rapid and reliable and provides an unambiguous measurement of absolute volumes in different osmotic environments. Because the osmolar concentration of sucrose does not counter-balance the osmolar concentrations of ions in the growth medium, we suggest that sucrose accumulation is one of the mechanisms involved in the process of adaptation to salt of Synechococcus 6311. The accumulation of sucrose in non-N2-fixing cyanobacteria such as Synechococcus 6311 and in N2-fixing cyanobacteria such as Nostoc muscorum suggests a common mechanism of osmoregulation of fresh water cyanobacteria in response to increasing NaCl concentrations in the growth medium. Images PMID:16593309

  14. Is the weight loss of hyperbaric habitation a disorder of osmoregulation

    NASA Technical Reports Server (NTRS)

    Raymond, L. W.; Raymond, N. S.; Frattiali, V. P.; Sode, J.; Leach, C. S.; Spaur, W. H.

    1980-01-01

    To examine the weight loss of hyperbaric helium-oxygen habitation, the exchange of liquids and calories was measured in six men who lived in this atmosphere for 32 d. The maximum pressure was 49.5 ATA. The men lost 3.7-10.1 kg, in spite of warm ambient (31-32 C) temperatures and adequate calories (2,737 kcal/d) provided for the sedentary ways of chamber living. Weight loss and a calculated fluid deficit were accompanied by significant hemoconcentration, shown by increases in serum proteins. These changes were followed by a rise in urinary aldosterone and vasopressin, but not thirst. Weight loss in hyperbaric atmospheres is probably multifactorial, but the data suggests an uncoupling of normal osmoregulation may have occured in the present set of subjects. This may have been due to altered lung mechanics, increased catecholamines, or effects of high pressure on cellular responses to vasopressin.

  15. Heme Distortions in Sperm-Whale Carbonmonoxy Myoglobin: Correlations between Rotational Strengths and Heme Distortions in MD-Generated Structures

    SciTech Connect

    KIEFL,CHRISTOPH; SCREERAMA,NARASIMHA; LU,YI; QIU,YAN; SHELNUTT,JOHN A.; WOODY,ROBERT W.

    2000-07-13

    The authors have investigated the effects of heme rotational isomerism in sperm-whale carbonmonoxy myoglobin using computational techniques. Several molecular dynamics simulations have been performed for the two rotational isomers A and B, which are related by a 180{degree} rotation around the {alpha}-{gamma} axis of the heme, of sperm-whale carbonmonoxy myoglobin in water. Both neutron diffraction and NMR structures were used as starting structures. In the absence of an experimental structure, the structure of isomer B was generated by rotating the heme in the structure of isomer A. Distortions of the heme from planarity were characterized by normal coordinate structural decomposition and by the angle of twist of the pyrrole rings from the heme plane. The heme distortions of the neutron diffraction structure were conserved in the MD trajectories, but in the NMR-based trajectories, where the heme distortions are less well defined, they differ from the original heme deformations. The protein matrix induced similar distortions on the heroes in orientations A and B. The results suggest that the binding site prefers a particular macrocycle conformation, and a 180{degree} rotation of the heme does not significantly alter the protein's preference for this conformation. The intrinsic rotational strengths of the two Soret transitions, separated according to their polarization in the heme plane, show strong correlations with the ruf-deformation and the average twist angle of the pyrrole rings. The total rotational strength, which includes contributions from the chromophores in the protein, shows a weaker correlation with heme distortions.

  16. Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

    PubMed

    Rivera-Ingraham, Georgina A; Barri, Kiam; Boël, Mélanie; Farcy, Emilie; Charles, Anne-Laure; Geny, Bernard; Lignot, Jehan-Hervé

    2016-01-01

    Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2 weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress.

  17. [Effects of abrupt salinity stress on osmoregulation of juvenile Rachycentron canadum].

    PubMed

    Xu, Li-Wen; Liu, Guang-Feng; Wang, Rui-Xuan; Su, You-Lu; Guo, Zhi-Xun; Feng, Juan

    2007-07-01

    Rachycentron canadum is a thriving mariculture species for offshore cage in southern Mainland and Taiwan of China, due to its rapid growth rate and high quality flesh. In this paper, the gill Na(+)-K+ ATPase (NKA) activity and iono- and osmoregulation of juvenile R. canadum were investigated in a 12 h stress of ambient salinities (0-45), and the results showed that after an abrupt transfer to the salinities of 0, 5, 15, 25, 37 (control) and 45, the death of juvenile R. canadum only occurred in salinity 0, with a mortality of 100% by the end of the experiment. In all treatments, the gill NKA activity and serum osmolality fluctuated in first 3 h, and then changed smoothly. The NKA activity varied with salinity grade in U shape, being significantly (P < 0.05) higher in salinity 5 and the lowest in salinity 15 in 12 h, while the serum osmolality (ranged 293-399 mOsmol x kg(-1)) presented a positive correlation with salinity. Serum [Na+] and [Cl-] concentration slightly increased with salinity within the period of 3-12 h, while serum [K+] displayed a reverse pattern. The isosmotic point was estimated as 328.2 mOsm x kg(-1) and corresponded to salinity 11.48. The isoionic points for serum [Na+], [K+] and [Cl-] were estimated as 155.2, 6.16, and 137.1 mmol x L(-1), which corresponded to the salinities of 10.68, 20.44 and 8.41, respectively. It was concluded that R. canadum could be characterized physiologically as a "higher-NKA-in-hyposmotic media" marine euryhaline teleost with the capability of rapid and effective hyper/hypo iono- and osmoregulation.

  18. The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa.

    PubMed

    Wegele, Rosalina; Tasler, Ronja; Zeng, Yuhong; Rivera, Mario; Frankenberg-Dinkel, Nicole

    2004-10-29

    For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.

  19. Single or functionalized fullerenes interacting with heme group

    SciTech Connect

    Costa, Wallison Chaves; Diniz, Eduardo Moraes

    2014-09-15

    The heme group is responsible for iron transportation through the bloodstream, where iron participates in redox reactions, electron transfer, gases detection etc. The efficiency of such processes can be reduced if the whole heme molecule or even the iron is somehow altered from its original oxidation state, which can be caused by interactions with nanoparticles as fullerenes. To verify how such particles alter the geometry and electronic structure of heme molecule, here we report first principles calculations based on density functional theory of heme group interacting with single C{sub 60} fullerene or with C{sub 60} functionalized with small functional groups (−CH{sub 3}, −COOH, −NH{sub 2}, −OH). The calculations shown that the system heme + nanoparticle has a different spin state in comparison with heme group if the fullerene is functionalized. Also a functional group can provide a stronger binding between nanoparticle and heme molecule or inhibit the chemical bonding in comparison with single fullerene results. In addition heme molecule loses electrons to the nanoparticles and some systems exhibited a geometry distortion in heme group, depending on the binding energy. Furthermore, one find that such nanoparticles induce a formation of spin up states in heme group. Moreover, there exist modifications in density of states near the Fermi energy. Although of such changes in heme electronic structure and geometry, the iron atom remains in the heme group with the same oxidation state, so that processes that involve the iron might not be affected, only those that depend on the whole heme molecule.

  20. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  1. The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

    PubMed Central

    Wu, Bo; Novelli, Jacopo; Foster, Jeremy; Vaisvila, Romualdas; Conway, Leslie; Ingram, Jessica; Ganatra, Mehul; Rao, Anita U.; Hamza, Iqbal; Slatko, Barton

    2009-01-01

    Background Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. Methods and Findings Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a ∼600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5

  2. 21 CFR 862.1410 - Iron (non-heme) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Iron (non-heme) test system. 862.1410 Section 862....1410 Iron (non-heme) test system. (a) Identification. An iron (non-heme) test system is a device intended to measure iron (non-heme) in serum and plasma. Iron (non-heme) measurements are used in...

  3. 21 CFR 862.1410 - Iron (non-heme) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Iron (non-heme) test system. 862.1410 Section 862....1410 Iron (non-heme) test system. (a) Identification. An iron (non-heme) test system is a device intended to measure iron (non-heme) in serum and plasma. Iron (non-heme) measurements are used in...

  4. 21 CFR 862.1410 - Iron (non-heme) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Iron (non-heme) test system. 862.1410 Section 862....1410 Iron (non-heme) test system. (a) Identification. An iron (non-heme) test system is a device intended to measure iron (non-heme) in serum and plasma. Iron (non-heme) measurements are used in...

  5. The Biosynthesis of Capuramycin-type Antibiotics

    PubMed Central

    Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D.; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J.; Spork, Anatol P.; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S.; Van Lanen, Steven G.

    2015-01-01

    A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5′-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5′-aldehyde transaldolase were uncovered, suggesting that C–C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5′-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. PMID:25855790

  6. A study of the electrical polarization of Sepia officinalis yolk envelope, a role for Na+/K+-ATPases in osmoregulation?

    PubMed Central

    Bonnaud, Laure; Franko, Delphine; Vouillot, Léna; Bouteau, François

    2013-01-01

    The cuttlefish Sepia officinalis mate and spawn in the intertidal zone where eggs are exposed during low tide to osmotic stress. Embryonic outer yolk sac is a putative site for osmoregulation of young S. officinalis embryos. By using electrophysiological recordings and immunostaining we showed, (i) that the chorion is only a passive barrier for ions, since large molecules could not pass through it, (ii) that a complex transepithelial potential difference occurs through the yolk epithelium, (iii) that ionocyte-like cells and Na+/K+-ATPases were localized in the yolk epithelium and (iv) that ouabain sensitive Na+/K+-ATPase activity could participate to this yolk polarization. These data warrant further study on the role of ion transport systems of this epithelium in the osmoregulation processes in S. officinalis embryos. PMID:24505501

  7. Proline 107 Is a Major Determinant in Maintaining the Structure of the Distal Pocket and Reactivity of the High-Spin Heme of MauG

    SciTech Connect

    Feng, Manliang; Jensen, Lyndal M.R.; Yukl, Erik T.; Wei, Xiaoxi; Liu, Aimin; Wilmot, Carrie M.; Davidson, Victor L.

    2012-05-09

    The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H{sub 2}O{sub 2} and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

  8. Time-resolved heme protein intermediates

    NASA Astrophysics Data System (ADS)

    Rousseau, Denis

    2005-03-01

    To determine the enzymatic mechanisms of heme proteins, it is necessary to identify the intermediates along the catalytic pathway and measure the times of their formation and decay. Resonance Raman scattering spectra are especially powerful for obtaining such information as the electronic structure of the heme group and the nature of the ligand coordinated to the heme iron atom may be monitored. The oxygen intermediates of two physiologically important enzymes will be presented. Nitric oxide synthase (NOS) uses oxygen to convert arginine to NO and citrulline; and cytochrome c oxidase (CcO) reduces oxygen to water to support oxidative phosphorylation. The fate or the oxygen in each of these enzymes has been followed by resonance Raman scattering. In NOS the oxygen is slowly converted to an activated species that then reacts fast, whereas in CcO the oxygen is rapidly converted to a reactive species that subsequently reacts slowly. The properties of the intermediates and the origin of the differences between these enzymes will be discussed.

  9. Fe-heme conformations in ferric myoglobin.

    PubMed Central

    Longa, S D; Pin, S; Cortès, R; Soldatov, A V; Alpert, B

    1998-01-01

    X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry. PMID:9826636

  10. Mimicking Heme Enzymes in the Solid State: Metal-Organic Materials with Selectively Encapsulated Heme

    SciTech Connect

    Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

    2011-06-13

    To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal–organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a “ship-in-a-bottle” fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

  11. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  12. Trypanosome Glycosylphosphatidylinositol Biosynthesis

    PubMed Central

    Kinoshita, Taroh

    2009-01-01

    Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis. PMID:19724691

  13. Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine.

    PubMed Central

    Smith, L T; Smith, G M; Madkour, M A

    1990-01-01

    We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed. PMID:2254260

  14. Heme compounds as iron sources for nonpathogenic Rhizobium bacteria.

    PubMed Central

    Noya, F; Arias, A; Fabiano, E

    1997-01-01

    Many animal-pathogenic bacteria can use heme compounds as iron sources. Like these microorganisms, rhizobium strains interact with host organisms where heme compounds are available. Results presented in this paper indicate that the use of hemoglobin as an iron source is not restricted to animal-pathogenic microorganisms. We also demonstrate that heme, hemoglobin, and leghemoglobin can act as iron sources under iron-depleted conditions for Rhizobium meliloti 242. Analysis of iron acquisition mutant strains indicates that siderophore-, heme-, hemoglobin-, and leghemoglobin-mediated iron transport systems expressed by R. meliloti 242 share at least one component. PMID:9139934

  15. Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

    PubMed

    Horáková, Eva; Changmai, Piya; Paris, Zdeněk; Salmon, Didier; Lukeš, Julius

    2015-11-01

    ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei. PMID:26277108

  16. Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

    PubMed

    Horáková, Eva; Changmai, Piya; Paris, Zdeněk; Salmon, Didier; Lukeš, Julius

    2015-11-01

    ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei.

  17. A transcriptomic scan for potential candidate genes involved in osmoregulation in an obligate freshwater palaemonid prawn (Macrobrachium australiense)

    PubMed Central

    Rahi, Md. Lifat; Nguyen, Viet Tuan; Mather, Peter B.; Hurwood, David A.

    2016-01-01

    Background Understanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype) addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance) ability is the main mechanism to overcome the problems related to environmental salinity gradients. Methods To better understand how osmoregulatory performance in freshwater (FW) crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i), reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii), performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas) and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions. Results We obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp) with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO) terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million) ≥20 showed 1625 transcripts commonly expressed in all four libraries. Among the

  18. Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

    PubMed Central

    Kitatsuji, Chihiro; Izumi, Kozue; Nambu, Shusuke; Kurogochi, Masaki; Uchida, Takeshi; Nishimura, Shin-Ichiro; Iwai, Kazuhiro; O’Brian, Mark R.; Ikeda-Saito, Masao; Ishimori, Koichiro

    2016-01-01

    The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the “active site conversion” from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel “two-step self-oxidative modification” mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion. PMID:26729068

  19. Characterization of heme binding to recombinant α1-microglobulin

    PubMed Central

    Karnaukhova, Elena; Rutardottir, Sigurbjörg; Rajabi, Mohsen; Wester Rosenlöf, Lena; Alayash, Abdu I.; Åkerström, Bo

    2014-01-01

    Background: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme1 and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. Methods: To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. Results: The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with KD values in a μM range. Conclusions: This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins. PMID:25538624

  20. Heme Recognition By a Staphylococcus Aureus IsdE

    SciTech Connect

    Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.; Murphy, M.E.P.

    2009-06-03

    Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single {alpha}-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met{sup 78} and His{sup 229}. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His{sup 299} is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

  1. Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ.

    PubMed

    Celis, Arianna I; Streit, Bennett R; Moraski, Garrett C; Kant, Ravi; Lash, Timothy D; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; DuBois, Jennifer L

    2015-07-01

    A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out. PMID:26083961

  2. Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

    PubMed

    Ebrahimi, Aida; Alam, Muhammad Ashraful

    2016-06-28

    Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplication (growth) to reach a detectable threshold. The cells are confined in an evaporating droplet that serves as a dynamic microenvironment. Evaporation-induced increase in ionic concentration is reflected in a proportional increase of the droplet's osmotic pressure, which in turn, stimulates the osmoregulatory response from the cells. By monitoring the time-varying electrical conductance of evaporating droplets, bacterial cells are identified within a few minutes compared with several hours in growth-based methods. To show the versatility of the proposed method, we show detection of WT and genetically modified nonhalotolerant cells (Salmonella typhimurium) and dead vs. live differentiation of nonhalotolerant (such as Escherichia coli DH5α) and halotolerant cells (such as Staphylococcus epidermidis). Unlike the growth-based techniques, the assay time of the proposed method is independent of cell concentration or the bacteria type. The proposed label-free approach paves the road toward realization of a new class of real time, array-formatted electrical sensors compatible with droplet microfluidics for laboratory on a chip applications.

  3. Plasma membrane potential of the alga dunaliella, and its relation to osmoregulation.

    PubMed

    Oren-Shamir, M; Pick, U; Avron, M

    1990-06-01

    A fluorescent dye sensitive to membrane potential was used to follow the plasma-membrane potential in the unicellular halo-tolerant alga Dunaliella salina. The signal observed during dissipation of the plasma membrane potential by the addition of excess K(+) and valinomycin, or a protonophore, was taken as a measure of the preexisting potential. A resting potential of -85 to -100 millivolts (negative inside) was calculated. Following a hypertonic shock, the plasma membrane was rapidly hyperpolarized. This hyperpolarization was transient, and the algae resumed their resting potential about 30 minutes after the shock. The resting plasma membrane potential was decreased by vanadate and is concluded to be generated mostly by the plasma membrane ATPase of Dunaliella. The transient hyperpolarization following a hypertonic shock indicates, therefore, a transient activation of the ATPase. This is further corroborated by a rapid transient decrease in the intracellular ATP following a hypertonic shock and its inhibition by vanadate. It is suggested that activation of the plasma membrane ATPase may be the trigger for osmoregulation in Dunaliella. PMID:16667480

  4. Acute extracellular ethanol load does not produce hyponatremia by internal osmoregulation

    SciTech Connect

    Jackson, J.E.; Tzamaloukas, A.H.; Long, D.A.

    1986-03-05

    Hyponatremia is frequently present in subjects intoxicated with ethanol. To study whether an acute increase in extracellular osmolality by addition of ethanol creates any clinically appreciable osmotic shift of intracellular water extracellularly, they infused over 20 sec 11 mmol/kg of ethanol intravenously into 5 anesthetized dogs (2 with intact renal function, 3 anuric) and measured plasma sodium and ethanol concentrations and osmolality at frequent intervals for 100 min after the end of the infusion. For a range of ethanol concentration between 4 and 120 mmol/l, changes in osmolality were equal to ethanol concentration in plasma water (y = -0.49 + 1.06 x mosm/kg per mmol/l, r = 0.981, p < 0.01). Plasma sodium concentration remained unchanged from baseline throughout the experiments, even at 1 min post-infusion, when osmolality was 78 +/- 25 mosm/kg above the baseline. An acute increase in extracellular osmolality created by rapid intravenous infusion of a large dose of ethanol does not create any osmotic shift of intracellular water extracellularly, that can be detected by dilution of extracellular sodium. The mechanism of hyponatremia in ethanol intoxication is not internal osmoregulation, but abnormalities in external balance of body water and/or solute.

  5. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

    PubMed Central

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T.; Petalcorin, Mark I. R.; Dolphin, Colin T.

    2016-01-01

    ABSTRACT Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  6. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis.

    PubMed

    Peña-Villalobos, Isaac; Narváez, Cristóbal; Sabat, Pablo

    2016-01-01

    Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  7. Osmoregulation and salt gland Na, K-ATPase activity following exposure to the anticholinesterase fenthion

    USGS Publications Warehouse

    Rattner, B.A.; Fleming, W.J.; Murray, H.C.

    1982-01-01

    Salt gland function and osmoregulation in aquatic birds drinking hyperosmotic water has been suggested to be impaired by organophosphorus insecticides. To test this hypothesis, adult ducks (Anas rubripes) were provided various regimens of fresh or salt (1.5% NaCl) water (FW, SW) and mash containing vehicle or 21 ppm fenthion (Fn) on days 1-7 and 7-12 of this study. The 8 treatments (day 1-7:day 7-12) included :FW:FW, FW:FW+Fn, FW:SW, FW+Fn:SW, FW:SW+Fn, FW+Fn:SW+FN, SW;SW, and SW:5W+Fn. Ducks were bled by jugular venipuncture on days 1,7 and 12, and then sacrificed. Brain and salt gland acetylcholinesterase activities were substantially inhibited (44-52% and 14-26%) by Fn. However, plasma Na, Cl and osmolality, as indirect but cumulative indices of salt gland function, were uniformly elevated in all SW groups including those receiving Fn. In a second experiment, salt gland Na,K-ATPase activity was reduced after in vitro incubation with DDE (40 and 400 ?M; positive control), but was unaffected by Fn and its oxygen analog (0.04-400 ?M). The present findings suggest that environmentally realistic concentrations of organophosphorus insecticides do not affect osmoregulatory function in adult ducks.

  8. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis

    PubMed Central

    Peña-Villalobos, Isaac; Narváez, Cristóbal

    2016-01-01

    ABSTRACT Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  9. Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon Acipenser schrenckii

    NASA Astrophysics Data System (ADS)

    Zhao, Feng; Zhuang, Ping; Zhang, Longzhen; Hou, Junli

    2010-05-01

    We evaluated the ability of juvenile Amur sturgeon ( Acipenser schrenckii) to osmoregulate and grow in saltwater. Hatchery-reared juveniles (mean weight 106.8 g, 5-month old) were transferred from freshwater to 10, 20, and 25 salinity saltwater over a period of 20 d. We measured the growth, serum osmolality, ion concentrations, and Na+/K+-ATPase activity. In addition, we prepared samples of gill tissue to quantify morphological changes in gill ultrastructure. Rearing in up to 25 saltwater for 30 d had no significant effect on growth. Similarly, serum osmolality and ion concentrations were similar to levels reported in other teleosts following acclimation to saltwater. Serum osmolality and Na+, Cl- concentrations increased significantly with the initial increase in salinity. Afterwards, levels tended to stabilize and then decrease. Serum K+ levels did not change during acclimation to saltwater. Gill Na+/K+-ATPase activity increased initially as salinity was increased. However, the activity later decreased and, finally stabilized at 3.7±0.1 μmol Pi/mg·prot·h in 25 saltwater (1.6 times higher than the level in those in freshwater). In fish that were held only in freshwater, the chloride cells were located in the interlamellar regions of the filament and at the base of the lamella. Following acclimation to 25 saltwater for 30 d, the number and size of chloride cells increased significantly. Our results suggest that juvenile Amur sturgeon is able to tolerate, and grow in, relatively high concentrations of saltwater.

  10. Biological significance and applications of heme c proteins and peptides.

    PubMed

    Kleingardner, Jesse G; Bren, Kara L

    2015-07-21

    Hemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme c, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme c properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented. Proteins that bind heme c are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes c (cyts c) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts c, the functional significance of heme covalent attachment has remained elusive. One observation is that heme c reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme b, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts c and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme c lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt c. Another consequence of heme covalent attachment is the strong vibrational coupling seen

  11. Bone marrow: its contribution to heme catabolism.

    PubMed

    Mähönen, Y; Anttinen, M; Vuopio, P; Tenhunen, R

    1976-01-01

    Heme oxygenase (HO) and biliverdin reductase (BR), the two NADPH-dependent enzymes involved in the degradation of hemoglobin and its derivatives, were measured in bone marrow aspirates from 5 hematologically normal persons, 4 patients with chronic leucemia (CL), 11 patients with acute leucemia (AL), 8 patients with refractory sideroblastic anemia (RA), 7 patients with iron-deficiency anemia (IA), 5 patients with hemolytic anemia (HA), and 7 patients with secondary anemia (SA) to determine the enzymatic capacity of the bone marrow in different hematologic disorders for heme catabolism. HO activity in the bone marrow of normal persons was 0.42 +/- 0.28 (SD) nmoles bilirubin/10 mg protein/min; in CL, 2.15 +/- 1.34; in AL, 0.39 +/- 0.25; in RA, 0.58 +/- 0.37; in IA, 0.41 +/- 0.28; in HA, 2.56 +/- 1.40; and in SA, 1.72 +/- 1.06. BR activity, respectively, was in normal persons 8.7 +/- 2.4 (SD) nmoles bilirubin/10 mg protein/min; in CL, 13.6 +/- 9.1; in AL, 3.8 +/- 3.1 in RA, 5.1 +/- 2.7; in IA, 5.5 +/- 3.7; in HA, 17.0 +/- 7.2; and in SA, 10.5 +/- 4.2. On the basis of these findings it seems evident that both oxygenase and biliverdin reductase activities of the bone marrow are capable of adaptive regulation. The physiologic role of bone marrow in heme catabolism seems to be of significant importance.

  12. Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

    PubMed Central

    Kranz, Robert G.; Richard-Fogal, Cynthia; Taylor, John-Stephen; Frawley, Elaine R.

    2009-01-01

    Summary: Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments. PMID:19721088

  13. Staphylococcus lugdunensis IsdG liberates iron from host heme.

    PubMed

    Haley, Kathryn P; Janson, Eric M; Heilbronner, Simon; Foster, Timothy J; Skaar, Eric P

    2011-09-01

    Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization.

  14. Role of heme in bromine-induced lung injury.

    PubMed

    Lam, Adam; Vetal, Nilam; Matalon, Sadis; Aggarwal, Saurabh

    2016-06-01

    Bromine (Br2 ) gas inhalation poses an environmental and occupational hazard resulting in high morbidity and mortality. In this review, we underline the acute lung pathology (within 24 h of exposure) and potential therapeutic interventions that may be utilized to mitigate Br2 -induced human toxicity. We discuss our latest published data, which suggest that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. Our study was based on previous findings that demonstrated that Br2 upregulates the heme-degrading enzyme heme oxygenase-1 (HO-1), which converts toxic heme into bilverdin. Interestingly, following Br2 inhalation, heme levels were indeed elevated in bronchoalveolar lavage fluid, plasma, and whole lung tissue in C57BL/6 mice. High heme levels correlated with increased lung oxidative stress, lung inflammation, respiratory acidosis, lung edema, higher airway resistance, and mortality. However, therapeutic reduction of heme levels, by either scavenging with hemopexin or degradation by HO-1, improved lung function and survival. Therefore, heme attenuation may prove a useful adjuvant therapy to treat patients after Br2 exposure. PMID:27244263

  15. ARSENITE INDUCTION OF HEME OXYGENASE AS A BIOMARKER

    EPA Science Inventory

    ARSENITE INDUCTION OF HEME OXYGENASE AS A BIOMARKER

    Useful biomarkers of arsenic effects in both experimental animals and humans are needed. Arsenate and arsenite are good inducers of rat hepatic and renal heme oxygenase (HO); monomethylarsonic acid (MMA) and dimethylarsi...

  16. Role of heme in bromine-induced lung injury.

    PubMed

    Lam, Adam; Vetal, Nilam; Matalon, Sadis; Aggarwal, Saurabh

    2016-06-01

    Bromine (Br2 ) gas inhalation poses an environmental and occupational hazard resulting in high morbidity and mortality. In this review, we underline the acute lung pathology (within 24 h of exposure) and potential therapeutic interventions that may be utilized to mitigate Br2 -induced human toxicity. We discuss our latest published data, which suggest that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. Our study was based on previous findings that demonstrated that Br2 upregulates the heme-degrading enzyme heme oxygenase-1 (HO-1), which converts toxic heme into bilverdin. Interestingly, following Br2 inhalation, heme levels were indeed elevated in bronchoalveolar lavage fluid, plasma, and whole lung tissue in C57BL/6 mice. High heme levels correlated with increased lung oxidative stress, lung inflammation, respiratory acidosis, lung edema, higher airway resistance, and mortality. However, therapeutic reduction of heme levels, by either scavenging with hemopexin or degradation by HO-1, improved lung function and survival. Therefore, heme attenuation may prove a useful adjuvant therapy to treat patients after Br2 exposure.

  17. Mechanisms of Peroxynitrite Interactions with Heme Proteins

    PubMed Central

    Su, Jia; Groves, John T.

    2010-01-01

    Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. In this Forum we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve nitric oxide and peroxynitrite. We have recently described direct evidence that ferrylMb and NO2 are both produced during the reaction of PN and metmyolgobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [FeIV=O .NO2]. This caged pair reacts mainly via internal return with a rate constant kr to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, kobs of heme-mediated PN decomposition. Freely-diffusing NO2, which is liberated concomitantly from the radical pair (ke), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a β-sheet structure, at the expense of turns and helices at low pH. It is proposed that a short β-sheet segment, comprising residues 37-39 and 58-61, extends itself into the large 37-61 loop when the latter is destabilized by protonation of H26, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in

  18. Multi-heme proteins: Nature's electronic multi-purpose tool

    PubMed Central

    Bewley, Kathryn D.; Ellis, Katie E.; Firer-Sherwood, Mackenzie A.; Elliott, Sean J.

    2013-01-01

    While iron is often a limiting nutrient to Biology, when the element is found in the form of heme cofactors (iron protoporphyrin IX), living systems have exceled at modifying and tailoring the chemistry of the metal. In the context of proteins and enzymes, heme cofactors are increasingly found in stoichiometries greater than one, where a single protein macromolecule contains more than one heme unit. When paired or coupled together, these protein associated heme groups perform a wide variety of tasks, such as redox communication, long range electron transfer and storage of reducing/oxidizing equivalents. Here, we review recent advances in the field of multi-heme proteins, focusing on emergent properties of these complex redox proteins, and strategies found in Nature where such proteins appear to be modular and essential components of larger biochemical pathways. PMID:23558243

  19. Multi-heme proteins: nature's electronic multi-purpose tool.

    PubMed

    Bewley, Kathryn D; Ellis, Katie E; Firer-Sherwood, Mackenzie A; Elliott, Sean J

    2013-01-01

    While iron is often a limiting nutrient to Biology, when the element is found in the form of heme cofactors (iron protoporphyrin IX), living systems have excelled at modifying and tailoring the chemistry of the metal. In the context of proteins and enzymes, heme cofactors are increasingly found in stoichiometries greater than one, where a single protein macromolecule contains more than one heme unit. When paired or coupled together, these protein associated heme groups perform a wide variety of tasks, such as redox communication, long range electron transfer and storage of reducing/oxidizing equivalents. Here, we review recent advances in the field of multi-heme proteins, focusing on emergent properties of these complex redox proteins, and strategies found in Nature where such proteins appear to be modular and essential components of larger biochemical pathways. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

  20. Posttranslational stability of the heme biosynthetic enzyme ferrochelatase is dependent on iron availability and intact iron-sulfur cluster assembly machinery.

    PubMed

    Crooks, Daniel R; Ghosh, Manik C; Haller, Ronald G; Tong, Wing-Hang; Rouault, Tracey A

    2010-01-28

    Mammalian ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, possesses an iron-sulfur [2Fe-2S] cluster that does not participate in catalysis. We investigated ferrochelatase expression in iron-deficient erythropoietic tissues of mice lacking iron regulatory protein 2, in iron-deficient murine erythroleukemia cells, and in human patients with ISCU myopathy. Ferrochelatase activity and protein levels were dramatically decreased in Irp2(-/-) spleens, whereas ferrochelatase mRNA levels were increased, demonstrating posttranscriptional regulation of ferrochelatase in vivo. Translation of ferrochelatase mRNA was unchanged in iron-depleted murine erythroleukemia cells, and the stability of mature ferrochelatase protein was also unaffected. However, the stability of newly formed ferrochelatase protein was dramatically decreased during iron deficiency. Ferrochelatase was also severely depleted in muscle biopsies and cultured myoblasts from patients with ISCU myopathy, a disease caused by deficiency of a scaffold protein required for Fe-S cluster assembly. Together, these data suggest that decreased Fe-S cluster availability because of cellular iron depletion or impaired Fe-S cluster assembly causes reduced maturation and stabilization of apo-ferrochelatase, providing a direct link between Fe-S biogenesis and completion of heme biosynthesis. We propose that decreased heme biosynthesis resulting from impaired Fe-S cluster assembly can contribute to the pathogenesis of diseases caused by defective Fe-S cluster biogenesis. PMID:19965627

  1. Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

    PubMed

    Jiang, Yongying; Trnka, Michael J; Medzihradszky, Katalin F; Ouellet, Hugues; Wang, Yongqiang; Ortiz de Montellano, Paul R

    2009-03-01

    To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.

  2. Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments.

    PubMed

    Fujita, Yuichi; Tsujimoto, Ryoma; Aoki, Rina

    2015-01-01

    Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes. PMID:25830590

  3. Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments

    PubMed Central

    Fujita, Yuichi; Tsujimoto, Ryoma; Aoki, Rina

    2015-01-01

    Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes. PMID:25830590

  4. Effect of low temperature on chlorophyll biosynthesis in albinism line of wheat (Triticum aestivum) FA85.

    PubMed

    Liu, Xiao-Gang; Xu, Hong; Zhang, Jing-Ya; Liang, Guang-Wang; Liu, Ying-Tuan; Guo, Ai-Guang

    2012-07-01

    The 'stage albinism line of winter wheat' FA85 exhibits a severe block in chlorophyll (Chl) biosynthesis with prolonged low-temperature treatment. The correlations between leaf color and low temperature provide more comprehensive understanding of low temperature as an environmental signal that regulate the metabolic changes in the entire Chl-synthesizing pathway. In this study, we investigated differences in Chl biosynthesis between leaves of Aibian1 and FA85 by measuring their Chl precursors and heme content, transcripts for key genes of Chl biosynthesis and key enzyme activities. With prolonged low-temperature treatment, the Chl content gradually decreased, but Chl precursors, including protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide (Pchlide), simultaneously accumulated. Parallel to the decline in Chl content, the protoporphyrin IX distribution toward Chl synthesis was less than that in heme synthesis in the leaves of FA85. Corresponding to the change of protoporphyrin IX distribution, the relative changes in magnesium chelatase (EC 6.6.1.1) and ferrochelatase (EC 4.99.1.1) activities in the leaves of FA85 also indirectly reflected channeling of the metabolic flow into heme rather than Chl. A drastic loss in the transcripts for Pchlide oxidoreductase (EC 1.3.1.33) and Chl synthase (EC 2.5.1.62) accounted for a decrease in the metabolic flux and the re-direction of metabolites. The high-level accumulations of Chl precursors and traces of Chl in the leaves of FA85 suggest that a severe block between the steps from Pchlide to Chl formation during Chl biosynthesis is partially derived from the transcriptional downregulation of Pchlide oxidoreductase and Chl synthase.

  5. Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

    PubMed Central

    Gryczynski, Z; Tenenholz, T; Bucci, E

    1992-01-01

    Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear. PMID:1420905

  6. Absorption in the Q-band region by isolated ferric heme+ and heme+(histidine) in vacuo.

    PubMed

    Wyer, Jean Ann; Brøndsted Nielsen, Steen

    2010-08-28

    Absorption by heme proteins is determined by the heme microenvironment that is often vacuumlike (hydrophobic pocket). Here we provide absorption spectra in the Q-band region of isolated ferric heme(+) and heme(+)(histidine) ions in vacuo to be used as references in protein biospectroscopy. Ions were photoexcited in an electrostatic storage ring and their decay monitored in time. Both ions display a triple band structure with maxima at 500, 518, and 530 nm. Previous attempts to study four-coordinate Fe(III)-heme(+) were hampered by the strong affinity of Fe(3+) for water and anions. Absorption at higher wavelengths is also measured, which is ascribed to charge-transfer transitions from the porphyrin to the iron. Finally, our data serve to benchmark theoretical calculations. PMID:20815568

  7. Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

    PubMed Central

    Navascués, Joaquín; Pérez-Rontomé, Carmen; Gay, Marina; Marcos, Manuel; Yang, Fei; Walker, F. Ann; Desbois, Alain; Abián, Joaquín; Becana, Manuel

    2012-01-01

    Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O2 transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O2 and facilitate its diffusion to the N2-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lbam, Lbcm, and Lbdm, of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O2 binding. PMID:22308405

  8. Hereditary Tyrosinemia and the Heme Biosynthetic Pathway. PROFOUND INHIBITION OF δ-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY BY SUCCINYLACETONE

    PubMed Central

    Sassa, Shigeru; Kappas, Attallah

    1983-01-01

    Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of δ-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 × 10-7 M to 3 × 10-7 M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic

  9. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases.

    PubMed

    Michener, Joshua K; Nielsen, Jens; Smolke, Christina D

    2012-11-20

    Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression. PMID:23129650

  10. Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H; Stuehr, Dennis J

    2012-10-30

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

  11. Nickel induction of microsomal heme oxygenase activity in rodents

    SciTech Connect

    Sunderman, F.W. Jr.; Reid, M.C.; Bibeau, L.M.; Linden, J.V.

    1983-01-01

    Heme oxygenase activity was measured in tissues of rats killed after administration of NiCl/sub 2/ or Ni/sub 3/S/sub 2/. Induction of renal heme oxygenase activity occurred 6 hr after NiCl/sub 2/ injection (0.25 mmol/kg sc), reached a maximum of five to six times the baseline activity at 17 hr, and remained significantly increased at 72 hr. Heme oxygenase activities were also increased in liver, lung, and brain at 17 hr after the NiCl/sub 2/ injection; heme oxygenase activities in spleen and intestinal mucosa were unchanged. The effects of NiCl/sub 2/ on heme oxygenase activities in kidney and liver were dose-related from 0.06 to 0.75 mmol/kg, sc. Three Ni chelators were administered (1 mmol/kg, im) prior to injection of NiCl/sub 2/ (0.25 mmol/kg, sc); d-penicillamine partially prevented Ni induction of renal heme oxygenase activity; triethylenetetramine had no effect; sodium diethyldithiocarbamate enhanced the Ni induction of renal heme oxygenase activity (three times greater than NiCl/sub 2/ alone). Intrarenal injection of Ni/sub 3/S/sub 2/ (10 mg/rat) caused induction of renal heme oxygenase activity at 1 week but not at 2, 3, or 4 weeks; no correlation was observed between induction of renal heme oxygenase activity and erythropoietin-mediated erythrocytosis. Hypoxia (10% O/sub 2/, 12 hr/day, 7 days) did not affect renal heme oxygenase activity. Induction of renal heme oxygenase activity was observed in mice, hamsters, and guinea pigs killed 17 hr after injection of NiCl/sub 2/ (0.25 mmol/kg, sc). These studies established (a) the time course, dose-effect, organ selectivity, and species susceptibility relationships for Ni induction of microsomal heme oxygenase activity, (b) the effects of Ni chelators, and (c) the lack of relationship between induction of renal heme oxygenase activity and the erythrocytosis that develops after intrarenal injection of Ni/sub 3/S/sub 2/.

  12. Molecular-Genetic Analysis of Osmoregulation, Osmotic Adjustment and Growth in Arabidopsis

    SciTech Connect

    Elizabeth Bray

    2009-05-04

    The molecular mechanism by which plants control cellular solute and water content, called osmoregulation, is critical for cell expansion and survival, particularly in response to cellular water deficit or low water potential. Two loci, lwr1 and lwr2 (low water potential response), that affect osmoregulation were isolated as part of a larger screen to identify mutants with alterations in low water potential-induced proline accumulation. When seedlings of lwr2 were exposed to a steady low water potential stress over a period of several days using PEG-infused agar plates, the mutants had lesser proline accumulation and osmotic adjustment than the wild type, Ben. A mapping population was raised but it was not possible to reliably identify the mutants in the population and thus could not be used for mapping. The mutant lwr1 was mapped and the gene identified. The mutant lwr1 had greater accumulation of proline, higher total solute content, greater osmotic adjustment at low water potential, altered abscisic acid content, and increased sensitivity to applied abscisic acid with respect to Pro content than the wild type, Ben, when the water potential was decreased over a period of several days using PEG-infused agar plates. lwr1 also had altered growth and morphology, including defects in trichome branching with the majority of the trichomes having a single point, shortened siliques which were crooked, and significantly lengthened time to flowering. Using bulk segregant analysis, the lwr1 loci was mapped to the lower arm of chromosome II, near the marker nga168. Further detailed fine mapping located the mutation to the gene PKL, At2g25170, which was previously identified as a gene involved in altered root development. PKL encodes a chromatin remodeling factor. The mutation in lwr1 introduced a stop codon in the 14th exon of At2g25170. The mutant was not complemented by 4 other known mutants having a disrupted PKL gene confirming the placement of this mutation in lwr1. In

  13. Transcriptome Analysis of Portunus trituberculatus in Response to Salinity Stress Provides Insights into the Molecular Basis of Osmoregulation

    PubMed Central

    Lv, Jianjian; Liu, Ping; Wang, Yu; Gao, Baoquan; Chen, Ping; Li, Jian

    2013-01-01

    Background The swimming crab, Portunus trituberculatus, which is naturally distributed in the coastal waters of Asia-Pacific countries, is an important farmed species in China. Salinity is one of the most important abiotic factors that influence not only the distribution and abundance of crustaceans, it is also an important factor for artificial propagation of the crab. To better understand the interaction between salinity stress and osmoregulation, we performed a transcriptome analysis in the gills of Portunus trituberculatus challenged with salinity stress, using the Illumina Deep Sequencing technology. Results We obtained 27,696,835, 28,268,353 and 33,901,271 qualified Illumina read pairs from low salinity challenged (LC), non-challenged (NC), and high salinity challenged (HC) Portunus trituberculatus cDNA libraries, respectively. The overall de novo assembly of cDNA sequence data generated 94,511 unigenes, with an average length of 644 bp. Comparative genomic analysis revealed that 1,705 genes differentially expressed in salinity stress compared to the controls, including 615 and 1,516 unigenes in NC vs LC and NC vs HC respectively. GO functional enrichment analysis results showed some differentially expressed genes were involved in crucial processes related to osmoregulation, such as ion transport processes, amino acid metabolism and synthesis processes, proteolysis process and chitin metabolic process. Conclusion This work represents the first report of the utilization of the next generation sequencing techniques for transcriptome analysis in Portunus trituberculatus and provides valuable information on salinity adaptation mechanism. Results reveal a substantial number of genes modified by salinity stress and a few important salinity acclimation pathways, which will serve as an invaluable resource for revealing the molecular basis of osmoregulation in Portunus trituberculatus. In addition, the most comprehensive sequences of transcripts reported in this study

  14. Transcriptome Changes in Eriocheir sinensis Megalopae after Desalination Provide Insights into Osmoregulation and Stress Adaption in Larvae

    PubMed Central

    Hui, Min; Liu, Yuan; Song, Chengwen; Li, Yingdong; Shi, Guohui; Cui, Zhaoxia

    2014-01-01

    Eriocheir sinensis, an extremely invasive alien crab species, has important economic value in China. It encounters different salinities during its life cycle, and at the megalopal stage it faces a turning point regarding the salinity in its environment. We applied RNA sequencing to E. sinensis megalopae before (MB) and after (MA) desalination, resulting in the discovery of 21,042 unigenes and 908 differentially expressed genes (DEGs, 4.32% of the unigenes). The DEGs primarily belonged to the Gene Ontology groups “Energy metabolism,” “Oxidoreductase activity,” “Translation,” “Transport,” “Metabolism,” and “Stress response.” In total, 33 DEGs related to transport processes were found, including 12 proton pump genes, three ATP-binding cassettes (ABCs), 13 solute carrier (SLC) family members, two sweet sugar transporter (ST) family members and three other substance transporters. Mitochondrial genes as well as genes involved in the tricarboxylic acid cycle, glycolytic pathway, or β-oxidation pathway, which can generate energy in the form of ATP, were typically up-regulated in MA. 11 unigenes related to amino acid metabolism and a large number of genes related to protein synthesis were differentially expressed in MB and MA, indicating that E. sinensis possibly adjusts its concentration of free amino acid osmolytes for hyper-osmoregulation. Additionally, 33 salinity and oxidative stress induced genes were found to be differentially expressed, such as the LEA2, HSPs, GST and coagulation factor genes. Notably, LEA2 is an extremely hydrophilic protein that responds to desiccation and reported for the first time in crabs. Therefore, we suppose that when the environment is hypo-osmotic, the megalopae might compensate for ion loss via hyper-osmoregulation by consuming more energy, accompanied by a series of stress induced adaptions. This study provides the first genome-wide transcriptome analysis of E. sinensis megalopae for studying its osmoregulation

  15. Transcriptome changes in Eriocheir sinensis megalopae after desalination provide insights into osmoregulation and stress adaption in larvae.

    PubMed

    Hui, Min; Liu, Yuan; Song, Chengwen; Li, Yingdong; Shi, Guohui; Cui, Zhaoxia

    2014-01-01

    Eriocheir sinensis, an extremely invasive alien crab species, has important economic value in China. It encounters different salinities during its life cycle, and at the megalopal stage it faces a turning point regarding the salinity in its environment. We applied RNA sequencing to E. sinensis megalopae before (MB) and after (MA) desalination, resulting in the discovery of 21,042 unigenes and 908 differentially expressed genes (DEGs, 4.32% of the unigenes). The DEGs primarily belonged to the Gene Ontology groups "Energy metabolism," "Oxidoreductase activity," "Translation," "Transport," "Metabolism," and "Stress response." In total, 33 DEGs related to transport processes were found, including 12 proton pump genes, three ATP-binding cassettes (ABCs), 13 solute carrier (SLC) family members, two sweet sugar transporter (ST) family members and three other substance transporters. Mitochondrial genes as well as genes involved in the tricarboxylic acid cycle, glycolytic pathway, or β-oxidation pathway, which can generate energy in the form of ATP, were typically up-regulated in MA. 11 unigenes related to amino acid metabolism and a large number of genes related to protein synthesis were differentially expressed in MB and MA, indicating that E. sinensis possibly adjusts its concentration of free amino acid osmolytes for hyper-osmoregulation. Additionally, 33 salinity and oxidative stress induced genes were found to be differentially expressed, such as the LEA2, HSPs, GST and coagulation factor genes. Notably, LEA2 is an extremely hydrophilic protein that responds to desiccation and reported for the first time in crabs. Therefore, we suppose that when the environment is hypo-osmotic, the megalopae might compensate for ion loss via hyper-osmoregulation by consuming more energy, accompanied by a series of stress induced adaptions. This study provides the first genome-wide transcriptome analysis of E. sinensis megalopae for studying its osmoregulation and stress adaption

  16. Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

    PubMed Central

    Munro, P M; Gauthier, M J; Breittmayer, V A; Bongiovanni, J

    1989-01-01

    The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made. PMID:2675763

  17. A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.

    PubMed

    Wood, Janet M; Culham, Doreen E; Hillar, Alexander; Vernikovska, Yaroslava I; Liu, Feng; Boggs, Joan M; Keates, Robert A B

    2005-04-19

    Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism. PMID:15823022

  18. Effect of nutritional status on the osmoregulation of green sturgeon (Acipenser medirostris).

    PubMed

    Haller, Liran Y; Hung, Silas S O; Lee, Seunghyung; Fadel, James G; Lee, Jun-Ho; McEnroe, Maryann; Fangue, Nann A

    2015-01-01

    Anthropogenic climate change is linked to food web and salinity fluctuations in estuarine environments. Both decreased nutritional status and environmental salinity influence the physiological tolerance and health of fish populations; however, limited information on the interaction of these two factors and their physiological consequences is available. The green sturgeon (Acipenser medirostris) is a species of special concern in California, and the southern distinct population segment is listed as threatened. To test the hypothesis that poor nutrition negatively affects osmoregulation, juvenile green sturgeon (222 d posthatch) were randomly assigned to four feed restriction groups (12.5%, 25%, 50%, and 100% of the optimal feeding rate for 4 wk). Fish were then acutely exposed to 0-, 8-, 16-, or 32-ppt salinities and sampled at three time points (12, 72, or 120 h). Feed restriction significantly (P < 0.05) decreased specific growth rate, feed efficiency, condition factor, whole-body lipids, and protein content as well as plasma glucose, triglycerides, and proteins. Furthermore, feed restriction, salinity concentration, and salinity exposure time had significant effects on hematological indexes (hematocrit, hemoglobin), plasma values (osmolality, Na(+), K(+), Cl(-), glucose, lactate, cortisol), enzymatic activity (gill and pyloric ceca Na(+)/K(+) ATPase), and morphology of gill mitochondria-rich cells. The largest disturbances were observed at the highest salinity treatments across all feeding regimes. In addition, the interaction between feed restriction and acute salinity exposure at the highest salinity treatment resulted in high mortality rates during the first 72 h of salinity exposure. Evaluating the interactions of these environmental stressors and their implications on green sturgeon physiological tolerance will inform restoration and management efforts in rapidly changing estuarine environments.

  19. A novel tyrosine-heme C−O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins.

    PubMed

    Yan, Dao-Jing; Li, Wei; Xiang, Yu; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2015-01-01

    Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins. PMID:25392956

  20. A novel tyrosine-heme C−O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins.

    PubMed

    Yan, Dao-Jing; Li, Wei; Xiang, Yu; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2015-01-01

    Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins.

  1. In Vivo Detection of the Cyclic Osmoregulated Periplasmic Glucan of Ralstonia solanacearum by High-Resolution Magic Angle Spinning NMR

    NASA Astrophysics Data System (ADS)

    Wieruszeski, J.-M.; Bohin, A.; Bohin, J.-P.; Lippens, G.

    2001-07-01

    We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

  2. Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions

    PubMed Central

    Barr, Ian; Guo, Feng

    2016-01-01

    Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly. PMID:27390766

  3. Environmental chemical exposures and disturbances of heme synthesis.

    PubMed Central

    Daniell, W E; Stockbridge, H L; Labbe, R F; Woods, J S; Anderson, K E; Bissell, D M; Bloomer, J R; Ellefson, R D; Moore, M R; Pierach, C A; Schreiber, W E; Tefferi, A; Franklin, G M

    1997-01-01

    Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria. PMID:9114276

  4. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

    SciTech Connect

    Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C. . E-mail: mcpaes@uerj.br

    2007-03-30

    Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.

  5. On the fate of extracellular hemoglobin and heme in brain.

    PubMed

    Lara, Flavio A; Kahn, Suzana A; da Fonseca, Anna Cc; Bahia, Carlomagno P; Pinho, João Pc; Graca-Souza, Aurélio V; Houzel, Jean C; de Oliveira, Pedro L; Moura-Neto, Vivaldo; Oliveira, Marcus F

    2009-06-01

    Intracerebral hemorrhage (ICH) is a major cause of disability in adults worldwide. The pathophysiology of this syndrome is complex, involving both inflammatory and redox components triggered by the extravasation of blood into the cerebral parenchyma. Hemoglobin, heme, and iron released therein seem be important in the brain damage observed in ICH. However, there is a lack of information concerning hemoglobin traffic and metabolism in brain cells. Here, we investigated the fate of hemoglobin and heme in cultured neurons and astrocytes, as well as in the cortex of adult rats. Hemoglobin was made traceable by conjugation to Alexa 488, whereas a fluorescent heme analogue (tin-protoporphyrin IX) was prepared to allow heme tracking. Using fluorescence microscopy we observed that neurons were more efficient in uptake hemoglobin and heme than astrocytes. Exposure of cortical neurons to hemoglobin or heme resulted in an oxidative stress condition. Viability assays showed that neurons were more susceptible to both hemoglobin and heme toxicity than astrocytes. Together, these results show that neurons, rather than astrocytes, preferentially take up hemoglobin-derived products, indicating that these cells are actively involved in the ICH-associated brain damage.

  6. Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme

    DOE PAGES

    Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.; Sharma, Ajay; Hoffman, Brian M.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2015-05-05

    Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂₋ and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs).more » While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second

  7. Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme

    SciTech Connect

    Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.; Sharma, Ajay; Hoffman, Brian M.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2015-05-05

    Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂₋ and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts

  8. Molecular dynamics simulations of heme reorientational motions in myoglobin.

    PubMed Central

    Henry, E R

    1993-01-01

    Molecular dynamics simulations of 2-ns duration were performed on carbonmonoxymyoglobin and deoxymyoglobin in vacuo to study the reorientational dynamics of the heme group. The heme in both simulations undergoes reorientations of approximately 5 degrees amplitude on a subpicosecond time scale, which produce a rapid initial decay in the reorientational correlation function to about 0.99. The heme also experiences infrequent changes in average orientation of approximately 10 degrees amplitude, which lead to a larger slow decay of the reorientational correlation function over a period of hundreds of picoseconds. The simulations have not converged with respect to these infrequent transitions. However, an estimate of the order parameter for rapid internal motions of the heme from those orientations which are sampled by the simulations suggests that the subnanosecond orientational dynamics of the heme accounts for at least 30% of the unresolved initial anisotropy decay observed in the nanosecond time-resolved optical absorption experiments on myoglobin reported by Ansari et al. in a companion paper (Ansari, A., C.M. Jones, E.R. Henry, J. Hofrichter, and W.A. Eaton. 1992. Biophys. J. 64:852-868.). A more complete sampling of the accessible heme orientations would most likely increase this fraction further. The simulation of the liganded molecule also suggests that the conformational dynamics of the CO ligand may contribute significantly to discrepancies between the ligand conformation as probed by x-ray diffraction and by infrared-optical photoselection experiments. The protein back-bone explores multiple conformations during the simulations, with the largest structural changes appearing in the E and F helices, which are in contact with the heme. The variations in the heme orientation correlate with the conformational dynamics of the protein on a time scale of hundreds of picoseconds, suggesting that the heme orientation may provide a useful probe of dynamical processes

  9. The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding.

    PubMed

    Kanadani, Masahiro; Sato, Takehiro; Hino, Tomoya; Nagano, Shingo; Ozaki, Shin-ichi

    2015-10-01

    Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π-π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg-heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.

  10. A selective inhibitor of heme biosynthesis in endosymbiotic bacteria elicits antifilarial activity in vitro.

    PubMed

    Lentz, Christian S; Halls, Victoria; Hannam, Jeffrey S; Niebel, Björn; Strübing, Uta; Mayer, Günter; Hoerauf, Achim; Famulok, Michael; Pfarr, Kenneth M

    2013-02-21

    Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms in vitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.

  11. Metal ion facilitated dissociation of heme from b-type heme proteins.

    PubMed

    Mauk, Marcia R; Rosell, Federico I; Mauk, A Grant

    2009-11-25

    Addition of Ni(2+), Cu(2+), or Zn(2+) (10-40 equiv) to metMb in sodium bicarbonate buffer (25 degrees C) at alkaline pH (7.8-9.5) results in a time-dependent (2-6 h) change in the electronic absorption spectrum of the protein that is consistent with dissociation of the heme from the active site and that can be largely reversed by addition of EDTA. Similar treatment of cytochrome b(5), indoleamine 2,3-dioxygenase, and cytochrome P450(cam) (in the presence or absence of camphor) produces a similar spectroscopic response. Elution of metMb treated with Ni(2+) in this manner over an anion exchange column in buffer containing Ni(2+) affords apo-myoglobin without exposure to acidic pH or organic solvents as usually required. Bovine liver catalase, in which the heme groups are remote from the surface of the protein, and horseradish peroxidase, which has four disulfide bonds and just three histidyl residues, exhibit a much smaller spectroscopic response. We propose that formation of carbamino groups by reaction of bicarbonate with protein amino groups promotes both protein solubility and the interaction of the protein with metal ions, thereby avoiding precipitation while destabilizing the interaction of heme with the protein. From these observations, bicarbonate buffers may be of value in the study of nonmembrane proteins of limited solubility. PMID:19874033

  12. YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

    NASA Technical Reports Server (NTRS)

    Martin, E.; Lee, Y. C.; Murad, F.

    2001-01-01

    YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.

  13. Exploiting the Chromone Scaffold for the Development of Inhibitors of Corticosteroid Biosynthesis.

    PubMed

    Gobbi, Silvia; Hu, Qingzhong; Zimmer, Christina; Engel, Matthias; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Bisi, Alessandra

    2016-03-24

    The inhibition of corticosteroid biosynthesis could be considered as an emerging strategy to reduce their abnormally high levels, and in this framework CYP11B1 and CYP11B2 represent the most promising targets. In continuing our studies on flavonoid-like scaffolds as privileged structures in medicinal chemistry, in this paper we describe a small library of pyridyl- and imidazolylmethylchromones as potential inhibitors of these enzymes. Testing results proved that position 3 of the chromone scaffold is the most favorable for the introduction of the heme-coordinating heterocycles and, among them, the 4-imidazolyl moiety is the most convenient for the interaction with the heme iron of the selected cytochromes. A low nanomolar inhibitor of CYP11B1 (5c) was obtained, endowed with reasonable selectivity toward CYP11B2 and able to better discriminate with respect to CYP17 and CYP19.

  14. Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

    SciTech Connect

    Nagao, Satoshi; Hirai, Yueki; Kawano, Shin; Imai, Kiyohiro; Suzuki, Akihiro; Yamamoto, Yasuhiko . E-mail: yamamoto@chem.tsukuba.ac.jp

    2007-03-16

    A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using {sup 19}F NMR and the O{sub 2} binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in {alpha}- and {beta}- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O{sub 2} affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O{sub 2} affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O{sub 2} affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.

  15. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes.

    PubMed

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed.

  16. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

    PubMed Central

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed. PMID:24782769

  17. The Role of Heme Chirality in the Circular Dichroism of Heme Proteins

    NASA Astrophysics Data System (ADS)

    Woody, Robert W.; Pescitelli, Gennaro

    2014-07-01

    The rotational strength (R) of the Soret transition in sperm-whale myoglobin (SW Mb), the hemoglobin from Chironomus thummi thummi (CTT Hb), and human hemoglobin (hHb) has been calculated using 20 high-resolution (< 1:5 Å) crystal structures. The intrinsic rotational strength due to heme non-planarity was calculated using π-electron theory and time-dependent density functional theory (TDDFT). Calculations on model protoporphyrins with a planar nucleus and with various torsional angles for the 2- and 4-vinyl substituents showed maximum R of ±0.70 Debye-Bohr magneton (1 DBM = 0.9273 · 10-38 cgs units). Viewing the heme so that the 2- and 4-vinyls are in a counterclockwise relationship, if a vinyl points toward the viewer, it contributes positively to R. Calculations of the intrinsic R for explicit heme geometries of SW Mb, CTT Hb, and hHb gave averages of 0.40±0.09, ±0:44±0.04, and +0.32±0.11 DBM, respectively. Coupling of the Soret transition with aromatic side-chain and peptide backbone transitions was also considered. For SW Mb, the magnitudes of the contributions decreased in the order Rint > Raro > Rpep. For CTT Hb and hHB, the orders were, respectively, Rint > Rpep > Raro and Rint > Raro ≈ Rpep. Human Hb ɑ chains showed the same trend as CTT Hb. Only in the hHb β chains did Raro predominate, with the order Raro > Rint > Rpep. The total predicted Rtot for SW Mb, CTT Hb, and hHb averaged +0.77±0.10 (0.56 - 0.80), -0.37±0.12 (-0.5), and +0.31±0.17 DBM (0.23 - 0.50), respectively. (Values in parentheses are experimental values.) Thus, contrary to the currently accepted view, coupling with aromatic side-chain or peptide transitions is not the dominant factor in the Soret circular dichroism (CD) of these proteins. The Soret CD is dominated by intrinsic CD of the heme chromophore, of which vinyl torsion is the major determinant. This result suggests an explanation for the large effect of heme isomerism on the Soret CD of Mb and Hb. Rotation about the

  18. Feasibility of combining spectra with texture data of multispectral imaging to predict heme and non-heme iron contents in pork sausages.

    PubMed

    Ma, Fei; Qin, Hao; Shi, Kefu; Zhou, Cunliu; Chen, Conggui; Hu, Xiaohua; Zheng, Lei

    2016-01-01

    To precisely determine heme and non-heme iron contents in meat product, the feasibility of combining spectral with texture features extracted from multispectral imaging data (405-970 nm) was assessed. In our study, spectra and textures of 120 pork sausages (PSs) treated by different temperatures (30-80 °C) were analyzed using different calibration models including partial least squares regression (PLSR) and LIB support vector machine (Lib-SVM) for predicting heme and non-heme iron contents in PSs. Based on a combination of spectral and textural features, optimized PLSR models were obtained with determination coefficient (R(2)) of 0.912 for heme and of 0.901 for non-heme iron prediction, which demonstrated the superiority of combining spectra with texture data. Results of satisfactory determination and visualization of heme and non-heme iron contents indicated that multispectral imaging could serve as a feasible approach for online industrial applications in the future. PMID:26212953

  19. Feasibility of combining spectra with texture data of multispectral imaging to predict heme and non-heme iron contents in pork sausages.

    PubMed

    Ma, Fei; Qin, Hao; Shi, Kefu; Zhou, Cunliu; Chen, Conggui; Hu, Xiaohua; Zheng, Lei

    2016-01-01

    To precisely determine heme and non-heme iron contents in meat product, the feasibility of combining spectral with texture features extracted from multispectral imaging data (405-970 nm) was assessed. In our study, spectra and textures of 120 pork sausages (PSs) treated by different temperatures (30-80 °C) were analyzed using different calibration models including partial least squares regression (PLSR) and LIB support vector machine (Lib-SVM) for predicting heme and non-heme iron contents in PSs. Based on a combination of spectral and textural features, optimized PLSR models were obtained with determination coefficient (R(2)) of 0.912 for heme and of 0.901 for non-heme iron prediction, which demonstrated the superiority of combining spectra with texture data. Results of satisfactory determination and visualization of heme and non-heme iron contents indicated that multispectral imaging could serve as a feasible approach for online industrial applications in the future.

  20. RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly.

    PubMed

    Raza, Amir; Malik, Hassan Jamil; Shafiq, Muhammad; Amin, Imran; Scheffler, Jodi A; Scheffler, Brian E; Mansoor, Shahid

    2016-01-01

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA) against both aquaporin (AQP) and a sucrase gene, alpha glucosidase (AGLU). These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR) was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci) and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops. PMID:27105353

  1. Osmoregulation by juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, in hypo- and hyper-saline waters.

    PubMed

    Cramp, R L; Hansen, M J; Franklin, C E

    2015-07-01

    While there is a considerable body of work describing osmoregulation by elasmobranchs in brackish and saltwater, far fewer studies have investigated osmoregulation in hypersaline waters. We examined osmo- and ionoregulatory function and plasticity in juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, exposed to three experimental salinities (25, 34 and 40‰) for two weeks. C. punctatum inhabits sheltered coastal areas and bays which can naturally become hypersaline as a consequence of evaporation of water but can also become hyposaline during flood events. We hypothesised that C. punctatum would demonstrate a phenotypically plastic osmoregulatory physiology. Plasma osmolality, urea, Na(+) and Cl(-) levels increased significantly with increasing environmental salinity. Rectal gland and branchial sodium-potassium ATPase (NKA) activities were unaffected by salinity. Using immunohistochemistry and Western Blotting we found evidence for the presence of the key ion-regulatory proteins vacuolar H(+)-ATPase (VHA), pendrin (Cl(-)/HCO₃(-) co-transporter) and the Na(+)-H(+) exchanger isoform 3 (NHE3) in discrete cells within the branchial epithelia. These results indicate that C. punctatum is a partially euryhaline elasmobranch able to maintain osmo- and ionoregulatory function between environmental salinities of 25‰ and 40‰. As suggested for other elasmobranchs, the gills of C. punctatum likely play a limited role in maintaining Na(+) homeostasis over the salinity range studied, but may play an important role in acid-base balance. PMID:25868436

  2. Trade-offs in osmoregulation and parallel shifts in molecular function follow ecological transitions to freshwater in the Alewife

    USGS Publications Warehouse

    Velotta, Jonathan P.; McCormick, Stephen; Schultz, Eric T.

    2015-01-01

    Adaptation to freshwater may be expected to reduce performance in seawater because these environments represent opposing selective regimes. We tested for such a trade-off in populations of the Alewife (Alosa pseudoharengus). Alewives are ancestrally anadromous, and multiple populations have been independently restricted to freshwater (landlocked). We conducted salinity challenge experiments, whereby juvenile Alewives from one anadromous and multiple landlocked populations were exposed to freshwater and seawater on acute and acclimation timescales. In response to acute salinity challenge trials, independently derived landlocked populations varied in the degree to which seawater tolerance has been lost. In laboratory-acclimation experiments, landlocked Alewives exhibited improved freshwater tolerance, which was correlated with reductions in seawater tolerance and hypo-osmotic balance, suggesting that trade-offs in osmoregulation may be associated with local adaptation to freshwater. We detected differentiation between life-history forms in the expression of an ion-uptake gene (NHE3), and in gill Na+/K+-ATPase activity. Trade-offs in osmoregulation, therefore, may be mediated by differentiation in ion-uptake and salt-secreting pathways.

  3. RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly

    PubMed Central

    Raza, Amir; Malik, Hassan Jamil; Shafiq, Muhammad; Amin, Imran; Scheffler, Jodi A.; Scheffler, Brian E.; Mansoor, Shahid

    2016-01-01

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA) against both aquaporin (AQP) and a sucrase gene, alpha glucosidase (AGLU). These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR) was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci) and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops. PMID:27105353

  4. Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.

    PubMed

    Serrano, Laetitia; Blanvillain, Gaëlle; Soyez, Daniel; Charmantier, Guy; Grousset, Evelyse; Aujoulat, Fabien; Spanings-Pierrot, Céline

    2003-03-01

    This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na(+) concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)-related peptides, including stereoisomers (L-CHH and D-Phe(3) CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe(3) CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe(3) CHH fraction significantly increased the hemolymph osmolality and Na(+) content 24 h after injection. Two other CHH-related peptides caused a smaller increase in Na(+) concentration. No significant variation was observed in hemolymph Cl(-) concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe(3) CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.

  5. Measurements and modeling of water transport and osmoregulation in a single kidney cell using optical tweezers and videomicroscopy

    NASA Astrophysics Data System (ADS)

    Lúcio, A. D.; Santos, R. A.; Mesquita, O. N.

    2003-10-01

    With an optical tweezer installed in our optical microscope we grab a single Madin Darby Canine kidney cell and keep it suspended in the medium without touching the glass substrate or other cells. Since the optically trapped cell remains with a closely round shape, we can directly measure its volume by using videomicroscopy with digital image analysis. We submit this cell to a hyperosmotic shock (up-shock) and video record the process: the cell initially shrinks due to osmotic efflux of water and after a while, due to regulatory volume increase (RVI), an osmoregulation response, it inflates again (water influx) until it reaches a new volume (the regulatory volume VR). In addition to considering standard osmotic water transport, we model RVI using a simple phenomenological model. We obtain an expression for cell volume variation as a function of time that fits very well with our experimental data, where two characteristic times appear naturally: one related to water transport and the other related to RVI. From the fit we obtain water permeability, osmolyte influx rate for RVI, and regulatory volume. With the addition of the hormone vasopressin, water permeability increases while the regulatory volume decreases until inhibition of RVI. In summary, we present a technique to measure directly volume changes of a single isolated kidney cell under osmotic shock and a phenomenological analysis of water transport that takes into account osmoregulation.

  6. Osmoregulation by juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, in hypo- and hyper-saline waters.

    PubMed

    Cramp, R L; Hansen, M J; Franklin, C E

    2015-07-01

    While there is a considerable body of work describing osmoregulation by elasmobranchs in brackish and saltwater, far fewer studies have investigated osmoregulation in hypersaline waters. We examined osmo- and ionoregulatory function and plasticity in juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, exposed to three experimental salinities (25, 34 and 40‰) for two weeks. C. punctatum inhabits sheltered coastal areas and bays which can naturally become hypersaline as a consequence of evaporation of water but can also become hyposaline during flood events. We hypothesised that C. punctatum would demonstrate a phenotypically plastic osmoregulatory physiology. Plasma osmolality, urea, Na(+) and Cl(-) levels increased significantly with increasing environmental salinity. Rectal gland and branchial sodium-potassium ATPase (NKA) activities were unaffected by salinity. Using immunohistochemistry and Western Blotting we found evidence for the presence of the key ion-regulatory proteins vacuolar H(+)-ATPase (VHA), pendrin (Cl(-)/HCO₃(-) co-transporter) and the Na(+)-H(+) exchanger isoform 3 (NHE3) in discrete cells within the branchial epithelia. These results indicate that C. punctatum is a partially euryhaline elasmobranch able to maintain osmo- and ionoregulatory function between environmental salinities of 25‰ and 40‰. As suggested for other elasmobranchs, the gills of C. punctatum likely play a limited role in maintaining Na(+) homeostasis over the salinity range studied, but may play an important role in acid-base balance.

  7. Structure of the Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme and enzymatic inactivation by mutation of the heme coordinating residue His-193

    SciTech Connect

    Suits,M.; Jaffer, N.; Jia, Z.

    2006-01-01

    Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 {angstrom} resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a {beta}-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain, assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the {alpha}-meso carbon position where O{sub 2} is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.

  8. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP.

  9. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP. PMID:27270708

  10. Osmoregulation in an avian nectarivore, the whitebellied sunbird Nectarinia talatala: response to extremes of diet concentration.

    PubMed

    Fleming, P A; Nicolson, S W

    2003-06-01

    Water intake of nectarivores is intrinsically linked to nectar concentration. Osmoregulation in whitebellied sunbirds Nectarinia talatala (body mass 9.3+/-0.1 g, mean +/- S.D., N=7), was examined by feeding them sucrose solutions, equivalent to extreme diet concentrations (0.07-2.5 mol l(-1) sucrose; 2-65% w/w), with and without supplementary drinking water. Total water gain was 33-515% of body mass daily. Cloacal fluid (CF) volume increased with diet dilution from 0.4% to 309% of body mass while increases in evaporative water loss (obtained by difference) were also recorded. Osmolality of CF demonstrated the largest scope yet recorded for a bird and was significantly correlated with water flux: mean values were 6-460 mosm kg(-1) H(2)O (minimum 3, maximum 1900 mosm kg(-1)). When supplementary water was provided, its consumption by birds fed concentrated diets (2.5 mol l(-1) sucrose) led to a dramatic reduction in CF osmolality, from 461+/-253 to 80+/-119 mosm kg(-1) fluid. Sunbirds maintained energy balance on sucrose diets varying tenfold in concentration, from 0.25 to 2.5 mol l(-1); however, on extremely dilute diets (0.07 and 0.1 mol l(-1) sucrose, lower than natural nectar concentrations) their inability to maintain energy balance was probably due to excess preformed water. Total osmotic excretion and concentrations of Na(+) and K(+) increased with high water fluxes, and are a possible physiological constraint for nectarivorous birds on artificial dilute diets devoid of electrolytes. Even low electrolyte levels in nectars may be adequate to replace these losses, but other physiological limitations to the intake of dilute nectars are increased energetic costs of solute recovery, increased heat loss and interference with digestive processes. Sunbirds therefore deal with sugar solutions spanning the range of nectar concentrations by shutting down water excretion on concentrated diets, or, on dilute diets, by producing extremely dilute CF with some of the lowest

  11. FORUM: Bioinspired Heme, Heme/non-heme Diiron, Heme/copper and Inorganic NOx Chemistry: ·NO(g) Oxidation, Peroxynitrite-Metal Chemistry and ·NO(g) Reductive Coupling

    PubMed Central

    Schopfer, Mark P.; Wang, Jun; Karlin, Kenneth D.

    2010-01-01

    The focus of this Forum review highlights work from our own laboratories and those of others in the area of biochemical and biologically inspired inorganic chemistry dealing with nitric oxide (nitrogen monoxide, ·NO(g)) and its biological roles and reactions. The latter focus is on (i) oxidation of ·NO(g) to nitrate by nitric oxide dioxygenases (NOD’s), and (ii) reductive coupling of two molecules of ·NO(g) to give N2O(g). In the former case, NOD’s are described and the highlighting of possible peroxynitrite-heme intermediates and consequences of this are given by discussion of recent works with myoglobin and a synthetic heme model system for NOD action. Summaries of recent copper complex chemistries with ·NO(g) and O2(g) leading to peroxynitrite species are given. The coverage of biological reductive coupling of ·NO(g) deals with bacterial nitric oxide reductases (NOR’s) with heme/non-heme diiron active sites, and on heme/Cu oxidases such as cytochrome c oxidase which can mediate the same chemistry. Recent designed protein and synthetic model compound (heme/non-heme diiron or heme/copper) as functional mimics are discussed in some detail. We also highlight examples from the chemical literature, not necessarily involving biologically relevant metal ions, which describe the oxidation of ·NO(g) to nitrate (or nitrite) and possible peroxynitrite intermediates, or reductive coupling of ·NO(g) to give nitrous oxide. PMID:20666386

  12. Isocyanides inhibit human heme oxygenases at the verdoheme stage.

    PubMed

    Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

    2009-09-22

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

  13. Chromosomal localization of the human heme oxygenase genes: Heme oxygenase-1 (HMOX1) maps to chromosome 22q12 and heme oxygenase-2 (HMOX2) maps to chromosome 16p13. 3

    SciTech Connect

    Kutty, R.K.; Kutty, G.; Rodriguez, I.R.; Chader, G.J.; Wiggert, B. )

    1994-04-01

    Heme oxygenase catalyzes the oxidation of heme to biliverdin, the precursor of the bile pigment bilirubin, and carbon monoxide, a putative neurotransmitter. The authors have employed polymerase chain reaction and fluorescence in situ hybridization to determine the chromosome localization of the genes coding for the two known heme oxygenase isozymes. Heme oxygenase-1 (HMOX1), the inducible form, was localized to human chromosome 22q12, while heme oxygenase-2 (HMOX2), the constitutive form, was localized to chromosome 16p13.3. 14 refs., 3 figs.

  14. BIOSYNTHESIS OF YEAST CAROTENOIDS

    PubMed Central

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  15. Biosynthesis of pulcherriminic acid

    PubMed Central

    MacDonald, J. C.

    1965-01-01

    1. Candida pulcherrima was grown on a complex medium to which various compounds had been added to determine their effect on the biosynthesis of pulcherriminic acid. Most of the pulcherriminic acid synthesized by C. pulcherrima PRL2019 was derived from the l-[1-14C]leucine added to the medium. 2. The cyclic dipeptide of l-leucine (cyclo-l-leucyl-l-leucyl) was shown, by trapping experiments involving cycloleucyl-leucyl isomers, to be synthesized by strain PRL2019. Cyclo-l-leucyl-l-leucyl was derived from l-leucine and was converted into pulcherriminic acid. Cyclo-l-leucyl-l-leucyl was a precursor of pulcherriminic acid in strain PRL2007 also. 3. The results supported the hypothesis that pulcherriminic acid is derived from l-leucine and that cyclo-l-leucyl-l-leucyl is an intermediate in the biosynthesis. PMID:5837792

  16. EPR of Mononuclear Non-Heme Iron Proteins

    PubMed Central

    Gaffney, Betty J.

    2010-01-01

    Flexible geometry of three- to six-protein side-chain ligands to non-heme iron in proteins is the basis for widely diverse reactivites ranging from iron transport to redox chemistry. The gap between fixed states determined by x-ray analysis can be filled by spectroscopic study of trapped intermediates. EPR is a versatile and relatively quick approach to defining intermediate states in terms of the geometry and electronic structures of iron. A number of examples in which the iron chemistry of non-heme proteins is understood through x-ray structures at subbond length resolution, refined calculations, and spectroscopy exist now. Some examples in which EPR has provided unique insight are summarized in Table 1. Assignment and quantitative evaluation of the EPR resonances in ferric, non-heme iron sites is the focus of the first section of this review. An earlier chapter in this series provides more background on the theory specific to EPR of S = 5/2 metal ions [1]. Besides EPR spectra of ferric mononuclear sites, EPR of ferrous iron coupled to a spin 1/2 radical, as it pertains to the categories mononuclear and non-heme, will also be covered, in the second half of this chapter. Examples include the quinone-ferrous interactions in photosynthetic reaction centers and nitric oxide complexes with non-heme ferrous iron. Other recent reviews of the biochemistry and spectroscopy of non-heme iron proteins provide additional background [2-6]. PMID:20428459

  17. A heme-binding domain controls regulation of ATP-dependent potassium channels.

    PubMed

    Burton, Mark J; Kapetanaki, Sofia M; Chernova, Tatyana; Jamieson, Andrew G; Dorlet, Pierre; Santolini, Jérôme; Moody, Peter C E; Mitcheson, John S; Davies, Noel W; Schmid, Ralf; Raven, Emma L; Storey, Nina M

    2016-04-01

    Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

  18. Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans

    PubMed Central

    Rao, Anita U.; Cerqueira, Gustavo C.; Mitreva, Makedonka; El-Sayed, Najib M.; Krause, Michael; Hamza, Iqbal

    2010-01-01

    Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 µM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival. PMID:20686661

  19. HutZ is required for efficient heme utilization in Vibrio cholerae.

    PubMed

    Wyckoff, Elizabeth E; Schmitt, Michael; Wilks, Angela; Payne, Shelley M

    2004-07-01

    Vibrio cholerae, the causative agent of cholera, requires iron for growth. One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V. cholerae. These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane. However, little is known about the fate of the heme after it enters the cell. In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization. hutZ (open reading frame [ORF] VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon. A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity. Heme oxygenase mutants of both Corynebacterium diphtheriae and C. ulcerans fail to use heme as an iron source. When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect. Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein. HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

  20. The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

    PubMed

    Mercurio, Sonia; Petrillo, Sara; Chiabrando, Deborah; Bassi, Zuni Irma; Gays, Dafne; Camporeale, Annalisa; Vacaru, Andrei; Miniscalco, Barbara; Valperga, Giulio; Silengo, Lorenzo; Altruda, Fiorella; Baron, Margaret H; Santoro, Massimo Mattia; Tolosano, Emanuela

    2015-06-01

    Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

  1. De Novo Assembly and Characterization of Narrow-Ridged Finless Porpoise Renal Transcriptome and Identification of Candidate Genes Involved in Osmoregulation

    PubMed Central

    Ruan, Rui; Guo, Ai-Huan; Hao, Yu-Jiang; Zheng, Jin-Song; Wang, Ding

    2015-01-01

    During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and the marine East Asian finless porpoise (N. a. sunameri, NAS), provide excellent subjects to understand the genetic basis of osmoregulatory divergence between freshwater and marine mammals. The kidney plays an important and well-established role in osmoregulation in marine mammals and thus, herein, we utilized RNA-seq to characterize the renal transcriptome and preliminarily analyze the divergence between the NAA and the NAS. Approximately 48.98 million clean reads from NAS and 49.40 million clean reads from NAA were obtained by RNA-Seq. And 73,449 (NAS) and 68,073 (NAA) unigenes were assembled. Among these annotations, 22,231 (NAS) and 21,849 (NAA) unigenes were annotated against the NCBI nr protein database. The ion channel complex GO term and four pathways were detected as relevant to osmoregulation by GO and KEGG pathway classification of these annotated unigenes. Although the endangered status of the study species prevented analysis of biological replicates, we identified nine differentially expressed genes (DEGs) that may be vital in the osmoregulation of the narrow-ridged finless porpoise and worthwhile for future studies. Of these DEGs, the differential expression and distribution of the aquaporin-2 (AQP2) in the collecting duct were verified using immunohistochemical experiments. Together, this work is the first report of renal transcriptome sequencing in cetaceans, and it will provide a valuable resource for future molecular genetics studies on cetacean osmoregulation. PMID:25608655

  2. Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti.

    PubMed Central

    Smith, L T; Pocard, J A; Bernard, T; Le Rudulier, D

    1988-01-01

    Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared. PMID:3290197

  3. Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti

    SciTech Connect

    Smith, L.T.; Pocard, J.A.; Bernard, T.; Le Rudulier, D.

    1988-07-01

    Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, the authors used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, /sup 14/C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.

  4. Facile heme vinyl posttranslational modification in a hemoglobin.

    PubMed

    Preimesberger, Matthew R; Wenke, Belinda B; Gilevicius, Lukas; Pond, Matthew P; Lecomte, Juliette T J

    2013-05-21

    Iron-protoporphyrin IX, or b heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the c-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its b heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid b/c heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of Synechocystis GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117-2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine-heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection. PMID:23607716

  5. Geometric and Electronic Structure Contributions to Function in Non-Heme Iron Enzymes

    PubMed Central

    Solomon, Edward I.; Light, Kenneth M.; Liu, Lei V.; Srnec, Martin; Wong, Shaun D.

    2014-01-01

    Conspectus Mononuclear non-heme Fe (NHFe) enzymes play key roles in antibiotic biosynthesis, hypoxic response, DNA repair, anticancer therapy and many other biological processes. On a molecular level these enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation and electrophilic aromatic substitution (EAS). Most of these enzymes use an FeII site to activate dioxygen. These ferrous active sites had been inaccessible to traditional spectroscopic methods. A methodology has been developed that provides detailed geometric and electronic structure insight for these NHFeII active sites. This has defined a general mechanistic strategy utilized by a wide range of these enzymes to control O2 activation by FeII coordination unsaturation only in the presence of cosubstrates to limit autooxidation and self-hydroxylation. Depending on the type of enzyme, O2 activation either involves a 2e− reduced FeIII–OOH intermediate or a 4e− reduced FeIV=O intermediate. The nature of these intermediates has been defined in terms of geometric structure using nuclear resonance vibrational spectroscopy (NRVS) and electronic structure using magnetic circular dichroism (MCD) to define the frontier molecular orbitals (FMOs) that control reactivity. For FeIII–OOH intermediates the anticancer drug Activated Bleomycin is shown to be the non-heme Fe analog of compound 0 in heme (e.g. P450) chemistry but undergoes different reactivity where the low-spin (LS) FeIII–OOH can directly abstract an H atom from DNA. It is also shown that the transition states of LS and high-spin (HS) FeIII–OOH are fundamentally different in that the former goes through a hydroxyl radical while the latter is activated for EAS without O-O cleavage, which is important in one class of NHFe enzymes that utilizes a HS FeIII–OOH intermediate in dioxygenation. For FeIV=O intermediates the LS form is shown to have a π-type FMO activated for attack

  6. Geometric and electronic structure contributions to function in non-heme iron enzymes.

    PubMed

    Solomon, Edward I; Light, Kenneth M; Liu, Lei V; Srnec, Martin; Wong, Shaun D

    2013-11-19

    Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)═O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for

  7. Computational prediction of heme-binding residues by exploiting residue interaction network.

    PubMed

    Liu, Rong; Hu, Jianjun

    2011-01-01

    Computational identification of heme-binding residues is beneficial for predicting and designing novel heme proteins. Here we proposed a novel method for heme-binding residue prediction by exploiting topological properties of these residues in the residue interaction networks derived from three-dimensional structures. Comprehensive analysis showed that key residues located in heme-binding regions are generally associated with the nodes with higher degree, closeness and betweenness, but lower clustering coefficient in the network. HemeNet, a support vector machine (SVM) based predictor, was developed to identify heme-binding residues by combining topological features with existing sequence and structural features. The results showed that incorporation of network-based features significantly improved the prediction performance. We also compared the residue interaction networks of heme proteins before and after heme binding and found that the topological features can well characterize the heme-binding sites of apo structures as well as those of holo structures, which led to reliable performance improvement as we applied HemeNet to predicting the binding residues of proteins in the heme-free state. HemeNet web server is freely accessible at http://mleg.cse.sc.edu/hemeNet/. PMID:21991319

  8. Malaria impairs resistance to Salmonella through heme- and heme oxygenase-dependent dysfunctional granulocyte mobilization

    PubMed Central

    Cunnington, A. J.; de Souza, J.B.; Walther, R-M.; Riley, E. M.

    2011-01-01

    In sub-Saharan Africa, invasive non-Typhoid Salmonella (NTS) is a common and often fatal complication of Plasmodium falciparum infection. Induction of heme oxygenase-1 (HO-1) mediates tolerance to the cytotoxic effects of heme during malarial hemolysis but might impair resistance to NTS by limiting production of bactericidal reactive oxygen species. We show that co-infection of mice with Plasmodium yoelii 17XNL (Py17XNL) and S. typhimurium causes acute, fatal bacteremia with increased bacterial load; features reproduced by phenylhydrazine hemolysis or hemin administration. S. typhimurium localized predominantly in granulocytes. Py17XNL, phenylhydrazine and hemin caused premature mobilization of granulocytes from bone marrow with a quantitative defect in the oxidative burst. Inhibition of HO by tin protoporphyrin abrogated the impairment of resistance to S. typhimurium by hemolysis. Thus a mechanism of tolerance to one infection, malaria, impairs resistance to another, NTS. Furthermore, HO inhibitors may be useful adjunctive therapy for NTS infection in the context of hemolysis. PMID:22179318

  9. Human Heme Oxygenase-1 Efficiently Catabolizes Heme in the Absence of Biliverdin Reductase

    PubMed Central

    Huber, Warren J.; Backes, Wayne L.

    2010-01-01

    Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR. PMID:20679134

  10. Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: enzymes activity and plasma ions.

    PubMed

    Silva, Alexandre O F da; Martinez, Cláudia B R

    2014-11-01

    Cadmium (Cd) is a trace element that is very toxic to fish. It is commonly found in surface waters contaminated with industrial effluents. When dissolved in water, Cd can rapidly cause physiological changes in the gills and kidneys of freshwater fish. The objective of this study was to evaluate the acute effects of Cd on the osmoregulation of the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to Cd at two concentrations [1 (Cd1) and 10 (Cd10) μgL(-1)] for 24 and 96h. The effects of Cd were evaluated through the analysis of ions (Na(+), K(+), Ca(2+), and Cl(-)) and plasma osmolality, and by measuring the activities of enzymes involved in osmoregulation obtained from the gills and kidney. Fish exposed to Cd for 24 and 96h showed a decrease in Na(+)/K(+)-ATPase activity in the gills and kidney. The activity of carbonic anhydrase decreased in the gills after 24h and in both tissues after 96h of Cd exposure. The gill Ca(2+)-ATPase activity also decreased with Cd exposure, with a concomitant drop in the plasma concentration of Ca(2+). Despite the hypocalcemia, there were no changes in the concentration of the ions Na(+), K(+), and Cl(-) or in plasma osmolality. Among the enzymes involved in ion transport, H(+)-ATPase was the only enzyme that showed increased activity in gills, whereas its activity in kidney remained unchanged. The results of this study demonstrate that waterborne Cd at the maximum concentrations set by Brazilian guidelines for freshwater affects the gills and kidney functions of P. lineatus. Acute exposure to Cd resulted in the decrease of the activity of enzymes, which culminated with the loss of the fish's ability to regulate the levels of calcium in the blood, leading to hypocalcemia.

  11. The role of tegumental aquaporin from the human parasitic worm, Schistosoma mansoni, in osmoregulation and drug uptake

    PubMed Central

    Faghiri, Zahra; Skelly, Patrick J.

    2009-01-01

    Schistosomes are parasitic platyhelminths that constitute an important public health problem globally. Infection is characterized by the presence of adult worms within the vasculature of their hosts, where they can reside for many years. The worms are covered by an unusual dual lipid bilayer through which they import nutrients. How the parasites import other vital molecules, such as water, is not known. Recent proteomic analysis of the schistosome tegumental membranes revealed the presence of an aquaporin homologue at the host-interactive surface whose cDNA we have cloned and characterized. The cDNA encodes a predicted 304-aa protein (SmAQP) that is found largely in the parasite tegument by immunolocalization and is most highly expressed in the intravascular life stages. Treatment of parasites with short interfering RNAs targeting the SmAQP gene results in potent (>90%) suppression. These suppressed parasites resist swelling when placed in hypotonic medium, unlike their control counterparts, which rapidly double in volume. In addition, SmAQP-suppressed parasites, unlike controls, resist shrinkage when incubated in hyperosmotic solution. While suppressed parasites exhibit lower viability in culture relative to controls and exhibit a stunted appearance following prolonged suppression, they are nonetheless more resistant to killing by the drug potassium antimonyl tartrate (PAT). This is likely because SmAQP acts as a conduit for this drug, as is the case for aquaporins in other systems. These experiments reveal a heretofore unrecognized role of the schistosome tegument in controlling water and drug movement into the parasites and highlight the importance of the tegument in parasite osmoregulation and drug uptake.—Faghiri, Z., Skelly, P. J. The role of tegumental aquaporin from the human parasitic worm, Schistosoma mansoni, in osmoregulation and drug uptake. PMID:19364765

  12. Thermodynamic analysis of hydration in human serum heme-albumin

    SciTech Connect

    Baroni, Simona; Pariani, Giorgio; Fanali, Gabriella; Longo, Dario; Ascenzi, Paolo; Aime, Silvio; Fasano, Mauro

    2009-07-31

    Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 {sup o}C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = {sup 5}/{sub 2} manifold. Values of correlation times for tensor fluctuation ({tau}{sub v}) and chemical exchange of water molecules ({tau}{sub M}) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46 {+-} 0.2 kJ mol{sup -1}, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with {Delta}H = 68 {+-} 28 kJ mol{sup -1} and {Delta}S = 200 {+-} 80 J mol{sup -1} K{sup -1}. These results highlight the role of the water solvent in heme-HSA structure-function relationships.

  13. Dietary iron controls circadian hepatic glucose metabolism through heme synthesis.

    PubMed

    Simcox, Judith A; Mitchell, Thomas Creighton; Gao, Yan; Just, Steven F; Cooksey, Robert; Cox, James; Ajioka, Richard; Jones, Deborah; Lee, Soh-Hyun; King, Daniel; Huang, Jingyu; McClain, Donald A

    2015-04-01

    The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling.

  14. Heme oxygenase activity correlates with serum indices of iron homeostasis in healthy nonsmokers

    EPA Science Inventory

    Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirm...

  15. Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad: recent developments in enzymology and modeling studies.

    PubMed

    Bruijnincx, Pieter C A; van Koten, Gerard; Klein Gebbink, Robertus J M

    2008-12-01

    Iron-containing enzymes are one of Nature's main means of effecting key biological transformations. The mononuclear non-heme iron oxygenases and oxidases have received the most attention recently, primarily because of the recent availability of crystal structures of many different enzymes and the stunningly diverse oxidative transformations that these enzymes catalyze. The wealth of available structural data has furthermore established the so-called 2-His-1-carboxylate facial triad as a new common structural motif for the activation of dioxygen. This superfamily of mononuclear iron(ii) enzymes catalyzes a wide range of oxidative transformations, ranging from the cis-dihydroxylation of arenes to the biosynthesis of antibiotics such as isopenicillin and fosfomycin. The remarkable scope of oxidative transformations seems to be even broader than that associated with oxidative heme enzymes. Not only are many of these oxidative transformations of key biological importance, many of these selective oxidations are also unprecedented in synthetic organic chemistry. In this critical review, we wish to provide a concise background on the chemistry of the mononuclear non-heme iron enzymes characterized by the 2-His-1-carboxylate facial triad and to discuss the many recent developments in the field. New examples of enzymes with unique reactivities belonging to the superfamily have been reported. Furthermore, key insights into the intricate mechanistic details and reactive intermediates have been obtained from both enzyme and modeling studies. Sections of this review are devoted to each of these subjects, i.e. the enzymes, biomimetic models, and reactive intermediates (225 references).

  16. The role of hemoglobin heme loss in Heinz body formation: studies with a partially heme-deficient hemoglobin and with genetically unstable hemoglobins

    PubMed Central

    Jacob, Harry S.; Winterhalter, Kaspar H.

    1970-01-01

    A number of mutant hemoglobins are inordinately unstable, denaturing in circulating red cells into Heinz bodies, resulting in congenital Heinz body hemolytic anemia (CHBHA). We have emphasized that most such hemoglobins involve amino acid substitutions at sites neighboring the heme group of the β-polypeptide chain, and have shown that heme binding to globin is diminished thereby. Thus, hemes were progressively lost from four unstable hemoglobins (Köln, Hammersmith, San Francisco, and Zürich) as they precipitated into Heinz bodies at 50°C. The role of heme loss, especially from beta chains, in Heinz body formation was supported by studies with a hemoglobin synthesized to contain hemes only on its alpha chains (α2hemeβ20). The behavior of this compound, postulated to be an intermediary in the formation of Heinz bodies, mimicked that of the genetically unstable hemoglobins in several ways: (a) it precipitated at 50°C into typical coccoid Heinz bodies; (b) as also observed with CHBHA hemoglobins this denaturation was virtually prevented by the heme ligands, cyanide or carbon monoxide, which inhibit further heme loss; it was potentiated by oxidation of hemes to the ferri- state, which accentuates heme loss; (c) the thiol groups of α2hemeβ20 were hyperreactive, forming mixed disulfides with glutathione and membrane sulfhydryls at rates similar to those of CHBHA hemoglobins and 10 or more times that of normal hemoglobin A; (d) heme repletion of the protein molecules by the addition of crystalline hemin to either α2hemeβ20 or to the genetically unstable hemoglobins, prevented their precipitation into Heinz bodies and normalized their aberrant electrophoretic behaviors; and (e) during Heinz body formation at 50°C both α2hemeβ20 and the genetically unstable hemoglobins released free αheme-chains into solution, suggesting that the bulk of the whitish, Heinz body precipitate is naked β8-chains. We conclude that heme loss from mutant beta chains is an early step

  17. Fatty acid hydroperoxide lyase is a heme protein.

    PubMed

    Shibata, Y; Matsui, K; Kajiwara, T; Hatanaka, A

    1995-02-01

    Fatty acid hydroperoxide lyase (HPO lyase) is an enzyme that cleaves hydroperoxides of polyunsaturated fatty acids to form short chain aldehydes and omega-oxoacids. Spectrophotometric analyses of HPO lyase highly purified from green bell pepper fruits indicate that it is a heme protein. The heme species was revealed to be heme b (protoheme IX) from the absorption spectrum of the pyridine hemochromogen. Although the spectrum highly resembles that of a plant cytochrome P450, allene oxide synthase from flaxseed, CO treatment of the enzyme caused no appearance of a peak at 450 nm, which is an essential diagnostic feature of a cytochrome P450. Internal amino acid sequences determined with peptide fragments obtained from the lyase showed no homology with any reported sequences.

  18. Novel Insights into the Vasoprotective Role of Heme Oxygenase-1

    PubMed Central

    Marcantoni, Emanuela; Di Francesco, Luigia; Dovizio, Melania; Bruno, Annalisa; Patrignani, Paola

    2012-01-01

    Cardiovascular risk factors contribute to enhanced oxidative stress which leads to endothelial dysfunction. These events trigger platelet activation and their interaction with leukocytes and endothelial cells, thus contributing to the induction of chronic inflammatory processes at the vascular wall and to the development of atherosclerotic lesions and atherothrombosis. In this scenario, endogenous antioxidant pathways are induced to restrain the development of vascular disease. In the present paper, we will discuss the role of heme oxygenase (HO)-1 which is an enzyme of the heme catabolism and cleaves heme to form biliverdin and carbon monoxide (CO). Biliverdin is reduced enzymatically to the potent antioxidant bilirubin. Recent evidence supports the involvement of HO-1 in the antioxidant and antiinflammatory effect of cyclooxygenase(COX)-2-dependent prostacyclin in the vasculature. Moreover, the role of HO-1 in estrogen vasoprotection is emerging. Finally, possible strategies to develop novel therapeutics against cardiovascular disease by targeting the induction of HO-1 will be discussed. PMID:22518279

  19. Insights into heme structure from Soret excitation Raman spectroscopy.

    PubMed

    Callahan, P M; Babcock, G T

    1981-02-17

    Laser lines in resonance with the Soret band optical transitions of several heme proteins and heme model compounds have been used to obtain Raman spectra of these species. Correlations between the observed frequency of a polarized mode in the 1560-1600-cm-1 region and the heme iron spin and coordination geometry have been developed. The position of this band is also a function of the pattern of porphyrin pyrrole ring beta-carbon substitution, and therefore structural information can be extracted from the Raman data only after this dependence has been taken into account. Quantitative correlations between the frequency of this band and the porphyrin core size are presented for three commonly occurring classes of heme compounds: (a) protoheme derivatives, (b) iron porphyrins in which all ring positions are saturated, and (c) heme alpha species. A polarized mode in the 1470-1510-cm-1 region is also consistently enhanced upon Soret excitation of these compounds, but is relatively insensitive to peripheral substituents, and can be used in conjunction with the polarized mode described above to assign heme geometries. In the frequency region above 1600 cm-1, a vibration is observed which also responds to changes in porphyrin geometry. However, this band is sometimes obscured by vibrations of unsaturated beta-carbon substituents, particularly in the case of protoheme derivatives. The normal coordinate analysis developed by Abe and co-workers [Abe, M., Kitagawa, T., & Kyogoku, Y. (1978) J Chem. Phys. 69, 4526-4534] is used to rationalize the dependence of the various modes on porphyrin geometry and peripheral substitution.

  20. 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

  1. Human heme oxygenase oxidation of 5- and 15-phenylhemes.

    PubMed

    Wang, Jinling; Niemevz, Fernando; Lad, Latesh; Huang, Liusheng; Alvarez, Diego E; Buldain, Graciela; Poulos, Thomas L; de Montellano, Paul R Ortiz

    2004-10-01

    Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.

  2. Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus.

    PubMed

    Ouattara, Mahamoudou; Pennati, Andrea; Devlin, Darius J; Huang, Ya-Shu; Gadda, Giovanni; Eichenbaum, Zehava

    2013-10-15

    The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.

  3. Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

    PubMed Central

    Alves, Liliane R.; Costa, Elaine S.; Sorgine, Marcos H. F.; Nascimento-Silva, Maria Clara L.; Teodosio, Cristina; Bárcena, Paloma; Castro-Faria-Neto, Hugo C.; Bozza, Patrícia T.; Orfao, Alberto

    2011-01-01

    In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity. PMID:21765894

  4. AN INTEGRATED PHARMACOKINETIC AND PHARMACODYNAMIC STUDY OF ARSENITE ACTION 2. HEME OXYGENASE INDUCTION IN MICE

    EPA Science Inventory

    Heme oxygenase (HO) is the rate-limiting enzyme in heme degradation and its activity has a significant impact on intracellular heme pools. Rat studies indicate that HO induction is a sensitive, dose-dependent response to arsenite (AsIII) exposure in both liver and kidney. The o...

  5. Like iron in the blood of the people: the requirement for heme trafficking in iron metabolism

    PubMed Central

    Korolnek, Tamara; Hamza, Iqbal

    2014-01-01

    Heme is an iron-containing porphyrin ring that serves as a prosthetic group in proteins that function in diverse metabolic pathways. Heme is also a major source of bioavailable iron in the human diet. While the synthesis of heme has been well-characterized, the pathways for heme trafficking remain poorly understood. It is likely that heme transport across membranes is highly regulated, as free heme is toxic to cells. This review outlines the requirement for heme delivery to various subcellular compartments as well as possible mechanisms for the mobilization of heme to these compartments. We also discuss how these trafficking pathways might function during physiological events involving inter- and intra-cellular mobilization of heme, including erythropoiesis, erythrophagocytosis, heme absorption in the gut, as well as heme transport pathways supporting embryonic development. Lastly, we aim to question the current dogma that heme, in toto, is not mobilized from one cell or tissue to another, outlining the evidence for these pathways and drawing parallels to other well-accepted paradigms for copper, iron, and cholesterol homeostasis. PMID:24926267

  6. The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

    PubMed

    Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina; Hunter, Gregory A; Gakh, Oleksandr; Isaya, Grazia; Ferreira, Gloria C; Al-Karadaghi, Salam

    2016-05-27

    Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.

  7. The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

    PubMed

    Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina; Hunter, Gregory A; Gakh, Oleksandr; Isaya, Grazia; Ferreira, Gloria C; Al-Karadaghi, Salam

    2016-05-27

    Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species. PMID:27026703

  8. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  9. Biosynthesis of methanopterin

    SciTech Connect

    White, R.H. )

    1990-06-05

    The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. (methylene{sup 2}H)-6-(hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from (methyl-{sup 2}H{sub 3})methionine. Cells grown in the presence of (methylene-{sup 2}H)-6-(hydroxymethyl)pterin, (ethyl-{sup 2}H{sub 4})-6-(1 (RS)-hydroxyethyl)pterin, (methyl-{sup 2}H{sub 3})-6-(hydroxymethyl)-7-methylpterin, (ethyl-{sup 2}H{sub 4}, methyl-{sup 2}H{sub 3})-6-(1 (RS)-hydroxyethyl)-7-methylpterin, and (1-ethyl-{sup 3}H)-6-(1 (RS)-hydroxyethyl)-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic (methylene-{sup 3}H)-7,8-H{sub 2}-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H{sub 2}-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.

  10. Tearing down to build up: Metalloenzymes in the biosynthesis lincomycin, hormaomycin and the pyrrolo [1,4]benzodiazepines.

    PubMed

    Colabroy, Keri L

    2016-06-01

    The metabolic pathways for the production of lincomycin, hormaomycin and the antitumor pyrrolo [1,4] benzodiazepines share a vinyl substituted pyrroline carboxylic acid (3-vinyl-2,3-pyrroline-5-carboxylic acid, VPCA) as a common intermediate. Biosynthesis of this vinyl substituted pyrroline carboxylic acid intermediate requires a short, three-enzyme pathway containing two metalloenzymes: a heme-dependent l-tyrosine hydroxylase and a non-heme Fe(2+) dependent l-DOPA dioxygenase. The l-tyrosine hydroxylase is an unprecedented type of peroxidase that specifically monohydroxylates tyrosine, while the l-DOPA extradiol cleaving enzyme is a single-domain vicinal-oxygen-chelate (VOC) dioxygenase. The dioxygenase product subsequently undergoes an, as yet uncharacterized, C-C bond cleavage reaction. This mini-pathway demonstrates the use of metal-dependent chemistry typically associated with natural product degradation in order to build a compact, functionalized building block for larger, bioactive molecules.

  11. Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex.

    PubMed

    Girvan, Hazel M; Bradley, Justin M; Cheesman, Myles R; Kincaid, James R; Liu, Yilin; Czarnecki, Kazimierz; Fisher, Karl; Leys, David; Rigby, Stephen E J; Munro, Andrew W

    2016-09-13

    DGCR8 is the RNA-binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys(-)) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman, and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated from the substrate-binding site. UV-vis absorption spectra of the Fe(II) and Fe(II)-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron-nuclear double resonance spectroscopy data exclude histidine or water as axial ligands for ferric DGCR8 and favor bis-thiolate coordination in this form. UV-vis MCD and near-infrared MCD provide data consistent with this conclusion. UV-vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous-CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from those of previous studies of a different ferrous DGCR8 isoform.

  12. Kinetics of nitric oxide dissociation from five- and six-coordinate nitrosyl hemes and heme proteins, including soluble guanylate cyclase.

    PubMed

    Kharitonov, V G; Sharma, V S; Magde, D; Koesling, D

    1997-06-01

    Kinetics of NO dissociation were characterized for three five-coordinate systems, heme-NO, HSA-heme-NO (human serum albumin), GC-NO (soluble guanylate cyclase), and for the six-coordinate system, Im-heme-NO. Nitrosyl myoglobin was redetermined for comparison. Previously known, six-coordinate R and T state nitrosyl hemoglobins are also included in the comparison. The data indicate that NO dissociates more than 1000 times faster from five-coordinate model heme than it does from the six-coordinate analog. Such a negative trans-effect between NO and a proximal base is in sharp contrast to carboxy heme derivatives, in which ligand dissociation rates are greatly slowed in when a trans base is present. As a result of opposite trans-effects, six-coordinate carboxy and nitrosyl derivatives have comparable dissociation rates, even though the five-coordinate species are very different. In proteins, five- and six-coordinate forms do not show a large difference in dissociation rates. Part of the reason may be due to different probabilities for geminate recombination in the different proteins, but this cannot explain all the facts. There must also be influences of the protein structure on bond-breaking rate constants themselves. With the exception of hemoglobin in the T state, nitrosyl guanylate cyclase shows the highest NO dissociation rate constant, k(obs) = 6 x 10(-4) s(-1). This would yield a half-life of about 2 min at 37 degrees C for dissociation of NO from GC-NO, a number that has implications for the mechanism of regulation of the activity of this key heme enzyme.

  13. Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

    PubMed Central

    Kirby, Amy E.; Metzger, Daniel J.; Murphy, Erin R.; Connell, Terry D.

    2001-01-01

    Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent. PMID:11598070

  14. Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

    PubMed

    Pham, Dominique; Charmantier, Guy; Boulo, Viviane; Wabete, Nelly; Ansquer, Dominique; Dauga, Clément; Grousset, Evelyse; Labreuche, Yannick; Charmantier-Daures, Mireille

    2016-01-01

    The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

  15. Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

    PubMed Central

    Siitonen, Vilja; Selvaraj, Brinda; Niiranen, Laila; Lindqvist, Ylva; Schneider, Gunter; Metsä-Ketelä, Mikko

    2016-01-01

    Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon–carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5′′–C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)–dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5′′ atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4′′ carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution. PMID:27114534

  16. Genetic analysis of mouse strains with variable serum sodium concentrations identifies the Nalcn sodium channel as a novel player in osmoregulation

    PubMed Central

    Sinke, Anne P.; Caputo, Christina; Tsaih, Shirng-Wern; Yuan, Rong; Ren, Dejian; Deen, Peter M. T.

    2011-01-01

    In central osmoregulation, a 1–2% rise in plasma osmolality is detected by specialized osmoreceptors located in the circumventricular organs of the hypothalamus. A disturbance in this tightly regulated balance will result in either hyponatremia or hypernatremia, which are both common electrolyte disorders in hospitalized patients. Despite the high clinical importance of hypo- and hypernatremia and the fact that this vital process has been studied for many years, the genes and corresponding proteins involved in this process are just beginning to be identified. To identify novel genes involved in the (patho-)physiology of osmoregulation, we therefore employed haplotype association mapping on an aging group of 27 inbred mouse strains. Serum sodium concentrations were determined in all strains at 6, 12, and 18 mo of age, and high-resolution mapping was performed for males and females separately. We identified a total of five loci associated with the serum sodium concentration of which the locus on chromosome 14, containing only one known gene (Nalcn), showed the strongest correlation. Within this locus three different haplotypes could be distinguished, which associated with different average serum sodium levels. The association of Nalcn with sodium levels was confirmed by analysis of heterozygous Nalcn knockout mice, which displayed hypernatremia compared with wild-type littermates. Our study demonstrates that Nalcn associates with serum sodium concentrations in mice and indicates that Nalcn is an important novel player in osmoregulation. PMID:21177381

  17. Alteration of the regiospecificity of human heme oxygenase-1 by unseating of the heme but not disruption of the distal hydrogen bonding network.

    PubMed

    Wang, Jinling; Evans, John P; Ogura, Hiroshi; La Mar, Gerd N; Ortiz de Montellano, Paul R

    2006-01-10

    Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane. PMID:16388581

  18. Analysis of a heme-dependent signal transduction system in Corynebacterium diphtheriae: deletion of the chrAS genes results in heme sensitivity and diminished heme-dependent activation of the hmuO promoter.

    PubMed

    Bibb, Lori A; King, Natalie D; Kunkle, Carey A; Schmitt, Michael P

    2005-11-01

    The Corynebacterium diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme as an iron source. Transcription of hmuO is activated by heme or hemoglobin and repressed by iron and DtxR. Previous studies with Escherichia coli showed that heme-dependent transcriptional activation of an hmuO promoter-lacZ fusion was dependent on the cloned C. diphtheriae chrA and chrS genes (chrAS), which encode the response regulator and sensor kinase, respectively, of a two-component signal transduction system. In this study, nonpolar deletions in the chrAS genes were constructed on the chromosome of C. diphtheriae. Mutations in chrAS resulted in marked reduction in heme-dependent transcription of hmuO, which indicates that the ChrA/S system is a key regulator at the hmuO promoter. However, low but significant levels of heme-specific transcriptional activity were observed at the hmuO promoter in the chrAS mutants, suggesting that an additional heme-dependent activator is involved in hmuO expression. The chrAS mutants were also sensitive to heme, which was observed only in stationary-phase cultures and correlated with reduced cell viability. The heme sensitivity of the mutants was not due to reduced expression of hmuO, and these results suggest that additional factors controlled by the ChrA/S system may be involved in protection against heme toxicity. Transcriptional analysis of the chrAS operon revealed that it was not autoregulated or affected by iron or heme levels.

  19. Pentachlorophenol (PCP) degradation using heme and hydrogen peroxide

    SciTech Connect

    Chen, S.T.

    1996-11-01

    Investigations of pentachlorophenol (PCP) degradation using both biotic and abiotic methods have been extensively studied. Due to the hydrophobic nature of PCP and its toxicity, the performance of the biotic treatments varies from site to site and is not satisfactory in most cases. An abiotic method for oxidative PCP degradation in soil under unsaturated conditions and a neutral pH was found. Hydrogen peroxide was used as an oxidant and heme as a catalyst. A mechanism was proposed to describe the possible reaction of heme and peroxide at the presence of PCP. In order to ensure that heme and peroxide are the most important factors during the reaction, two screening tests were run. In order to find the best conditions of PCP degradation using heme and peroxide, a statistical technique, so-called response surface methodology (RSM), was employed and the best conditions for PCP degradation in soil were determined. In order to examine the rate and extent of PCP degradation, kinetic studies were conducted and the results showed that about 70% of PCP was degraded within the first two hours and up to 80% of PCP was degraded within one day. Up to 17% of the PCP was mineralized to carbon dioxide as well. A scaled-up experiment was also studied to confirm the results in the laboratory. The result of the scaled-up experiment showed not much difference between the laboratory and the scaled-up experiments.

  20. Mechanism of reaction of chlorite with mammalian heme peroxidases.

    PubMed

    Jakopitsch, Christa; Pirker, Katharina F; Flemmig, Jörg; Hofbauer, Stefan; Schlorke, Denise; Furtmüller, Paul G; Arnhold, Jürgen; Obinger, Christian

    2014-06-01

    This study demonstrates that heme peroxidases from different superfamilies react differently with chlorite. In contrast to plant peroxidases, like horseradish peroxidase (HRP), the mammalian counterparts myeloperoxidase (MPO) and lactoperoxidase (LPO) are rapidly and irreversibly inactivated by chlorite in the micromolar concentration range. Chlorite acts as efficient one-electron donor for Compound I and Compound II of MPO and LPO and reacts with the corresponding ferric resting states in a biphasic manner. The first (rapid) phase is shown to correspond to the formation of a MPO-chlorite high-spin complex, whereas during the second (slower) phase degradation of the prosthetic group was observed. Cyanide, chloride and hydrogen peroxide can block or delay heme bleaching. In contrast to HRP, the MPO/chlorite system does not mediate chlorination of target molecules. Irreversible inactivation is shown to include heme degradation, iron release and decrease in thermal stability. Differences between mammalian peroxidases and HRP are discussed with respect to differences in active site architecture and heme modification.

  1. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    EPA Science Inventory

    An ELISA assay for heme oxygenase (HO-l )

    Abstract

    A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  2. Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

    PubMed

    Azuma, Junya; Wong, Ronald J; Morisawa, Takeshi; Hsu, Mark; Maegdefessel, Lars; Zhao, Hui; Kalish, Flora; Kayama, Yosuke; Wallenstein, Matthew B; Deng, Alicia C; Spin, Joshua M; Stevenson, David K; Dalman, Ronald L; Tsao, Philip S

    2016-01-01

    Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

  3. Nitrite attenuated hypochlorous acid-mediated heme degradation in hemoglobin.

    PubMed

    Lu, Naihao; Li, Jiayu; Ren, Xiaoming; Tian, Rong; Peng, Yi-Yuan

    2015-08-01

    Hypochlorous acid (HOCl) is elevated in many inflammatory diseases and causes the accumulation of free iron. Through the Fenton reaction, free iron has the ability to generate free radicals and subsequently is toxic. Recent studies have demonstrated that HOCl participates in heme destruction of hemoglobin (Hb) and free iron release. In this study, it was showed that nitrite (NO2(-)) could prevent HOCl-mediated Hb heme destruction and free iron release. Also, NO2(-) prevented HOCl-mediated loss of Hb peroxidase activity. After the NO2(-)/HOCl treatment, Tyr 42 in α-chain was found to be nitrated in Hb, attenuating the electron transferring abilities of phenolic compounds. The protective effects of NO2(-) on HOCl-induced heme destruction were attributed to its reduction of ferryl Hb and/or direct scavenging of HOCl. Therefore, NO2(-) could show protective effects in some inflammatory diseases by preventing HOCl-mediated heme destruction of hemoproteins and free iron release. PMID:26051522

  4. ARSENIC INDUCTION OF HEME OXYGENASE AS A BIOMARKER

    EPA Science Inventory


    Useful biomarkers of arsenic effects in both experimental animals and humans are needed. Arsenate and arsenite are good inducers of rat hepatic and renal heme oxygenase (HO); monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are not. Therefore, HO enzyme induction ...

  5. Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

    PubMed

    Azuma, Junya; Wong, Ronald J; Morisawa, Takeshi; Hsu, Mark; Maegdefessel, Lars; Zhao, Hui; Kalish, Flora; Kayama, Yosuke; Wallenstein, Matthew B; Deng, Alicia C; Spin, Joshua M; Stevenson, David K; Dalman, Ronald L; Tsao, Philip S

    2016-01-01

    Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease. PMID:26894432

  6. Structural and spectroscopic characterisation of a heme peroxidase from sorghum.

    PubMed

    Nnamchi, Chukwudi I; Parkin, Gary; Efimov, Igor; Basran, Jaswir; Kwon, Hanna; Svistunenko, Dimitri A; Agirre, Jon; Okolo, Bartholomew N; Moneke, Anene; Nwanguma, Bennett C; Moody, Peter C E; Raven, Emma L

    2016-03-01

    A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.

  7. The Synthetic Analogs of Oxygen-Binding Heme Proteins.

    ERIC Educational Resources Information Center

    Suslick, Kenneth S.; Reinert, Thomas J.

    1985-01-01

    Discusses model studies aimed at elucidating various ways in which molecular oxygen interacts with metalloproteins. The focus is on the chemistry of iron(II) porphyrins and their adducts with nitrogenous bases, carbon monoxide, and dioxygen, which are most relevant to the functional proteries of the heme proteins, hemoglobin, and myoglobin. (JN)

  8. A mononuclear non-heme-iron dioxygen-carrying protein?

    PubMed

    Attia, Amr A A; Silaghi-Dumitrescu, Radu

    2016-09-01

    The ability of mononuclear non-heme iron complexes to function as molecular oxygen transporters is investigated by density functional theory. The factors governing the efficiency of the reversible binding of dioxygen at the active site of the dinuclear non-heme iron enzyme hemerythrin, including antiferromagnetic coupling and the conversion of dioxygen to hydroperoxo by a proton coupled 2-electron transfer mechanism, are revisited and considered as possible tools in mononuclear non-heme complexes. Several mononuclear non-heme model complexes, including active sites of enzymes already known to interact with dioxgenic ligands, are constructed and the molecular oxygen transportation capabilities of these complexes are examined computationally. The high-spin nature of the ground state of these complexes implies an intrinsic kinetic lability of the oxy structures, as also evident from potential energy surface calculations towards iron-dioxygen cleavage. Proton affinities as calibrated with reference compounds showed that these complexes are highly unlikely to undergo protonation to form hydroperoxo-like adducts. Mixed superoxo descriptions of the dissociated dioxygenic ligands in all complexes add to the overall conclusion that these model structures are significantly disadvantaged in any attempt to be employed for molecular oxygen transportation. PMID:27607306

  9. Electrochemical titration of the cytochrome hemes in the Rhodopseudomonas viridis reaction center. Cyclic equilibrium titrations yield midpoint potentials without evidence for heme cooperativity.

    PubMed

    Fritz, F; Moss, D A; Mäntele, W

    1992-02-01

    The redox and spectral characteristics of the 4-heme cytochrome c unit of the photochemical reaction center from Rhodopseudomonas viridis were studied by a combination of protein electrochemistry and spectroscopy using an ultra thin-layer spectroelectrochemical cell. Quantitative and reversible reduction of the high-potential and the low-potential hemes was performed in cyclic titrations to record the optical difference spectra in the alpha-band region. The titration of the absorbance from the high-potential hemes can be approximated with a sum of 2 Nernst functions with Em = 0.113 V and Em = 0.175 V. The corresponding titration of the absorbance from the low-potential hemes yielded Em = -0.257 V and Em = -0.175 V (all potentials quoted vs. Ag/AgC1/3 M KCl; add 0.208 V for potentials vs. standard hydrogen electrode). The high-potential hemes equilibrate rapidly and titrate identically for oxidative and reductive titrations. Under identical conditions, the low-potential hemes exhibit a hysteresis, thus indicating much slower equilibration with the applied potential. Cyclic titrations with increasing equilibration periods, however, indicate the disappearance of the hysteresis for equilibration periods approximately twice as long as for the high-potential hemes. We take this as evidence for a slower internal equilibration, but against a cooperativity of the low-potential hemes as observed for other multi-heme cytochromes.

  10. Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin.

    PubMed

    Klatt, P; Pfeiffer, S; List, B M; Lehner, D; Glatter, O; Bächinger, H P; Werner, E R; Schmidt, K; Mayer, B

    1996-03-29

    Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

  11. The crystal structure of the extracellular 11-heme cytochrome UndA reveals a conserved 10-heme motif and defined binding site for soluble iron chelates.

    PubMed

    Edwards, Marcus J; Hall, Andrea; Shi, Liang; Fredrickson, James K; Zachara, John M; Butt, Julea N; Richardson, David J; Clarke, Thomas A

    2012-07-01

    Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure has also been crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

  12. The Crystal Structure of the Extracellular 11-heme Cytochrome UndA Reveals a Conserved 10-heme Motif and Defined Binding Site for Soluble Iron Chelates.

    SciTech Connect

    Edwards, Marcus; Hall, Andrea; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David; Clarke, Thomas A.

    2012-07-03

    Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure is the first outer membrane cytochrome to be crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

  13. The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases.

    PubMed

    Cheesman, M R; Zumft, W G; Thomson, A J

    1998-03-17

    EPR spectra at liquid helium temperatures and MCD spectra at room temperature and 4.2 K are presented for fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The MCD spectra show that the enzyme contains three heme groups at equivalent concentrations but distinctive in their axial coordination. Two, in the low-spin ferric state at all temperatures, give rise to infrared charge-transfer transitions which show the hemes to have bis-histidine and histidine-methionine ligation, respectively. The EPR spectra show them to be magnetically isolated. The third heme has an unusual temperature-dependent spin state and spectroscopic features which are consistent with histidine-hydroxide coordination. No EPR signals have been detected from this heme. Together with its unusual near-infrared MCD, this suggests a spin-spin interaction between this heme and another paramagnet. The three hemes account for only 75% of the iron content, and it is concluded that the additional paramagnet is a mononuclear ferric ion. These results provide further evidence that NOR is indeed structurally related to heme-copper oxidases and that it contains a heme/non-heme iron spin-coupled pair at the active site.

  14. The effect of proteins from animal source foods on heme iron bioavailability in humans.

    PubMed

    Pizarro, Fernando; Olivares, Manuel; Valenzuela, Carolina; Brito, Alex; Weinborn, Valerie; Flores, Sebastián; Arredondo, Miguel

    2016-04-01

    Forty-five women (35-45 year) were randomly assigned to three iron (Fe) absorption sub-studies, which measured the effects of dietary animal proteins on the absorption of heme Fe. Study 1 was focused on heme, red blood cell concentrate (RBCC), hemoglobin (Hb), RBCC+beef meat; study 2 on heme, heme+fish, chicken, and beef; and study 3 on heme and heme+purified animal protein (casein, collagen, albumin). Study 1: the bioavailability of heme Fe from Hb was similar to heme only (∼13.0%). RBCC (25.0%) and RBCC+beef (21.3%) were found to be increased 2- and 1.6-fold, respectively, when compared with heme alone (p<0.05). Study 2: the bioavailability from heme alone (10.3%) was reduced (p<0.05) when it was blended with fish (7.1%) and chicken (4.9%), however it was unaffected by beef. Study 3: casein, collagen, and albumin did not affect the bioavailability of Fe. Proteins from animal source foods and their digestion products did not enhance heme Fe absorption.

  15. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease

    PubMed Central

    Camus, Stéphane M.; De Moraes, João A.; Bonnin, Philippe; Abbyad, Paul; Le Jeune, Sylvain; Lionnet, François; Loufrani, Laurent; Grimaud, Linda; Lambry, Jean-Christophe; Charue, Dominique; Kiger, Laurent; Renard, Jean-Marie; Larroque, Claire; Le Clésiau, Hervé; Tedgui, Alain; Bruneval, Patrick; Barja-Fidalgo, Christina; Alexandrou, Antigoni; Tharaux, Pierre-Louis; Boulanger, Chantal M.

    2015-01-01

    Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. PMID:25827830

  16. Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization.

    PubMed

    Draganova, Elizabeth B; Adrian, Seth A; Lukat-Rodgers, Gudrun S; Keutcha, Cyrianne S; Schmitt, Michael P; Rodgers, Kenton R; Dixon, Dabney W

    2016-10-01

    The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV-visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT-CO. PMID:27561288

  17. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

    PubMed

    Camus, Stéphane M; De Moraes, João A; Bonnin, Philippe; Abbyad, Paul; Le Jeune, Sylvain; Lionnet, François; Loufrani, Laurent; Grimaud, Linda; Lambry, Jean-Christophe; Charue, Dominique; Kiger, Laurent; Renard, Jean-Marie; Larroque, Claire; Le Clésiau, Hervé; Tedgui, Alain; Bruneval, Patrick; Barja-Fidalgo, Christina; Alexandrou, Antigoni; Tharaux, Pierre-Louis; Boulanger, Chantal M; Blanc-Brude, Olivier P

    2015-06-11

    Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

  18. Induction of heme oxygenase in intestinal epithelial cells: studies in Caco-2 cell cultures.

    PubMed

    Cable, J W; Cable, E E; Bonkovsky, H L

    1993-12-01

    Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.

  19. Heme Signaling Impacts Global Gene Expression, Immunity and Dengue Virus Infectivity in Aedes aegypti

    PubMed Central

    Bottino-Rojas, Vanessa; Talyuli, Octávio A. C.; Jupatanakul, Natapong; Sim, Shuzhen; Dimopoulos, George; Venancio, Thiago M.; Bahia, Ana C.; Sorgine, Marcos H.; Oliveira, Pedro L.; Paiva-Silva, Gabriela O.

    2015-01-01

    Blood-feeding mosquitoes are exposed to high levels of heme, the product of hemoglobin degradation. Heme is a pro-oxidant that influences a variety of cellular processes. We performed a global analysis of heme-regulated Aedes aegypti (yellow fever mosquito) transcriptional changes to better understand influence on mosquito physiology at the molecular level. We observed an iron- and reactive oxygen species (ROS)-independent signaling induced by heme that comprised genes related to redox metabolism. By modulating the abundance of these transcripts, heme possibly acts as a danger signaling molecule. Furthermore, heme triggered critical changes in the expression of energy metabolism and immune response genes, altering the susceptibility towards bacteria and dengue virus. These findings seem to have implications on the adaptation of mosquitoes to hematophagy and consequently on their ability to transmit diseases. Altogether, these results may also contribute to the understanding of heme cell biology in eukaryotic cells. PMID:26275150

  20. Heme Signaling Impacts Global Gene Expression, Immunity and Dengue Virus Infectivity in Aedes aegypti.

    PubMed

    Bottino-Rojas, Vanessa; Talyuli, Octávio A C; Jupatanakul, Natapong; Sim, Shuzhen; Dimopoulos, George; Venancio, Thiago M; Bahia, Ana C; Sorgine, Marcos H; Oliveira, Pedro L; Paiva-Silva, Gabriela O

    2015-01-01

    Blood-feeding mosquitoes are exposed to high levels of heme, the product of hemoglobin degradation. Heme is a pro-oxidant that influences a variety of cellular processes. We performed a global analysis of heme-regulated Aedes aegypti (yellow fever mosquito) transcriptional changes to better understand influence on mosquito physiology at the molecular level. We observed an iron- and reactive oxygen species (ROS)-independent signaling induced by heme that comprised genes related to redox metabolism. By modulating the abundance of these transcripts, heme possibly acts as a danger signaling molecule. Furthermore, heme triggered critical changes in the expression of energy metabolism and immune response genes, altering the susceptibility towards bacteria and dengue virus. These findings seem to have implications on the adaptation of mosquitoes to hematophagy and consequently on their ability to transmit diseases. Altogether, these results may also contribute to the understanding of heme cell biology in eukaryotic cells.

  1. Diverse inhibitors of aflatoxin biosynthesis.

    PubMed

    Holmes, Robert A; Boston, Rebecca S; Payne, Gary A

    2008-03-01

    Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

  2. An investigation of hemopexin redox properties by spectroelectrochemistry: biological relevance for heme uptake.

    PubMed

    Flaherty, Meghan M; Rish, Kimberley R; Smith, Ann; Crumbliss, Alvin L

    2008-06-01

    Hemopexin (HPX) has two principal roles: it sequesters free heme in vivo for the purpose of preventing the toxic effects of this moiety, which is largely due to heme's ability to catalyze free radical formation, and it transports heme intracellularly thus limiting its availability as an iron source for pathogens. Spectroelectrochemistry was used to determine the redox potential for heme and meso-heme (mH) when bound by HPX. At pH 7.2, the heme-HPX assembly exhibits E (1/2) values in the range 45-90 mV and the mH-HPX assembly in the range 5-55 mV, depending on environmental electrolyte identity. The E (1/2) value exhibits a 100 mV positive shift with a change in pH from 7.2 to 5.5 for mH-HPX, suggesting a single proton dependent equilibrium. The E (1/2) values for heme-HPX are more positive in the presence of NaCl than KCl indicating that Na(+), as well as low pH (5.5) stabilizes ferro-heme-HPX. Furthermore, comparing KCl with K(2)HPO(4), the chloride salt containing system has a lower potential, indicating that heme-HPX is easier to oxidize. These physical properties related to ferri-/ferro-heme reduction are both structurally and biologically relevant for heme release from HPX for transport and regulation of heme oxygenase expression. Consistent with this, when the acidification of endosomes is prevented by bafilomycin then heme oxygenase-1 induction by heme-HPX no longer occurs. PMID:17712531

  3. Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

    PubMed

    Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

    2016-08-12

    Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined. PMID:27626297

  4. Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin*

    PubMed Central

    Ascenzi, Paolo; di Masi, Alessandra; Coletta, Massimo; Ciaccio, Chiara; Fanali, Gabriella; Nicoletti, Francesco P.; Smulevich, Giulietta; Fasano, Mauro

    2009-01-01

    Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role

  5. Serine biosynthesis and transport defects.

    PubMed

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. PMID:27161889

  6. Stereoselectivity in Polyphenol Biosynthesis

    NASA Technical Reports Server (NTRS)

    Lewis, Norman G.; Davin, Laurence B.

    1992-01-01

    Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.

  7. A Soret marker band for four-coordinate ferric heme proteins from absorption spectra of isolated Fe(III)-Heme+ and Fe(III)-Heme+(His) ions in vacuo.

    PubMed

    Lykkegaard, Morten Køcks; Ehlerding, Anneli; Hvelplund, Preben; Kadhane, Umesh; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted; Panja, Subhasis; Wyer, Jean Ann; Zettergren, Henning

    2008-09-10

    In this work, we report the absorption spectra in the Soret band region of isolated Fe(III)-heme+ and Fe(III)-heme+(His) ions in vacuo from action spectroscopy. Fe(III)-heme+ refers to iron(III) coordinated by the dianion of protoporphyrin IX. We find that the absorption of the five-coordinate complex is similar to that of pentacoordinate metmyoglobin variants with hydrophobic binding pockets except for an overall blueshift of about 16 nm. In the case of four-coordinate iron(III), the Soret band is similar to that of five-coordinate iron(III) but much narrower. These spectra serve as a benchmark for theoretical modeling and also serve to identify the coordination state of ferric heme proteins. To our knowledge this is the first unequivocal spectroscopic characterization of isolated 4c ferric heme in the gas phase. PMID:18700762

  8. Metabolite-driven Regulation of Heme Uptake by the Biliverdin IXβ/δ-Selective Heme Oxygenase (HemO) of Pseudomonas aeruginosa.

    PubMed

    Mouriño, Susana; Giardina, Bennett J; Reyes-Caballero, Hermes; Wilks, Angela

    2016-09-23

    Pseudomonas aeruginosa acquires extracellular heme via the Phu (Pseudomonas heme uptake) and Has (heme assimilation system) systems. We have previously shown the catalytic actions of heme oxygenase (HemO) along with the cytoplasmic heme transport protein PhuS control heme flux into the cell. To further investigate the role of the PhuS-HemO couple in modulating heme uptake, we have characterized two HemO variants, one that is catalytically inactive (HemO H26A/K34A/K132A or HemOin) and one that has altered regioselectivity (HemO N19K/K34A/F117Y/K132A or HemOα), producing biliverdin IXα (BVIXα). HemOα similar to wild type was able to interact and acquire heme from holo-PhuS. In contrast, the HemOin variant did not interact with holo-PhuS and showed no enzymatic activity. Complementation of a hemO deletion strain with the hemOin or hemOα variants in combination with [(13)C]heme isotopic labeling experiments revealed that the absence of BVIXβ and BVIXδ leads to a decrease in extracellular levels of hemophore HasA. We propose BVIXβ and/or BVIXδ transcriptionally or post-transcriptionally regulates HasA. Thus, coupling the PhuS-dependent flux of heme through HemO to feedback regulation of the cell surface signaling system through HasA allows P. aeruginosa to rapidly respond to fluctuating extracellular heme levels independent of the iron status of the cell. PMID:27493207

  9. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    NASA Astrophysics Data System (ADS)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  10. Natural selection and the genetic basis of osmoregulation in heteromyid rodents as revealed by RNA-seq.

    PubMed

    Marra, Nicholas J; Romero, Andrea; DeWoody, J Andrew

    2014-06-01

    One adaptation of ecological and evolutionary interest is the extraordinary ability of desert rodents to retain water during waste production. Much is known regarding the unique kidney physiology of kangaroo rats (Dipodomys spp.) and their ability to retain water during waste production, yet the genetic basis of these physiological adaptations is relatively unknown. Herein, we utilized RNA-seq data to conduct a comparative study to identify osmoregulatory genes expressed in heteromyid rodents. We sequenced kidney tissue from two temperate desert species (Dipodomys spectabilis and Chaetodipus baileyi) from two separate subfamilies of the Heteromyidae and compared these transcriptomes to a tropical mesic species (Heteromys desmarestianus) from a third subfamily. The evolutionary history of these subfamilies provided a robust phylogenetic control that allowed us to separate shared evolutionary history from convergence. Using two methods to detect differential expression (DE), we identified 1890 genes that showed consistent patterns of DE between the arid and mesic species. A three-species reciprocal BLAST analysis revealed 3511 sets of putative orthologues that, upon comparison to known Mus musculus sequences, revealed 323 annotated and full-length genic regions. Selection tests displayed evidence of positive selection (dn/ds > 1) on six genes in the two desert species and remained significant for one of these genes after correction for multiple testing. Thus, our data suggest that both the coding sequence and expression of genes have been shaped by natural selection to provide the genetic architecture for efficient osmoregulation in desert-adapted heteromyid rodents.

  11. Osmoregulation during Long-Term Fasting in Lungfish and Elephant Seal: Old and New Lessons for the Nephrologist.

    PubMed

    Rossier, Bernard C

    2016-01-01

    Vertebrates control the osmolality of their extra- and intra-cellular compartments despite large variations in salt and water intake. Aldosterone-dependent sodium reabsorption and vasopressin-dependent water transport in the distal nephron and collecting duct play a critical role in the final control of sodium and water balance. Long-term fasting (no eating, no drinking) represents an osmotic challenge for survival. Evolution has found very different solutions to meet this challenge. To illustrate this point, I will discuss osmoregulation of a mammal (elephant seal pup) and of a fish (lungfish) that are able to survive long-term fasting for months or even years. Homer W. Smith taught us how informative comparative anatomy and physiology of the kidney could help physiologists and nephrologists to better understand how the kidney works. In recent years, comparative genomics, transcriptomics and proteomics across the tree of life have led to the emergence of a new discipline, evolutionary medicine. In the near future, physiologists and nephrologists will benefit from this new field of investigation, thanks to its potential for the identification of novel drug targets and therapies.

  12. Osmoregulation during Long-Term Fasting in Lungfish and Elephant Seal: Old and New Lessons for the Nephrologist.

    PubMed

    Rossier, Bernard C

    2016-01-01

    Vertebrates control the osmolality of their extra- and intra-cellular compartments despite large variations in salt and water intake. Aldosterone-dependent sodium reabsorption and vasopressin-dependent water transport in the distal nephron and collecting duct play a critical role in the final control of sodium and water balance. Long-term fasting (no eating, no drinking) represents an osmotic challenge for survival. Evolution has found very different solutions to meet this challenge. To illustrate this point, I will discuss osmoregulation of a mammal (elephant seal pup) and of a fish (lungfish) that are able to survive long-term fasting for months or even years. Homer W. Smith taught us how informative comparative anatomy and physiology of the kidney could help physiologists and nephrologists to better understand how the kidney works. In recent years, comparative genomics, transcriptomics and proteomics across the tree of life have led to the emergence of a new discipline, evolutionary medicine. In the near future, physiologists and nephrologists will benefit from this new field of investigation, thanks to its potential for the identification of novel drug targets and therapies. PMID:26901864

  13. Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue.

    PubMed

    Zhang, Yan; Lamm, Randy; Pillonel, Christian; Lam, Stephen; Xu, Jin-Rong

    2002-02-01

    Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

  14. Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis.

    PubMed

    Francis, Warren R; Shaner, Nathan C; Christianson, Lynne M; Powers, Meghan L; Haddock, Steven H D

    2015-01-01

    The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr ("FYY"). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide "FYY" as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis. PMID:26125183

  15. Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis.

    PubMed

    Francis, Warren R; Shaner, Nathan C; Christianson, Lynne M; Powers, Meghan L; Haddock, Steven H D

    2015-01-01

    The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr ("FYY"). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide "FYY" as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis.

  16. Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis

    PubMed Central

    Francis, Warren R.; Shaner, Nathan C.; Christianson, Lynne M.; Powers, Meghan L.; Haddock, Steven H. D.

    2015-01-01

    The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr (“FYY”). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide “FYY” as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis. PMID:26125183

  17. Bioavailability of Heme Iron in Biscuit Filling Using Piglets as an Animal Model for Humans

    PubMed Central

    Quintero-Gutiérrez, Adrián Guillermo; González-Rosendo, Guillermina; Sánchez-Muñoz, Jonathan; Polo-Pozo, Javier; Rodríguez-Jerez, José Juan

    2008-01-01

    The objective of this work was to evaluate the bioavailability of heme iron added to biscuit filling. It comprised two stages: first, the development of the heme iron enriched biscuit filling; second, the evaluation of the bioavailability of the mineral in fattening piglets. Two groups were selected randomly and fed: a) Low iron feed and biscuits with heme iron supplemented filling; b) Normal feed (with ferrous sulphate). Weight and blood parameters were measured every fifteen days. Averages were compared after duplicate analyses. The filling had a creamy appearance, chocolate taste and smell, appropriate spreadability, heme iron content of 2.6 mg per gram and a shelf-life of a month. The heme iron supplemented pigs registered a greater (P<0.05) weight gain (27.8% more than the control group). Mortality in the heme iron group was 10%, compared to 50% in the control group. The amount of iron measured in the different compartment was greater in the heme group (3315 mg) than in the control group (2792 mg). However, the amount of iron consumed in the latter was greater. We show that an acceptable product with high heme iron content can be formulated, suitable for use as biscuit filling. The heme iron supplement produced better weight increase and lesser mortality in fattening pigs. The bioavailability of heme iron was 23% greater (P<0.05) compared to ferrous sulphate. PMID:18311330

  18. The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption.

    PubMed

    Weinborn, Valerie; Pizarro, Fernando; Olivares, Manuel; Brito, Alex; Arredondo, Miguel; Flores, Sebastián; Valenzuela, Carolina

    2015-10-30

    The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe) absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study). Study I focused on the effects of cereal proteins (zein, gliadin and glutelin) and study II on the effects of legume proteins (soy, pea and lentil) on heme Fe absorption. When heme was given alone (as a control), study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05). In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p < 0.05). These results suggest that with the exception of soy proteins, which decreased absorption, proteins derived from cereals and legumes do not affect heme Fe absorption.

  19. The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption

    PubMed Central

    Weinborn, Valerie; Pizarro, Fernando; Olivares, Manuel; Brito, Alex; Arredondo, Miguel; Flores, Sebastián; Valenzuela, Carolina

    2015-01-01

    The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe) absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study). Study I focused on the effects of cereal proteins (zein, gliadin and glutelin) and study II on the effects of legume proteins (soy, pea and lentil) on heme Fe absorption. When heme was given alone (as a control), study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05). In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p < 0.05). These results suggest that with the exception of soy proteins, which decreased absorption, proteins derived from cereals and legumes do not affect heme Fe absorption. PMID:26529009

  20. The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption.

    PubMed

    Weinborn, Valerie; Pizarro, Fernando; Olivares, Manuel; Brito, Alex; Arredondo, Miguel; Flores, Sebastián; Valenzuela, Carolina

    2015-11-01

    The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe) absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study). Study I focused on the effects of cereal proteins (zein, gliadin and glutelin) and study II on the effects of legume proteins (soy, pea and lentil) on heme Fe absorption. When heme was given alone (as a control), study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05). In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p < 0.05). These results suggest that with the exception of soy proteins, which decreased absorption, proteins derived from cereals and legumes do not affect heme Fe absorption. PMID:26529009

  1. Interaction of nitric oxide with human heme oxygenase-1.

    PubMed

    Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

    2003-01-24

    NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

  2. Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury

    PubMed Central

    Sawicki, Konrad Teodor; Shang, Meng; Wu, Rongxue; Chang, Hsiang-Chun; Khechaduri, Arineh; Sato, Tatsuya; Kamide, Christine; Liu, Ting; Naga Prasad, Sathyamangla V; Ardehali, Hossein

    2015-01-01

    Background Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. Methods and Results We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. Conclusions Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a

  3. Cyanide binding to human plasma heme-hemopexin: A comparative study

    SciTech Connect

    Ascenzi, Paolo; Leboffe, Loris; Polticelli, Fabio

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  4. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    PubMed Central

    de Visser, Sam P.; Stillman, Martin J.

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties. PMID:27070578

  5. Calculation of atom-centered partial charges for heme

    SciTech Connect

    Manchester, J.I.; Paulsen, M.D.; Ornstein, R.L.

    1994-05-01

    Atom-centered partial charges are calculated for the Fe-heme in cytochrome P450cam for use in molecular dynamics simulations of polar substrates bound in the active site of the enzyme. Charges are fit to the electrostatic potential produced by ab initio UHF wavefunctions for an Fe-porphine model. Basis set dependence of these charges is observed using the LANL1DZ, LANL2DZ and augmented 6-31G levels of theory. Upon geometry optimization of the enzyme, these charge sets cause varying degrees of distortion of the porphyrin from its crystallographically observed conformation. Scaling the charges calculated from the augmented 6-31G basis by 75% reduces the heme distortion while preserving reasonable interactions with a polar substrate. A comparison of the calculated charges with other published values is presented.

  6. Challenging Density Functional Theory Calculations with Hemes and Porphyrins.

    PubMed

    de Visser, Sam P; Stillman, Martin J

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol(-1)). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties. PMID:27070578

  7. Heme oxygenase-1: the "emerging molecule" has arrived.

    PubMed

    Morse, Danielle; Choi, Augustine M K

    2002-07-01

    Organisms on our planet have evolved in an oxidizing environment that is intrinsically inimical to life, and cells have been forced to devise means of protecting themselves. One of the defenses used most widely in nature is the enzyme heme oxygenase-1 (HO-1). This enzyme performs the seemingly lackluster function of catabolizing heme to generate bilirubin, carbon monoxide, and free iron. Remarkably, however, the activity of this enzyme results in profound changes in cells' abilities to protect themselves against oxidative injury. HO-1 has been shown to have anti-inflammatory, antiapoptotic, and antiproliferative effects, and it is now known to have salutary effects in diseases as diverse as atherosclerosis and sepsis. The mechanism by which HO-1 confers its protective effect is as yet poorly understood, but this area of invetsigation is active and rapidly evolving. This review highlights current information on the function of HO-1 and its relevance to specific pulmonary and cardiovascular diseases.

  8. Heme-based catalytic properties of human serum albumin

    PubMed Central

    Ascenzi, P; di Masi, A; Fanali, G; Fasano, M

    2015-01-01

    Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted. PMID:27551458

  9. Challenging Density Functional Theory Calculations with Hemes and Porphyrins.

    PubMed

    de Visser, Sam P; Stillman, Martin J

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol(-1)). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties.

  10. The di-heme family of respiratory complex II enzymes.

    PubMed

    Lancaster, C Roy D

    2013-05-01

    The di-heme family of succinate:quinone oxidoreductases is of particular interest, because its members support electron transfer across the biological membranes in which they are embedded. In the case of the di-heme-containing succinate:menaquinone reductase (SQR) from Gram-positive bacteria and other menaquinone-containing bacteria, this results in an electrogenic reaction. This is physiologically relevant in that it allows the transmembrane electrochemical proton potential Δp to drive the endergonic oxidation of succinate by menaquinone. In the case of the reverse reaction, menaquinol oxidation by fumarate, catalysed by the di-heme-containing quinol:fumarate reductase (QFR), evidence has been obtained that this electrogenic electron transfer reaction is compensated by proton transfer via a both novel and essential transmembrane proton transfer pathway ("E-pathway"). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory "E-pathway" appears to be required by all di-heme-containing QFR enzymes and results in the overall reaction being electroneutral. In addition to giving a brief overview of progress in the characterization of other members of this diverse family, this contribution summarizes key evidence and progress in identifying constituents of the "E-pathway" within the framework of the crystal structure of the QFR from the anaerobic epsilon-proteobacterium Wolinella succinogenes at 1.78Å resolution. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

  11. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  12. Functional imaging: monitoring heme oxygenase-1 gene expression in vivo

    NASA Astrophysics Data System (ADS)

    Zhang, Weisheng; Reilly-Contag, Pamela; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

  13. Insights into the biosynthesis and assembly of cryptophycean phycobiliproteins.

    PubMed

    Overkamp, Kristina E; Gasper, Raphael; Kock, Klaus; Herrmann, Christian; Hofmann, Eckhard; Frankenberg-Dinkel, Nicole

    2014-09-26

    Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.

  14. Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins♦

    PubMed Central

    Overkamp, Kristina E.; Gasper, Raphael; Kock, Klaus; Herrmann, Christian; Hofmann, Eckhard; Frankenberg-Dinkel, Nicole

    2014-01-01

    Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed. PMID:25096577

  15. The mechanism of spontaneous heme release from horseradish peroxidase isoenzyme A2.

    PubMed

    Smith, M L; Hjortsberg, K; Ohlsson, P I; Paul, K G

    1983-01-01

    We have investigated the spontaneous release of heme from chromatographically homogeneous horseradish peroxidase A2 at varying temperature, pH and iron ligands. A "biphasic" rate of heme release was observed under all conditions. Upon further purification of HRP A2, a component was isolated that was homogeneous by polyacrylamide gel electrophoresis and exhibited a single first order rate of heme release. The rate of the release increased with pH and temperature, decreased in the presence of cyanide and increased slightly in the presence of fluoride. These results are consistent with the idea that the rate of heme release is a measure of the flexibility of the protein lining the heme "pocket" with iron bonding playing a secondary, though important role in heme-protein interactions.

  16. Heme prevents amyloid beta peptide aggregation through hydrophobic interaction based on molecular dynamics simulation.

    PubMed

    Zhao, Li Na; Mu, Yuguang; Chew, Lock Yue

    2013-09-01

    Heme, which is abundant in hemoglobin and many other hemoproteins, is known to play an important role in electron transfer, oxygen transport, regulation of gene expression, and many other biological functions. With the belief that the aggregation of Aβ peptides forming higher order oligomers is one of the central pathological pathways in Alzheimer's disease, the formation of the Aβ-heme complex is essential as it inhibits Aβ aggregation and protects the neurons from degradation. In our studies, conventional molecular dynamics simulations were performed on the 1 Aβ + 1 heme and 2 Aβ + 4 hemes system, respectively, with the identification of several dominant binding motifs. We found that hydrophobic residues of the Aβ peptide have a high affinity to interact with heme instead of the histidine residue. We conclude that hydrophobic interaction plays a dominant role in the Aβ-heme complex formation which indirectly serves to physically prevent Aβ aggregation.

  17. Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins

    SciTech Connect

    Fonseca, Bruno M.; Tien, Ming; Rivera, Mario; Shi, Liang; Louro, Ricardo O.

    2012-04-02

    Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, present day methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg of pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins opening new possibilities in the characterization of this important class of proteins.

  18. Auxin biosynthesis and storage forms

    PubMed Central

    Strader, Lucia C.

    2013-01-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development. PMID:23580748

  19. Auxin biosynthesis and storage forms.

    PubMed

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  20. Unique Heme-Iron Coordination by the Hemoglobin Receptor IsdB of Staphylococcus aureus

    PubMed Central

    2011-01-01

    Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 Å resolution X-ray crystal structure of the IsdB-N2–heme complex. While the structure largely conforms to the eight-strand β-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s–1) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin. PMID:21574663

  1. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  2. Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins.

    PubMed

    Vincent, S H; Holeman, B; Cully, B C; Muller-Eberhard, U

    1986-01-27

    Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

  3. Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins.

    PubMed

    Ho, Winny W; Li, Huiying; Eakanunkul, Suntara; Tong, Yong; Wilks, Angela; Guo, Maolin; Poulos, Thomas L

    2007-12-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an "in" position where it can coordinate the heme iron to an "out" orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg(228) in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg(228), and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B(12)-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B(12), compared with ligands for FhuD, a peptide siderophore.

  4. Osmoregulation in the Hawaiian anchialine shrimp Halocaridina rubra (Crustacea: Atyidae): expression of ion transporters, mitochondria-rich cell proliferation and hemolymph osmolality during salinity transfers.

    PubMed

    Havird, Justin C; Santos, Scott R; Henry, Raymond P

    2014-07-01

    Studies of euryhaline crustaceans have identified conserved osmoregulatory adaptions allowing hyper-osmoregulation in dilute waters. However, previous studies have mainly examined decapod brachyurans with marine ancestries inhabiting estuaries or tidal creeks on a seasonal basis. Here, we describe osmoregulation in the atyid Halocaridina rubra, an endemic Hawaiian shrimp of freshwater ancestry from the islands' anchialine ecosystem (coastal ponds with subsurface freshwater and seawater connections) that encounters near-continuous spatial and temporal salinity changes. Given this, survival and osmoregulatory responses were examined over a wide salinity range. In the laboratory, H. rubra tolerated salinities of ~0-56‰, acting as both a hyper- and hypo-osmoregulator and maintaining a maximum osmotic gradient of ~868 mOsm kg(-1) H2O in freshwater. Furthermore, hemolymph osmolality was more stable during salinity transfers relative to other crustaceans. Silver nitrate and vital mitochondria-rich cell staining suggest all gills are osmoregulatory, with a large proportion of each individual gill functioning in ion transport (including when H. rubra acts as an osmoconformer in seawater). Additionally, expression of ion transporters and supporting enzymes that typically undergo upregulation during salinity transfer in osmoregulatory gills (i.e. Na(+)/K(+)-ATPase, carbonic anhydrase, Na(+)/K(+)/2Cl(-) cotransporter, V-type H(+)-ATPase and arginine kinase) were generally unaltered in H. rubra during similar transfers. These results suggest H. rubra (and possibly other anchialine species) maintains high, constitutive levels of gene expression and ion transport capability in the gills as a means of potentially coping with the fluctuating salinities that are encountered in anchialine habitats. Thus, anchialine taxa represent an interesting avenue for future physiological research.

  5. Inter-population differences in salinity tolerance and osmoregulation of juvenile wild and hatchery-born Sacramento splittail

    PubMed Central

    Verhille, Christine E.; Dabruzzi, Theresa F.; Cocherell, Dennis E.; Mahardja, Brian; Feyrer, Frederick; Foin, Theodore C.; Baerwald, Melinda R.; Fangue, Nann A.

    2016-01-01

    The Sacramento splittail (Pogonichthys macrolepidotus) is a minnow endemic to the highly modified San Francisco Estuary of California, USA and its associated rivers and tributaries. This species is composed of two genetically distinct populations, which, according to field observations and otolith strontium signatures, show largely allopatric distribution patterns as recently hatched juveniles. Juvenile Central Valley splittail are found primarily in the nearly fresh waters of the Sacramento and San Joaquin rivers and their tributaries, whereas San Pablo juveniles are found in the typically higher-salinity waters (i.e. up to 10‰) of the Napa and Petaluma Rivers. As the large salinity differences between young-of-year habitats may indicate population-specific differences in salinity tolerance, we hypothesized that juvenile San Pablo and Central Valley splittail populations differ in their response to salinity. In hatchery-born and wild-caught juvenile San Pablo splittail, we found upper salinity tolerances, where mortalities occurred within 336 h of exposure to 16‰ or higher, which was higher than the upper salinity tolerance of 14‰ for wild-caught juvenile Central Valley splittail. This, in conjunction with slower recovery of plasma osmolality, but not ion levels, muscle moisture or gill Na+,K+-ATPase activity, in Central Valley relative to San Pablo splittail during osmoregulatory disturbance provides some support for our hypothesis of inter-population variation in salinity tolerance and osmoregulation. The modestly improved salinity tolerance of San Pablo splittail is consistent with its use of higher-salinity habitats. Although confirmation of the putative adaptive difference through further studies is recommended, this may highlight the need for population-specific management considerations. PMID:27293743

  6. Inter-population differences in salinity tolerance and osmoregulation of juvenile wild and hatchery-born Sacramento splittail

    USGS Publications Warehouse

    Verhille, Christine E.; Dabruzzi, Theresa F.; Cocherell, Dennis E.; Mahardja, Brian; Feyrer, Frederick V.; Foin, Theodore C.; Baerwald, Melinda R.; Fangue, Nann A.

    2016-01-01

    The Sacramento splittail (Pogonichthys macrolepidotus) is a minnow endemic to the highly modified San Francisco Estuary of California, USA and its associated rivers and tributaries. This species is composed of two genetically distinct populations, which, according to field observations and otolith strontium signatures, show largely allopatric distribution patterns as recently hatched juveniles. Juvenile Central Valley splittail are found primarily in the nearly fresh waters of the Sacramento and San Joaquin rivers and their tributaries, whereas San Pablo juveniles are found in the typically higher-salinity waters (i.e. up to 10‰) of the Napa and Petaluma Rivers. As the large salinity differences between young-of-year habitats may indicate population-specific differences in salinity tolerance, we hypothesized that juvenile San Pablo and Central Valley splittail populations differ in their response to salinity. In hatchery-born and wild-caught juvenile San Pablo splittail, we found upper salinity tolerances, where mortalities occurred within 336 h of exposure to 16‰ or higher, which was higher than the upper salinity tolerance of 14‰ for wild-caught juvenile Central Valley splittail. This, in conjunction with slower recovery of plasma osmolality, but not ion levels, muscle moisture or gill Na+,K+-ATPase activity, in Central Valley relative to San Pablo splittail during osmoregulatory disturbance provides some support for our hypothesis of inter-population variation in salinity tolerance and osmoregulation. The modestly improved salinity tolerance of San Pablo splittail is consistent with its use of higher-salinity habitats. Although confirmation of the putative adaptive difference through further studies is recommended, this may highlight the need for population-specific management considerations.

  7. Structural characterization of a novel neuropeptide from the central nervous system of the leech Erpobdella octoculata. The leech osmoregulator factor.

    PubMed

    Salzet, M; Bulet, P; Weber, W M; Clauss, W; Verger-Bocquet, M; Malecha, J

    1996-03-22

    Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin

  8. Investigations on the osmoregulation of freshwater fish (Oreochromis niloticus) following exposures to metals (Cd, Cu) in differing hardness.

    PubMed

    Saglam, Dilek; Atli, Gülüzar; Canli, Mustafa

    2013-06-01

    Hardness is one of the most important factors in water chemistry as it affects fish physiology and metal toxicity. The aim of this study was to investigate osmoregulatory responses in the Nile tilapia Oreochromis niloticus exposed to copper and cadmium (1.0μg/mL) in soft water (SW) (hardness 80mg CaCO3/L and conductivity 1.77mS/cm) and hard water (HW) (hardness 320mg CaCO3/L and conductivity 5.80mS/cm) for 0, 1, 7 and 14 days. Following the exposures, Na(+)/K(+)-ATPase activity, ion and Cu levels in the gill, kidney and intestine were measured. There was no fish mortality within 14 days, except Cu exposure in SW which killed all fish between 8 and 12 days. Generally, Na(+)/K(+)-ATPase activity was altered by both metal exposures in the gill and kidney as it increased in HW condition, but decreased in SW condition. There were also alterations in Na(+)/K(+)-ATPase activity in the intestine as its activity generally decreased. Data, in general, showed that Cd was more effective on Na(+)/K(+)-ATPase activity comparing to Cu. However, ion levels altered mainly in the kidney and intestine. Tissue metal accumulation was higher in fish tissues from SW condition comparing to HW condition. Data represented here showed that the effects of metals differed in differing water hardness. This suggests that special attention should be paid to the water chemistry when natural monitoring studies are carried out. This study also suggests that the response of osmoregulation system of fish may be a sensitive indicator under stressful conditions in different natural waters.

  9. Inter-population differences in salinity tolerance and osmoregulation of juvenile wild and hatchery-born Sacramento splittail.

    PubMed

    Verhille, Christine E; Dabruzzi, Theresa F; Cocherell, Dennis E; Mahardja, Brian; Feyrer, Frederick; Foin, Theodore C; Baerwald, Melinda R; Fangue, Nann A

    2016-01-01

    The Sacramento splittail (Pogonichthys macrolepidotus) is a minnow endemic to the highly modified San Francisco Estuary of California, USA and its associated rivers and tributaries. This species is composed of two genetically distinct populations, which, according to field observations and otolith strontium signatures, show largely allopatric distribution patterns as recently hatched juveniles. Juvenile Central Valley splittail are found primarily in the nearly fresh waters of the Sacramento and San Joaquin rivers and their tributaries, whereas San Pablo juveniles are found in the typically higher-salinity waters (i.e. up to 10‰) of the Napa and Petaluma Rivers. As the large salinity differences between young-of-year habitats may indicate population-specific differences in salinity tolerance, we hypothesized that juvenile San Pablo and Central Valley splittail populations differ in their response to salinity. In hatchery-born and wild-caught juvenile San Pablo splittail, we found upper salinity tolerances, where mortalities occurred within 336 h of exposure to 16‰ or higher, which was higher than the upper salinity tolerance of 14‰ for wild-caught juvenile Central Valley splittail. This, in conjunction with slower recovery of plasma osmolality, but not ion levels, muscle moisture or gill Na(+),K(+)-ATPase activity, in Central Valley relative to San Pablo splittail during osmoregulatory disturbance provides some support for our hypothesis of inter-population variation in salinity tolerance and osmoregulation. The modestly improved salinity tolerance of San Pablo splittail is consistent with its use of higher-salinity habitats. Although confirmation of the putative adaptive difference through further studies is recommended, this may highlight the need for population-specific management considerations. PMID:27293743

  10. Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis

    PubMed Central

    Kunz, Hans-Henning; Gierth, Markus; Herdean, Andrei; Satoh-Cruz, Mio; Kramer, David M.; Spetea, Cornelia; Schroeder, Julian I.

    2014-01-01

    Multiple K+ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K+ transporters is limited. Potassium transport and a well-balanced K+ homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K+ transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na+/H+ antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis. PMID:24794527

  11. Alternate biosynthesis of valerenadiene and related sesquiterpenes.

    PubMed

    Paknikar, Shashikumar K; Kadam, Shahuraj H; Ehrlich, April L; Bates, Robert B

    2013-09-01

    It is proposed that the biosynthesis of the sesquiterpene valerenadiene, a key intermediate in the biosynthesis of a sedative in valerian, involves cyclopropane and not cyclobutane intermediates and includes as a key step a cyclopropylcarbinylcation-cyclopropylcarbinylcation rearrangement analogous to the one observed in the conversion of presqualene to squalene in triterpene and steroid biosynthesis. Similar mechanisms are proposed for the biosynthesis of the related sesquiterpenes pacifigorgiol, tamariscene and (+)-pacifigorgia-1,10-diene. PMID:24273843

  12. Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

    PubMed

    Servid, Amy E; McKay, Alison L; Davis, Cherry A; Garton, Elizabeth M; Manole, Andreea; Dobbin, Paul S; Hough, Michael A; Andrew, Colin R

    2015-06-01

    Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

  13. 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.

  14. In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

    DOEpatents

    Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H; Wang, Zhan; Lewis, Jared C

    2015-03-31

    The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

  15. A heme-binding domain controls regulation of ATP-dependent potassium channels

    PubMed Central

    Burton, Mark J.; Kapetanaki, Sofia M.; Chernova, Tatyana; Jamieson, Andrew G.; Dorlet, Pierre; Santolini, Jérôme; Mitcheson, John S.; Davies, Noel W.; Schmid, Ralf; Raven, Emma L.; Storey, Nina M.

    2016-01-01

    Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels. PMID:27006498

  16. Heme degradation upon production of endogenous hydrogen peroxide via interaction of hemoglobin with sodium dodecyl sulfate.

    PubMed

    Salehi, N; Moosavi-Movahedi, A A; Fotouhi, L; Yousefinejad, S; Shourian, M; Hosseinzadeh, R; Sheibani, N; Habibi-Rezaei, M

    2014-04-01

    In this study the hemoglobin heme degradation upon interaction with sodium dodecyl sulfate (SDS) was investigated using UV-vis and fluorescence spectroscopy, multivariate curve resolution analysis, and chemiluminescence method. Our results showed that heme degradation occurred during interaction of hemoglobin with SDS producing three fluorescent components. We showed that the hydrogen peroxide, produced during this interaction, caused heme degradation. In addition, the endogenous hydrogen peroxide was more effective in hemoglobin heme degradation compared to exogenously added hydrogen peroxide. The endogenous form of hydrogen peroxide altered oxyHb to aquamethemoglobin and hemichrome at low concentration. In contrast, the exogenous hydrogen peroxide lacked this ability under same conditions.

  17. Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters.

    PubMed

    Cabello-Donayre, María; Malagarie-Cazenave, Sophie; Campos-Salinas, Jenny; Gálvez, Francisco J; Rodríguez-Martínez, Alba; Pineda-Molina, Estela; Orrego, Lina M; Martínez-García, Marta; Sánchez-Cañete, María P; Estévez, Antonio M; Pérez-Victoria, José M

    2016-09-01

    Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets. PMID:27328668

  18. Nitrosylation of c heme in cd(1)-nitrite reductase is enhanced during catalysis.

    PubMed

    Rinaldo, Serena; Giardina, Giorgio; Cutruzzolà, Francesca

    2014-08-29

    The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs. It is now established that, during catalysis, cd1NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar "low" affinity for NO (KD=0.11 μM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.

  19. Insights into myeloperoxidase biosynthesis from its inherited deficiency.

    PubMed

    Nauseef, W M

    1998-09-01

    Myeloperoxidase (MPO) is a heme protein present in the granules of neutrophils and monocytes. The activated neutrophil releases MPO into the phagolysosome or into the extracellular space in response to a variety of agonists. During concomitant activation of the NADPH-dependent oxidase, the stimulated neutrophil also generates hydrogen peroxide, and in this way the MPO-hydrogen peroxide-halide system exerts its potent microbicidal activity. Recent interest in MPO has extended well beyond the domain of innate host defense against infection and includes generalized inflammatory diseases, atherosclerosis, and degenerative neurologic diseases. Search of the various data banks using the cDNA sequence for MPO has uncovered previously unsuspected relationships among peroxidatively active proteins in widely different species. In addition, application of the analytical tools of cell and molecular biology has allowed definition of specific genotypes underlying MPO deficiency and the impact of particular mutations on the fate of MPO precursors along the biosynthetic pathway. In parallel, such studies have allowed significant advances in understanding of the normal steps in MPO biosynthesis and intracellular targeting.

  20. Investigations of ultrafast ligand rebinding to heme and heme proteins using temperature and strong magnetic field perturbations

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenyu

    This thesis is written to summarize investigations of the mechanisms that underlie the kinetics of diatomic ligand rebinding to the iron atom of the heme group, which is chelated inside heme proteins. The family of heme proteins is a major object of studies for several branches of scientific research activity. Understanding the ligand binding mechanisms and pathways is one of the major goals for biophysics. My interests mainly focus on the physics of this ligand binding process. Therefore, to investigate the problem, isolated from the influence of the protein matrix, Fe-protophorphyrin IX is chosen as the prototype system in my studies. Myoglobin, the most extensively and intensively studied protein, is another ideal system that allows coupling the protein polypeptide matrix into the investigation. A technique to synchro-lock two laser pulse trains electronically is applied to our pump-probe spectroscopic studies. Based on this technique, a two color, fs/ps pump-probe system is developed which extends the temporal window for our investigation to 13ns and fills a gap existing in previous pump-probe investigations. In order to apply this newly-developed pump-probe laser system to implement systematic studies on the kinetics of diatomic ligand (NO, CO, O2) rebinding to heme and heme proteins, several experimental setups are utilized. In Chapter 1, the essential background knowledge, which helps to understand the iron-ligand interaction, is briefly described. In Chapter 2, in addition to a description of the preparation protocols of protein samples and details of the method for data analysis, three home-made setups are described, which include: a picosecond laser regenerative amplifier, a pump-probe application along the bore (2-inch in diameter) of a superconducting magnet and a temperature-controllable cryostat for spinning sample cell. Chapter 3 presents high magnetic field studies of several heme-ligand or protein-ligand systems. Pump-probe spectroscopy is used to

  1. Phermone biosynthesis activation in fire ants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN), was identified to stimulate sex pheromone biosynthesis in a moth. Since then, the physiological role, target site and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in mot...

  2. Calcium-Dependent Conformation of a Heme and Fingerprint Peptide of the Di-Heme Cytochrome c Peroxidase from Paracoccus Pantotrophus

    SciTech Connect

    PAULETA,SOFIA R.; LU,YI; GOODHEW,CELIA F.; MOURA,ISABEL; PETTIGREW,GRAHAM W.; SHELNUTT,JOHN A.

    2000-12-18

    The structural changes in the heme macrocycle and substituents caused by binding of Ca{sup 2+} to the diheme cytochrome c peroxidase from Paracoccuspantotrophus were clarified by resonance Raman spectroscopy of the inactive filly oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca{sup 2+}-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca{sup 2+}or Mg{sup 2+}. This increase in the heme distortion also explains the red shift in the Soret absorption band that occurs upon Ca{sup 2+} binding. Changes also occur in the low frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon CM{sup 2+} binding to site I. These structural changes, possibly enhanced in the semi-reduced form of the enzyme, may lead to loss of the sixth ligand at the peroxidatic heme and activation of the enzyme.

  3. Functional analysis of the zinc cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells.

    PubMed

    Defranoux, N; Gaisne, M; Verdière, J

    1994-03-01

    CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under heme-deficient conditions is dependent upon DNA binding. PMID:8152420

  4. The Evolution of Aflatoxin Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and O-methylsterigmatocysin (OMST), the respective penultimate and ultimate precursors of AF. Although ST, OMST, and ...

  5. Transcriptional control of flavonoid biosynthesis

    PubMed Central

    Li, Shutian

    2014-01-01

    Flavonoids are plant secondary polyphenolic metabolites and fulfil many vital biological functions, offering a valuable metabolic and genetic model for studying transcriptional control of gene expression. Arabidopsis thaliana mainly accumulates 3 types of flavonoids, including flavonols, anthocyanins, and proanthocyanidins (PAs). Flavonoid biosynthesis involves a multitude of well-characterized enzymatic and regulatory proteins. Three R2R3-MYB proteins (MYB11, MYB12, and MYB111) control flavonol biosynthesis via activating the early biosynthetic steps, whereas the production of anthocyanins and PAs requires the MYB-bHLH-WD40 (MBW) complex to activate the late biosynthetic genes. Additional regulators of flavonoid biosynthesis have recently come to light, which interact with R2R3-MYBs or bHLHs to organize or disrupt the formation of the MBW complex, leading to enhanced or compromised flavonoid production. This mini-review gives an overview of how these novel players modulate flavonoid metabolism and thus plant developmental processes and further proposes a fine-tuning mechanism to complete the complex regulatory network controlling flavonoid biosynthesis. PMID:24393776

  6. O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    SciTech Connect

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-03-04

    Research highlights: {yields} Human serum heme-albumin displays globin-like properties. {yields} O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin. {yields} Allosteric modulation of human serum heme-albumin reactivity. {yields} Rifampicin is an allosteric effector of human serum heme-albumin. {yields} Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O{sub 2}-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10{sup -5} and 8.3 x 10{sup -4} s{sup -1}, and h = 1.3 x 10{sup -4} and 8.5 x 10{sup -4} s{sup -1}, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 {sup o}C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O{sub 2} does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O{sub 2}-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  7. (-)-Menthol biosynthesis and molecular genetics.

    PubMed

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L; Wildung, Mark R

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil. PMID:16292524

  8. (-)-Menthol biosynthesis and molecular genetics

    NASA Astrophysics Data System (ADS)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  9. Membrane lipid modification by polyunsaturated fatty acids sensitizes oligodendroglial OLN-93 cells against oxidative stress and promotes up-regulation of heme oxygenase-1 (HSP32).

    PubMed

    Brand, Annette; Bauer, Nina G; Hallott, Amanda; Goldbaum, Olaf; Ghebremeskel, Kebreab; Reifen, Ram; Richter-Landsberg, Christiane

    2010-04-01

    Polyunsaturated fatty acids (PUFA) are highly abundant in brain tissue, and docosahexaenoic acid (DHA) might protect cells from oxidative stress (OS) during inflammation and demyelinating disorders, but also might exert pro-oxidant effects. Here we investigated if PUFA supplements lead to heat shock protein induction, altered cell survival properties and stress responses to OS exerted by hydrogen peroxide in oligodendroglial OLN-93 cells. The data show that supplements of various fatty acids (FA) with 18-22 carbons chain length and 2-6 double bonds led to PUFA enrichment in cellular membranes. Depending on the degree of desaturation, FA-supplements caused the up-regulation of heme oxygenase-1 (HSP32), a stress protein inducible by OS, and an increase in sensitivity to hydrogen peroxide-treatment. DHA, with the highest number of double bonds, was most effective. Co-treatment with DHA and the lipophilic vitamin E analogue alpha-tocopherol, suppressed heme oxygenase-1 up-regulation and cell survival was restored. Analysis of the lipid profile demonstrates that alpha-tocopherol not only has antioxidant capacities, but also directly modified the PUFA profile in cell membranes. Enrichment with higher omega-3, -6 and -9 PUFA and an increase in the biosynthesis rate of very long chain fatty acids, mainly changed the FA profile of ethanolamine and serine phosphoglycerides.

  10. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    PubMed Central

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2011-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

  11. Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Medeiros, Lia Carolina Soares; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Pignataro, Omar P.; Docampo, Roberto; Alonso, Guillermo D.

    2010-01-01

    Summary Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases. Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different phosphodiesterase (PDE) families. One of these PDEs, T. cruzi phosphodiesterase C2 (TcrPDEC2) has been characterized as a FYVE-domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labeling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in phosphodiesterase activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signaling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized phosphodiesterase involved in osmoregulation in T. cruzi. PMID:21166893

  12. Mechanism of the Mg-chelatase step in chlorophyll biosynthesis. Final technical report

    SciTech Connect

    Weinstein, Jon

    2002-05-02

    Mg-chelatase catalyzes the insertion of Mg2+ into protoporphyrin-IX (Proto) in the chlorophyll biosynthetic pathway. This is the first step unique to the chlorophyll pathway and is at the branchpoint between heme and chlorophyll biosynthesis. Previous work from our laboratory has shown that the enzyme from pea chloroplasts requires three distinct protein fractions (now known to contain the D, I and H subunits). The reaction requires ATP in two distinct steps: activation requiring two of the fractions (I and D) and metal ion insertion, requiring all three fractions. Work covered in this granting period includes the cloning and expression of the active form of one of the pea subunits and demonstration of the change in chromatographic behavior of the subunits upon activation with ATP.

  13. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    SciTech Connect

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K.

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  14. Paradoxical effects of heme arginate on survival of myocutaneous flaps

    PubMed Central

    Czopek, Alicja; Wigmore, Stephen J.; Kluth, David C.

    2013-01-01

    Ischemia reperfusion injury (IRI) contributes to partial flap and solid organ transplant failure. Heme-oxygenase 1 (HO-1) is an inducible, cytoprotective enzyme which protects against IRI in solid organ transplant models. Heme arginate (HA), a HO-1 inducer, is a promising, translatable, preconditioning agent. This study investigated the effects of preconditioning with HA on the clinical outcome of a myocutaneous IRI model. Forty male Lewis rats were randomized to intravenously receive 1) Control-NaCl, 2) HA, 3) HA and tin mesoporphyrin (SnMP), a HO-1 inhibitor; and 4) SnMP alone. Twenty-four hours later, an in situ transverse rectus abdominis myocutaneous flap was performed under isoflurane anesthesia. Viability of flaps was measured clinically and by laser-Doppler perfusion scanning. In vitro work on human epidermal keratinocytes (HEKa) assessed the effects of HA, SnMP, and the iron chelator desferrioxamine on 1) cytotoxicity, 2) intracellular reactive oxygen species (ROS) concentration, and 3) ROS-mediated DNA damage. In contrast to our hypothesis, HA preconditioning produced over 30% more flap necrosis at 48 h compared with controls (P = 0.02). HA-containing treatments produced significantly worse flap perfusion at all postoperative time points. In vitro work showed that HA is cytotoxic to keratinocytes. This cytotoxicity was independent of HO-1 and was mediated by the generation of ROS by free heme. In contrast to solid organ data, pharmacological preconditioning with HA significantly worsened clinical outcome, thus indicating that this is not a viable approach in free flap research. PMID:24089372

  15. Multicopper manganese oxidase accessory proteins bind Cu and heme.

    PubMed

    Butterfield, Cristina N; Tao, Lizhi; Chacón, Kelly N; Spiro, Thomas G; Blackburn, Ninian J; Casey, William H; Britt, R David; Tebo, Bradley M

    2015-12-01

    Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

  16. Pilot-scale tests of HEME and HEPA dissolution process

    SciTech Connect

    Qureshi, Z.H.; Strege, D.K.

    1994-06-01

    A series of pilot-scale demonstration tests for the dissolution of High Efficiency Mist Eliminators (HEME`s) and High Efficiency Particulate Airfilters (HEPA) were performed on a 1/5th linear scale. These fiberglass filters are to be used in the Defense Waste Processing Facility (DWPF) to decontaminate the effluents from the off-gases generated during the feed preparation process and vitrification. When removed, these filters will be dissolved in the Decontamination Waste Treatment Tank (DWTT) using 5 wt% NaOH solution. The contaminated fiberglass is converted to an aqueous stream which will be transferred to the waste tanks. The filter metal structure will be rinsed with process water before its disposal as low-level solid waste. The pilot-scale study reported here successfully demonstrated a simple one step process using 5 wt% NaOH solution. The proposed process requires the installation of a new water spray ring with 30 nozzles. In addition to the reduced waste generated, the total process time is reduced to 48 hours only (66% saving in time). The pilot-scale tests clearly demonstrated that the dissolution process of HEMEs has two stages - chemical digestion of the filter and mechanical erosion of the digested filter. The digestion is achieved by a boiling 5 wt% caustic solutions, whereas the mechanical break down of the digested filter is successfully achieved by spraying process water on the digested filter. An alternate method of breaking down the digested filter by increased air sparging of the solution was found to be marginally successful are best. The pilot-scale tests also demonstrated that the products of dissolution are easily pumpable by a centrifugal pump.

  17. Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer.

    PubMed

    Sunamura, Makoto; Duda, Dan G; Ghattas, Maivel H; Lozonschi, Lucian; Motoi, Fuyuhiko; Yamauchi, Jun-Ichiro; Matsuno, Seiki; Shibahara, Shigeki; Abraham, Nader G

    2003-01-01

    Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

  18. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    SciTech Connect

    Westberg, Johan A.; Jiang, Ji; Andersson, Leif C.

    2011-06-03

    Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  19. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages

    PubMed Central

    Philip, Mary; Chiu, Edison Y.; Hajjar, Adeline M.; Abkowitz, Janis L.

    2016-01-01

    Pathogenic bacteria have evolved multiple mechanisms to capture iron or iron-containing heme from host tissues or blood. In response, organisms have developed defense mechanisms to keep iron from pathogens. Very little of the body's iron store is available as free heme; rather nearly all body iron is complexed with heme or other proteins. The feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, exports heme from cells. It was unknown whether FLVCR regulates heme-iron availability after infection, but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection, we hypothesized that macrophages dynamically regulate FLVCR. We stimulated murine primary macrophages or macrophage cell lines with LPS and found that Flvcr is rapidly downregulated in a TLR4/MD2-dependent manner; TLR1/2 and TLR3 stimulation also decreased Flvcr expression. We identified several candidate TLR-activated transcription factors that can bind to the Flvcr promoter. Macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species. Our findings underscore the complexity of this regulation and point to a new role for FLVCR and heme export in macrophages responses to infection and inflammation. PMID:27006955

  20. Non-heme iron hydroperoxo species in superoxide reductase as a catalyst for oxidation reactions.

    PubMed

    Rat, S; Ménage, S; Thomas, F; Nivière, V

    2014-11-25

    The non-heme high-spin ferric iron hydroperoxo species formed in superoxide reductase catalyzes oxidative aldehyde deformylation through its nucleophilic character. This species also acts as an electrophile to catalyze oxygen atom transfer in sulfoxidation reactions, highlighting the oxidation potential of non-heme iron hydroperoxo species.

  1. Altered heme-mediated modulation of dendritic cell function in sickle cell alloimmunization

    PubMed Central

    Godefroy, Emmanuelle; Liu, Yunfeng; Shi, Patricia; Mitchell, W. Beau; Cohen, Devin; Chou, Stella T.; Manwani, Deepa; Yazdanbakhsh, Karina

    2016-01-01

    Transfusions are the main treatment for patients with sickle cell disease. However, alloimmunization remains a major life-threatening complication for these patients, but the mechanism underlying pathogenesis of alloimmunization is not known. Given the chronic hemolytic state characteristic of sickle cell disease, resulting in release of free heme and activation of inflammatory cascades, we tested the hypothesis that anti-inflammatory response to heme is compromised in alloimmunized sickle patients, increasing their risk of alloimmunization. Heme-exposed monocyte-derived dendritic cells from both non-alloimmunized sickle patients and healthy donors inhibited priming of pro-inflammatory CD4+ type 1 T cells, and exhibited significantly reduced levels of the maturation marker CD83. In contrast, in alloimmunized patients, heme did not reverse priming of pro-inflammatory CD4+ cells by monocyte-derived dendritic cells or their maturation. Furthermore, heme dampened NF-κB activation in non-alloimmunized, but not in alloimmunized monocyte-derived dendritic cells. Heme-mediated CD83 inhibition depended on Toll-like receptor 4 but not heme oxygenase 1. These data suggest that extracellular heme limits CD83 expression on dendritic cells in non-alloimmunized sickle patients through a Toll-like receptor 4-mediated pathway, involving NF-κB, resulting in dampening of pro-inflammatory responses, but that in alloimmunized patients this pathway is defective. This opens up the possibility of developing new therapeutic strategies to prevent sickle cell alloimmunization. PMID:27229712

  2. Measurements of Heme Relaxation and Ligand Recombination in Strong Magnetic Fields

    PubMed Central

    Zhang, Zhenyu; Benabbas, Abdelkrim; Ye, Xiong; Yu, Anchi; Champion, Paul M.

    2009-01-01

    Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 Tesla). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O2), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit. However, in the case of CO binding to heme in glycerol and O2 binding to myoglobin, we observe a small magnetic field dependent change in the early time amplitude of the optical response that is assigned to heme cooling. One possibility, consistent with this observation, is that there is a weak magnetic field dependence of the non-radiative branching ratio into the vibrationally hot electronic ground state during CO photolysis. Ancillary studies of the “spin-forbidden” CO binding reaction in a variety of heme compounds in the absence of magnetic field demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO binding to heme is not always retarded by unfavorable spin selection rules involving a double spin-flip superexchange mechanism. In fact, it appears that the small prefactor (~109s−1) found for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO rebinding. These results point to unresolved fundamental issues that underlie the theory of heme-ligand photolysis and rebinding. PMID:19588986

  3. Altered heme-mediated modulation of dendritic cell function in sickle cell alloimmunization.

    PubMed

    Godefroy, Emmanuelle; Liu, Yunfeng; Shi, Patricia; Mitchell, W Beau; Cohen, Devin; Chou, Stella T; Manwani, Deepa; Yazdanbakhsh, Karina

    2016-09-01

    Transfusions are the main treatment for patients with sickle cell disease. However, alloimmunization remains a major life-threatening complication for these patients, but the mechanism underlying pathogenesis of alloimmunization is not known. Given the chronic hemolytic state characteristic of sickle cell disease, resulting in release of free heme and activation of inflammatory cascades, we tested the hypothesis that anti-inflammatory response to heme is compromised in alloimmunized sickle patients, increasing their risk of alloimmunization. Heme-exposed monocyte-derived dendritic cells from both non-alloimmunized sickle patients and healthy donors inhibited priming of pro-inflammatory CD4(+) type 1 T cells, and exhibited significantly reduced levels of the maturation marker CD83. In contrast, in alloimmunized patients, heme did not reverse priming of pro-inflammatory CD4(+) cells by monocyte-derived dendritic cells or their maturation. Furthermore, heme dampened NF-κB activation in non-alloimmunized, but not in alloimmunized monocyte-derived dendritic cells. Heme-mediated CD83 inhibition depended on Toll-like receptor 4 but not heme oxygenase 1. These data suggest that extracellular heme limits CD83 expression on dendritic cells in non-alloimmunized sickle patients through a Toll-like receptor 4-mediated pathway, involving NF-κB, resulting in dampening of pro-inflammatory responses, but that in alloimmunized patients this pathway is defective. This opens up the possibility of developing new therapeutic strategies to prevent sickle cell alloimmunization.

  4. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  5. Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice

    PubMed Central

    Belcher, John D.; Mahaseth, Hemachandra; Welch, Thomas E.; Otterbein, Leo E.; Hebbel, Robert P.; Vercellotti, Gregory M.

    2006-01-01

    Transgenic sickle mice expressing βS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-κB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation–induced stasis, leukocyte-endothelium interactions, and NF-κB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-κB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation–induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice. PMID:16485041

  6. Cloning and Expression of cDNA for Rat Heme Oxygenase

    NASA Astrophysics Data System (ADS)

    Shibahara, Shigeki; Muller, Rita; Taguchi, Hayao; Yoshida, Tadashi

    1985-12-01

    Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in λ gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

  7. A hydrogen bond scaffold supported synthetic heme Fe(III)-O2(-) adduct.

    PubMed

    Mittra, Kaustuv; Chatterjee, Sudipta; Samanta, Subhra; Sengupta, Kushal; Bhattacharjee, Hridaynath; Dey, Abhishek

    2012-11-01

    A hydrogen bonded heme-Fe(III)-O(2)(-) adduct is stabilized and characterized using resonance Raman and EPR spectroscopy. The low O-O vibrations of this complex are quite different from those reported for other heme-Fe(III)-O(2)(-) adducts.

  8. Time-Resolved X-Ray Crystallography of Heme Proteins

    SciTech Connect

    Srajer, Vukica; Royer, Jr., William E.

    2008-04-29

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third-generation synchrotrons provide 100-ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have recently permitted observing for the first time allosteric transitions in real time for a cooperative dimeric hemoglobin.

  9. Time-resolved x-ray crystallography of heme proteins

    PubMed Central

    Royer, William E.

    2012-01-01

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third generation synchrotrons provide 100ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have permitted observing allosteric transitions in real time for a cooperative dimeric hemoglobin. PMID:18433638

  10. Altered cardiovascular reactivity and osmoregulation during hyperosmotic stress in adult rats developmentally exposed to polybrominated diphenyl ethers (PBDEs)

    SciTech Connect

    Shah, Ashini; Coburn, Cary G.; Watson-Siriboe, Abena; Whitley, Rebecca; Shahidzadeh, Anoush; Gillard, Elizabeth R.; Nichol, Robert; Leon-Olea, Martha; Gaertner, Mark; Kodavanti, Prasada Rao S.

    2011-10-15

    h (358.3 {+-} 12.4 mOsm/L) relative to 45 min post hyperosmotic injection (325.1 {+-} 11.4 mOsm/L). Impaired osmoregulation in PBDE-treated animals could not be attributed to decreased levels of plasma vasopressin. Our findings suggest that developmental exposure to PBDEs may disrupt cardiovascular reactivity and osmoregulatory responses to physiological activation in late adulthood. - Highlights: > We examined whether PBDE exposure could impact osmotic and cardiovascular regulation. > Hyperosmotic treatment yielded significant changes in systolic BP in PBDE exposed rats only. > PBDEs may disrupt cardiovascular and osmoregulatory responses to physiological activation.

  11. Systematic tuning of heme redox potentials and its effects on O2 reduction rates in a designed oxidase in myoglobin.

    PubMed

    Bhagi-Damodaran, Ambika; Petrik, Igor D; Marshall, Nicholas M; Robinson, Howard; Lu, Yi

    2014-08-27

    Cytochrome c Oxidase (CcO) is known to catalyze the reduction of O2 to H2O efficiently with a much lower overpotential than most other O2 reduction catalysts. However, methods by which the enzyme fine-tunes the reduction potential (E°) of its active site and the corresponding influence on the O2 reduction activity are not well understood. In this work, we report systematic tuning of the heme E° in a functional model of CcO in myoglobin containing three histidines and one tyrosine in the distal pocket of heme. By removing hydrogen-bonding interactions between Ser92 and the proximal His ligand and a heme propionate, and increasing hydrophobicity of the heme pocket through Ser92Ala mutation, we have increased the heme E° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme b in the CcO mimic with heme a analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher E° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O2 consumption studies on these CcO mimics revealed a strong enhancement in O2 reduction rates with increasing heme E°. Such methods of tuning the heme E° through a combination of secondary sphere mutations and heme substitutions can be applied to tune E° of other heme proteins, allowing for comprehensive investigations of the relationship between E° and enzymatic activity.

  12. Systematic tuning of heme redox potentials and its effects on O2 reduction rates in a designed oxidase in myoglobin.

    PubMed

    Bhagi-Damodaran, Ambika; Petrik, Igor D; Marshall, Nicholas M; Robinson, Howard; Lu, Yi

    2014-08-27

    Cytochrome c Oxidase (CcO) is known to catalyze the reduction of O2 to H2O efficiently with a much lower overpotential than most other O2 reduction catalysts. However, methods by which the enzyme fine-tunes the reduction potential (E°) of its active site and the corresponding influence on the O2 reduction activity are not well understood. In this work, we report systematic tuning of the heme E° in a functional model of CcO in myoglobin containing three histidines and one tyrosine in the distal pocket of heme. By removing hydrogen-bonding interactions between Ser92 and the proximal His ligand and a heme propionate, and increasing hydrophobicity of the heme pocket through Ser92Ala mutation, we have increased the heme E° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme b in the CcO mimic with heme a analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher E° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O2 consumption studies on these CcO mimics revealed a strong enhancement in O2 reduction rates with increasing heme E°. Such methods of tuning the heme E° through a combination of secondary sphere mutations and heme substitutions can be applied to tune E° of other heme proteins, allowing for comprehensive investigations of the relationship between E° and enzymatic activity. PMID:25076049

  13. Gibberellin biosynthesis in Gibberlla fujikuroi

    SciTech Connect

    Johnson, S.W.; Coolbaugh, R.C. )

    1989-04-01

    Gibberellins (GAs) are a group of plant growth hormones which were first isolated from the fungus Gibberella fujikuori. We have examined the biosynthesis of GAs in this fungus in liquid cultures using HPLC followed by GC-MS. Furthermore we have used cell-free enzyme extracts with {sup 14}C-labeled intermediates to examine the regulation of specific parts of the biosynthetic pathway. GA{sub 3} is the predominant GA in well aerated cultures. GA{sub 4} and GA{sub 7}, intermediates in GA{sub 3} biosynthesis, accumulate in cultures with low levels of dissolved oxygen, but are not detectable in more aerated cultures. Light stimulates GA production in G. fujikuroi cultures grown from young stock. Cell-free enzyme studies indicate that light has no effect on incorporation of mevalonic acid into kaurene, but does significantly stimulate the oxidation of kaurenoic acid.

  14. Lignification: Flexibility, Biosynthesis and Regulation.

    PubMed

    Zhao, Qiao

    2016-08-01

    Lignin is a complex phenolic polymer that is deposited in the secondary cell wall of all vascular plants. The evolution of lignin is considered to be a critical event during vascular plant development, because lignin provides mechanical strength, rigidity, and hydrophobicity to secondary cell walls to allow plants to grow tall and transport water and nutrients over a long distance. In recent years, great research efforts have been made to genetically alter lignin biosynthesis to improve biomass degradability for the production of second-generation biofuels. This global focus on lignin research has significantly advanced our understanding of the lignification process. Based on these advances, here I provide an overview of lignin composition, the biosynthesis pathway and its regulation. PMID:27131502

  15. Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements of heme lyase.

    PubMed

    Fülöp, Vilmos; Sam, Katharine A; Ferguson, Stuart J; Ginger, Michael L; Allen, James W A

    2009-05-01

    The principal physiological role of mitochondrial cytochrome c is electron transfer during oxidative phosphorylation. c-Type cytochromes are almost always characterized by covalent attachment of heme to protein through two thioether bonds between the heme vinyl groups and the thiols of cysteine residues in a Cys-Xxx-Xxx-Cys-His motif. Uniquely, however, members of the evolutionarily divergent protist phylum Euglenozoa, which includes Trypanosoma and Leishmania species, have mitochondrial cytochromes c with heme attached through only one thioether bond [to an (A/F)XXCH motif]; the implications of this for the cytochrome structures are unclear. Here we present the 1.55 A resolution X-ray crystal structure of cytochrome c from the trypanosomatid Crithidia fasciculata. Despite the fundamental difference in heme attachment and in the cytochrome c biogenesis machinery of the Euglenozoa, the structure is remarkably similar to that of typical (CXXCH) mitochondrial cytochromes c, both in overall fold and, other than the missing thioether bond, in the details of the heme attachment. Notably, this similarity includes the stereochemistry of the covalent heme attachment to the protein. The structure has implications for the maturation of c-type cytochromes in the Euglenozoa; it also hints at a distinctive redox environment in the mitochondrial intermembrane space of trypanosomes. Surprisingly, Saccharomyces cerevisiae cytochrome c heme lyase (the yeast cytochrome c biogenesis system) cannot efficiently mature Trypanosoma brucei cytochrome c or a CXXCH variant when expressed in the cytoplasm of Escherichia coli, despite their great structural similarity to yeast cytochrome c, suggesting that heme lyase requires specific recognition features in the apocytochrome. PMID:19459937

  16. /sup 1/H NMR probe for hydrogen bonding of distal residues to bound ligands in heme proteins: isotope effect on heme electronic structure of myoglobin

    SciTech Connect

    Lecomte, J.T.J.; La Mar, G.N.

    1987-11-11

    The authors demonstrate herein an isotope effect on the electronic structure of the heme in the low-spin, ferric cyanide complex of sperm whale myoglobin, metMbCN, which can be uniquely attributed to an H-bond between the distal (E7) histidyl imidazole and the bound ligand. The perturbation of the heme electronic structure arises directly from the isotope effect of the H bond between the distal His number7 and the bound ligand.

  17. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  18. Hemoglobin fructation promotes heme degradation through the generation of endogenous reactive oxygen species

    NASA Astrophysics Data System (ADS)

    Goodarzi, M.; Moosavi-Movahedi, A. A.; Habibi-Rezaei, M.; Shourian, M.; Ghourchian, H.; Ahmad, F.; Farhadi, M.; Saboury, A. A.; Sheibani, N.

    2014-09-01

    Protein glycation is a cascade of nonenzymatic reactions between reducing sugars and amino groups of proteins. It is referred to as fructation when the reducing monosaccharide is fructose. Some potential mechanisms have been suggested for the generation of reactive oxygen species (ROS) by protein glycation reactions in the presence of glucose. In this state, glucose autoxidation, ketoamine, and oxidative advance glycation end products (AGEs) formation are considered as major sources of ROS and perhaps heme degradation during hemoglobin glycation. However, whether fructose mediated glycation produces ROS and heme degradation is unknown. Here we report that ROS (H2O2) production occurred during hemoglobin fructation in vitro using chemiluminescence methods. The enhanced heme exposure and degradation were determined using UV-Vis and fluorescence spectrophotometry. Following accumulation of ROS, heme degradation products were accumulated reaching a plateau along with the detected ROS. Thus, fructose may make a significant contribution to the production of ROS, glycation of proteins, and heme degradation during diabetes.

  19. Serum iron increases with acute induction of hepatic heme oxygenase-1 in mice.

    PubMed

    Mostert, Volker; Nakayama, Akihiro; Austin, Lori M; Levander, Ximena A; Ferris, Christopher D; Hill, Kristina E; Burk, Raymond F

    2007-01-01

    Heme oxygenase (HO)-1 is induced by oxidative stress and protects against oxidant injury. We examined the effect of rapid induction of hepatic HO-1 on serum iron level. Serum iron was approximately doubled within 6 h when HO-1 was induced by phenobarbital treatment of selenium-deficient mice. Blocking heme synthesis with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) prevented the induction of HO-1 and the rise in serum iron. DDC did not block HO-1 induction by hemin. Inhibition of HO activity by tin protoporphyrin prevented a rise in serum iron that occurred following phorone treatment. These results indicate that heme synthesis or an exogenous source of heme is needed to allow induction of HO-1. Further, they link HO-1 induction with a rise in serum iron, suggesting that the iron resulting from catabolism of heme by HO-1 is released by the liver.

  20. Phosphatidylcholine biosynthesis and lipoprotein metabolism.

    PubMed

    Cole, Laura K; Vance, Jean E; Vance, Dennis E

    2012-05-01

    Phosphatidylcholine (PC) is the major phospholipid component of all plasma lipoprotein classes. PC is the only phospholipid which is currently known to be required for lipoprotein assembly and secretion. Impaired hepatic PC biosynthesis significantly reduces the levels of circulating very low density lipoproteins (VLDLs) and high density lipoproteins (HDLs). The reduction in plasma VLDLs is due in part to impaired hepatic secretion of VLDLs. Less PC within the hepatic secretory pathway results in nascent VLDL particles with reduced levels of PC. These particles are recognized as being defective and are degraded within the secretory system by an incompletely defined process that occurs in a post-endoplasmic reticulum compartment, consistent with degradation directed by the low-density lipoprotein receptor and/or autophagy. Moreover, VLDL particles are taken up more readily from the circulation when the PC content of the VLDLs is reduced, likely due to a preference of cell surface receptors and/or enzymes for lipoproteins that contain less PC. Impaired PC biosynthesis also reduces plasma HDLs by inhibiting hepatic HDL formation and by increasing HDL uptake from the circulation. These effects are mediated by elevated expression of ATP-binding cassette transporter A1 and hepatic scavenger receptor class B type 1, respectively. Hepatic PC availability has recently been linked to the progression of liver and heart disease. These findings demonstrate that hepatic PC biosynthesis can regulate the amount of circulating lipoproteins and suggest that hepatic PC biosynthesis may represent an important pharmaceutical target. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

  1. Biosynthesis of Fungal Indole Alkaloids

    PubMed Central

    Xu, Wei; Gavia, Diego J.; Tang, Yi

    2014-01-01

    This review provides a summary of recent research advances in elucidating the biosynthesis of fungal indole alkaloids. Different strategies used to incorporate and derivatize the indole/indoline moieties in various families of fungal indole alkaloids will be discussed, including tryptophan-containing nonribosomal peptides and polyketide-nonribosomal peptide hybrids; and alkaloids derived from other indole building blocks. This review also includes discussion regarding the downstream modifications that generate chemical and structural diversity among indole alkaloids. PMID:25180619

  2. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    PubMed

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  3. Pyrimidine Biosynthesis in Lactobacillus leichmannii

    PubMed Central

    Hutson, Judith Y.; Downing, Mancourt

    1968-01-01

    Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

  4. The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae.

    PubMed

    Hirotsu, Shoko; Chu, Grace C; Unno, Masaki; Lee, Dong-Sun; Yoshida, Tadashi; Park, Sam-Yong; Shiro, Yoshitsugu; Ikeda-Saito, Masao

    2004-03-19

    Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolution, respectively. The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices. The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the beta- and delta-meso axis of the porphyrin ring. A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule. This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs. In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex. Together with the kinked distal helix, this movement leaves only the alpha-meso carbon atom accessible to the iron-bound dioxygen. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage. This allows the enzyme to oxygenate selectively at the alpha-meso carbon in HmuO catalysis. PMID:14645223

  5. Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

    PubMed

    Uppal, Sheetal; Salhotra, Shikha; Mukhi, Nitika; Zaidi, Fatima Kamal; Seal, Manas; Dey, Somdatta Ghosh; Bhat, Rajiv; Kundu, Suman

    2015-01-23

    Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the "hemoglobin (Hb) superfamily." The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His(117) and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His(117) is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute. PMID:25451928

  6. Duodenal Absorption and Tissue Utilization of Dietary Heme and Nonheme Iron Differ in Rats123

    PubMed Central

    Cao, Chang; Thomas, Carrie E.; Insogna, Karl L.; O'Brien, Kimberly O.

    2014-01-01

    Background: Dietary heme contributes to iron intake, yet regulation of heme absorption and tissue utilization of absorbed heme remains undefined. Objectives: In a rat model of iron overload, we used stable iron isotopes to examine heme- and nonheme-iron absorption in relation to liver hepcidin and to compare relative utilization of absorbed heme and nonheme iron by erythroid (RBC) and iron storage tissues (liver and spleen). Methods: Twelve male Sprague-Dawley rats were randomly assigned to groups for injections of either saline or iron dextran (16 or 48 mg Fe over 2 wk). After iron loading, rats were administered oral stable iron in the forms of 57Fe-ferrous sulfate and 58Fe-labeled hemoglobin. Expression of liver hepcidin and duodenal iron transporters and tissue stable iron enrichment was determined 10 d postdosing. Results: High iron loading increased hepatic hepcidin by 3-fold and reduced duodenal expression of divalent metal transporter 1 (DMT1) by 76%. Nonheme-iron absorption was 2.5 times higher than heme-iron absorption (P = 0.0008). Absorption of both forms of iron was inversely correlated with hepatic hepcidin expression (heme-iron absorption: r = −0.77, P = 0.003; nonheme-iron absorption: r = −0.80, P = 0.002), but hepcidin had a stronger impact on nonheme-iron absorption (P = 0.04). Significantly more 57Fe was recovered in RBCs (P = 0.02), and more 58Fe was recovered in the spleen (P = 0.01). Conclusions: Elevated hepcidin significantly decreased heme- and nonheme-iron absorption but had a greater impact on nonheme-iron absorption. Differential tissue utilization of heme vs. nonheme iron was evident between erythroid and iron storage tissues, suggesting that some heme may be exported into the circulation in a form different from that of nonheme iron. PMID:25332470

  7. Imatinib binding to human serum albumin modulates heme association and reactivity.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; Trezza, Viviana; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2014-10-15

    Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo. PMID:25057771

  8. Covalent linkage of prosthetic heme to CYP4 family P450 enzymes.

    PubMed

    Henne, K R; Kunze, K L; Zheng, Y M; Christmas, P; Soberman, R J; Rettie, A E

    2001-10-30

    An extensive body of research on the structural properties of cytochrome P450 enzymes has established that these proteins possess a b-type heme prosthetic group which is noncovalently bound at the active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backbone and heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releases free heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, and SDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4A subfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROS R2 column under mild acidic conditions caused dissociation of less than one-third of the heme from the protein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresis conditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidenced by an intact protein mass value of 59,217 +/- 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensive staining of this approximately 60 kDa protein with tetramethylbenzidine/H(2)O(2) following SDS-PAGE. In addition, treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographically distinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicative of the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of a novel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s and their heme catalytic center.

  9. 1,2,3-Triazole-Heme Interactions in Cytochrome P450: Functionally Competent Triazole-Water- Heme Complexes

    PubMed Central

    Conner, Kip P.; Vennam, Preethi; Woods, Caleb M.; Krzyaniak, Matthew D.; Bowman, Michael K.; Atkins, William M.

    2012-01-01

    In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ) the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among CYP inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via ‘click’ chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and DFT computational studies were performed with unsusbstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent – 1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme – ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism based inactivator, 17-α-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low spin ferric heme iron (type II) in contrast to 17EE, which yields a high spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to 17EE, and with different regioselectivity. Surprisingly, CW EPR and HYSCORE EPR spectroscopy indicate that the 17-click does not displace water from the 6th axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model where 17-click pendant 1,2,3-TRZ hydrogen bonds with the 6th axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4•17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra. PMID:22809252

  10. Heme oxygenase-1 regulates mitochondrial quality control in the heart

    PubMed Central

    Hull, Travis D.; Boddu, Ravindra; Guo, Lingling; Tisher, Cornelia C.; Traylor, Amie M.; Patel, Bindiya; Joseph, Reny; Prabhu, Sumanth D.; Suliman, Hagir B.; Piantadosi, Claude A.; Agarwal, Anupam; George, James F.

    2016-01-01

    The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control. PMID:27110594

  11. Resilience of the Iron Environment in Heme Proteins

    PubMed Central

    Leu, Bogdan M.; Zhang, Yong; Bu, Lintao; Straub, John E.; Zhao, Jiyong; Sturhahn, Wolfgang; Alp, E. Ercan; Sage, J. Timothy

    2008-01-01

    Conformational flexibility is essential to the functional behavior of proteins. We use an effective force constant introduced by Zaccai, the resilience, to quantify this flexibility. Site-selective experimental and computational methods allow us to determine the resilience of heme protein active sites. The vibrational density of states of the heme Fe determined using nuclear resonance vibrational spectroscopy provides a direct experimental measure of the resilience of the Fe environment, which we compare quantitatively with values derived from the temperature dependence of atomic mean-squared displacements in molecular dynamics simulations. Vibrational normal modes in the THz frequency range dominate the resilience. Both experimental and computational methods find a higher resilience for cytochrome c than for myoglobin, which we attribute to the increased number of covalent links to the peptide in the former protein. For myoglobin, the resilience of the iron environment is larger than the average resilience previously determined for hydrogen sites using neutron scattering. Experimental results suggest a slightly reduced resilience for cytochrome c upon oxidation, although the change is smaller than reported in previous Mössbauer investigations on a bacterial cytochrome c, and is not reproduced by the simulations. Oxidation state also has no significant influence on the compressibility calculated for cyt c, although a slightly larger compressibility is predicted for myoglobin. PMID:18835904

  12. Heme oxygenase-1 comes back to endoplasmic reticulum

    SciTech Connect

    Kim, Hong Pyo; Pae, Hyun-Ock; Back, Sung Hun; Chung, Su Wol; Woo, Je Moon; Son, Yong; Chung, Hun-Taeg

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  13. Tryptophan-to-heme electron transfer in ferrous myoglobins

    PubMed Central

    Monni, Roberto; Al Haddad, André; van Mourik, Frank; Auböck, Gerald; Chergui, Majed

    2015-01-01

    It was recently demonstrated that in ferric myoglobins (Mb) the fluorescence quenching of the photoexcited tryptophan 14 (*Trp14) residue is in part due to an electron transfer to the heme porphyrin (porph), turning it to the ferrous state. However, the invariance of *Trp decay times in ferric and ferrous Mbs raises the question as to whether electron transfer may also be operative in the latter. Using UV pump/visible probe transient absorption, we show that this is indeed the case for deoxy-Mb. We observe that the reduction generates (with a yield of about 30%) a low-valence Fe–porphyrin π [FeII(porph●−)] -anion radical, which we observe for the first time to our knowledge under physiological conditions. We suggest that the pathway for the electron transfer proceeds via the leucine 69 (Leu69) and valine 68 (Val68) residues. The results on ferric Mbs and the present ones highlight the generality of Trp–porphyrin electron transfer in heme proteins. PMID:25902517

  14. Heatstroke Effect on Brain Heme Oxygenase-1 in Rats

    PubMed Central

    Wen, Ya-Ting; Liu, Tsung-Ta; Lin, Yuh-Feng; Chen, Chun-Chi; Kung, Woon-Man; Huang, Chi-Chang; Lin, Tien-Jen; Wang, Yuan-Hung; Wei, Li

    2015-01-01

    Exposure to high environmental temperature leading to increased core body temperature above 40°C and central nervous system abnormalities such as convulsions, delirium, or coma is defined as heat stroke. Studies in humans and animals indicate that the heat shock responses of the host contribute to multiple organ injury and death during heat stroke. Heme oxygenase-1 (HO-1)—a stress-responsive enzyme that catabolizes heme into iron, carbon monoxide, and biliverdin—has an important role in the neuroprotective mechanism against ischemic stroke. Here, we investigated the role of endogenous HO-1 in heat-induced brain damage in rats. RT-PCR results revealed that levels of HO-1 mRNA peaked at 0 h after heat exposure and immunoblot analysis revealed that the maximal protein expression occurred at 1 h post-heat exposure. Subsequently, we detected the HO-1 expression in the cortical brain cells and revealed the neuronal cell morphology. In conclusion, HO-1 is a potent protective molecule against heat-induced brain damage. Manipulation of HO-1 may provide a potential therapeutic approach for heat-related diseases. PMID:26392811

  15. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  16. Heme oxygenase-1 as a potential therapeutic target for hepatoprotection.

    PubMed

    Farombi, Ebenezer Olatunde; Surh, Young Joon

    2006-09-30

    Heme oxygenase (HO), the rate limiting enzyme in the breakdown of heme into carbon monoxide (CO), iron and bilirubin, has recently received overwhelming research attention. To date three mammalian HO isozymes have been identified, and the only inducible form is HO-1 while HO-2 and HO-3 are constitutively expressed. Advances in unveiling signal transduction network indicate that a battery of redox-sensitive transcription factors, such as activator protein-1 (AP-1), nuclear factor-kappa B (NF-kappaB) and nuclear factor E2-related factor-2 (Nrf2), and their upstream kinases including mitogen-activated protein kinases play an important regulatory role in HO-1 gene induction. The products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin have been shown to exert protective effects in several organs against oxidative and other noxious stimuli. In this context, it is interesting to note that induction of HO-1 expression contributes to protection against liver damage induced by several chemical compounds such as acetaminophen, carbon tetrachloride and heavy metals, suggesting HO-1 induction as an important cellular endeavor for hepatoprotection. The focus of this review is on the significance of targeted induction of HO-1 as a potential therapeutic strategy to protect against chemically-induced liver injury as well as hepatocarcinogenesis. PMID:17002867

  17. Heatstroke Effect on Brain Heme Oxygenase-1 in Rats.

    PubMed

    Wen, Ya-Ting; Liu, Tsung-Ta; Lin, Yuh-Feng; Chen, Chun-Chi; Kung, Woon-Man; Huang, Chi-Chang; Lin, Tien-Jen; Wang, Yuan-Hung; Wei, Li

    2015-01-01

    Exposure to high environmental temperature leading to increased core body temperature above 40°C and central nervous system abnormalities such as convulsions, delirium, or coma is defined as heat stroke. Studies in humans and animals indicate that the heat shock responses of the host contribute to multiple organ injury and death during heat stroke. Heme oxygenase-1 (HO-1)-a stress-responsive enzyme that catabolizes heme into iron, carbon monoxide, and biliverdin-has an important role in the neuroprotective mechanism against ischemic stroke. Here, we investigated the role of endogenous HO-1 in heat-induced brain damage in rats. RT-PCR results revealed that levels of HO-1 mRNA peaked at 0 h after heat exposure and immunoblot analysis revealed that the maximal protein expression occurred at 1 h post-heat exposure. Subsequently, we detected the HO-1 expression in the cortical brain cells and revealed the neuronal cell morphology. In conclusion, HO-1 is a potent protective molecule against heat-induced brain damage. Manipulation of HO-1 may provide a potential therapeutic approach for heat-related diseases. PMID:26392811

  18. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death

    PubMed Central

    Kwon, Min-Young; Park, Eunhee

    2015-01-01

    The oncogenic RAS-selective lethal small molecule Erastin triggers a unique iron-dependent form of nonapoptotic cell death termed ferroptosis. Ferroptosis is dependent upon the production of intracellular iron-dependent reactive oxygen species (ROS), but not other metals. However, key regulators remain unknown. The heme oxygenase (HO) is a major intracellular source of iron. In this study, the role of heme oxygenase in Erastin-triggered ferroptotic cancer cell death has been investigated. Zinc protoporphyrin IX (ZnPP), a HO-1 inhibitor, prevented Erastin-triggered ferroptotic cancer cell death. Furthermore, Erastin induced the protein and mRNA levels of HO-1 in HT-1080 fibrosarcoma cells. HO-1+/+ and HO-1−/− fibroblast, HO-1 overexpression, and chycloheximide-treated experiments revealed that the expression of HO-1 has a decisive effects in Erastin-triggered cell death. Hemin and CO-releasing molecules (CORM) promote Erastin-induced ferroptotic cell death, not by biliverdin and bilirubin. In addition, hemin and CORM accelerate the HO-1 expression in the presence of Erastin and increase membranous lipid peroxidation. Thus, HO-1 is an essential enzyme for iron-dependent lipid peroxidation during ferroptotic cell death. PMID:26405158

  19. Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif.

    PubMed

    Hanano, Abdulsamie; Burcklen, Michel; Flenet, Martine; Ivancich, Anabella; Louwagie, Mathilde; Garin, Jérôme; Blée, Elizabeth

    2006-11-01

    A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme. PMID:16956885

  20. The Magnesium Chelation Step in Chlorophyll Biosynthesis

    SciTech Connect

    Gregory L. Dilworth, Ph.D Chemical Sciences, Geosciences and Biosciences Division Office of Basis Energy Sciences, greg.dilworth@science.doe.gov

    2001-01-17

    The progress described in this report encompasses work supported by DOE grant DE-FG09-89ER13989 for the period 2/15/92 to the present 6/14/94. The goals of the project were to continue investigating the enzymology of Mg-chelatase and to investigate the co-regulation of heme and chlorophyll formation in intact plastids. During this period the laboratory had additional support (two years) from USDA to investigate heme metabolism in chloroplasts. This report is arranged so that the progress is described by reference to manuscripts which are published, under review or in preparation.

  1. Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis.

    PubMed

    Honsho, Masanori; Abe, Yuichi; Fujiki, Yukio

    2015-11-27

    Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

  2. Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD.

    PubMed

    Graves, Amanda B; Morse, Robert P; Chao, Alex; Iniguez, Angelina; Goulding, Celia W; Liptak, Matthew D

    2014-06-16

    Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon. PMID

  3. Ontogeny of osmoregulation in embryos of intertidal crabs (Hemigrapsus sexdentatus and H. crenulatus, Grapsidae, Brachyura): putative involvement of the embryonic dorsal organ.

    PubMed

    Seneviratna, Deepani; Taylor, H H

    2006-04-01

    This study examined whether the existence of hyperosmotic internal fluids in embryos of euryhaline crabs (Hemigrapsus sexdentatus and H. crenulatus) in dilute seawater reflects osmotic isolation due to impermeability of the egg envelope, as proposed for other decapods, or active osmoregulation. When ovigerous crabs with eggs at gastrula stage were transferred from 100% seawater (osmolality 1000 mmol kg(-1)) to 50% seawater, embryogenesis and hatching of zoea were completed normally, but were delayed. Hatching failed if the transfer to 50% seawater occurred before gastrulation, and embryogenesis was abnormal in 25% seawater. In 100% seawater, embryos at all stages were internally hyperosmotic by 150-250 mmol kg(-1). On transfer to 50% seawater, osmolality initially decreased but remained 200-350 mmol kg(-1) hyperosmotic to the medium for several weeks until hatching. High efflux rates of tritium-labelled water (t((1/2)) 16-75 min) and (22)Na (t(1/2) 109-374 min) from H. crenulatus embryos were inconsistent with the osmotic isolation hypothesis. It is concluded that post-gastrula embryos were actively hyper-osmoregulating. The diffusional water permeability of the embryos decreased during development while the sodium efflux rate increased 10-fold. Very rapidly exchanging pools of water and sodium (t(1/2) a few seconds to minutes) probably corresponded to peri-embryonic fluid and implied that the egg envelope was a negligible barrier to diffusion of water and salts. Higher Na(+)/K(+)-ATPase activities in late embryos of H. crenulatus incubated in 50% seawater than in embryos incubated in full strength seawater were consistent with an acclimation response. An area of the embryonic surface located over the yolk in the region of the embryonic dorsal organ stained with AgNO(3). Staining appeared at gastrulation, persisted throughout development and was lost at hatching. Deposits of AgCl between the outer and inner membranes, identified by X-ray microanalysis, suggest that

  4. Osmoregulation in methanogens

    SciTech Connect

    Roberts, M.F.

    1993-01-01

    Our major goal of our work has been to develop and use NMR techniques to study how methanogenic archaebacteria deal with osmotic stress with the hope of providing insights into increasing the salt tolerance of other cells. The project has three main sections: (i) in vivo studies of methanogens; (ii) use of [sup l3]C- and [sup l5]N- labeled potential precursors and in vitro analyses of specific label uptake for elucidation of osmolyte dynamics and biosynthetic pathways of osmolytes in these organisms, and isolation of key biosynthetic enzymes; and (iii) collaborative studies on identification of organic solutes in other methanogens.

  5. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli

    PubMed Central

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V.; Patel, Bhumit A.; Yeh, Syun-Ru; Rousseau, Dennis L.; Crane, Brian R.

    2010-01-01

    Over-expression of heme binding proteins in E. coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His- ligated hemes. PMID:20303407

  6. Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic, Vibrational, and Conformational Relaxation.

    PubMed

    Karunakaran, Venugopal

    2015-12-21

    The excited-state dynamics of ferric cytochrome c (Cyt c), an important electron-transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump-probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited-state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.

  7. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    SciTech Connect

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  8. Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.

    PubMed

    Zhao, Yuan; Du, Ke-Jie; Gao, Shu-Qin; He, Bo; Wen, Ge-Bo; Tan, Xiangshi; Lin, Ying-Wu

    2016-03-01

    Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network.

  9. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  10. Crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate. Conformational change of the distal helix during the heme cleavage reaction.

    PubMed

    Sugishima, Masakazu; Sakamoto, Hiroshi; Higashimoto, Yuichiro; Noguchi, Masato; Fukuyama, Keiichi

    2003-08-22

    The crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate (biliverdin(Fe)-HO-1), the immediate precursor of the final product, biliverdin, has been determined at a 2.4-A resolution. The electron density in the heme pocket clearly showed that the tetrapyrrole ring of heme is cleaved at the alpha-meso edge. Like the heme bound to HO-1, biliverdin-iron chelate is located between the distal and proximal helices, but its accommodation state seems to be less stable in light of the disordering of the solvent-exposed propionate and vinyl groups. The middle of the distal helix is shifted away from the center of the active site in biliverdin(Fe)-HO-1, increasing the size of the heme pocket. The hydrogen-bonding interaction between Glu-29 and Gln-38, considered to restrain the orientation of the proximal helix in the heme-HO-1 complex, was lost in biliverdin(Fe)-HO-1, leading to relaxation of the helix. Biliverdin has a distorted helical conformation; the lactam oxygen atom of its pyrrole ring-A interacted with Asp-140 through a hydrogen-bonding solvent network. Because of the absence of a distal water ligand, the iron atom is five-coordinated with His-25 and four pyrrole nitrogen atoms. The coordination geometry deviates considerably from a square pyramid, suggesting that the iron may be readily dissociated. We speculate that the opened conformation of the heme pocket facilitates sequential product release, first iron then biliverdin, and that because of biliverdin's increased flexibility, iron release triggers its slow dissociation. PMID:12794075

  11. Allocation of Heme Is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells

    PubMed Central

    Espinas, Nino A.; Kobayashi, Koichi; Sato, Yasushi; Mochizuki, Nobuyoshi; Takahashi, Kaori; Tanaka, Ryouichi; Masuda, Tatsuru

    2016-01-01

    Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions. PMID:27630653

  12. Allocation of Heme Is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells

    PubMed Central

    Espinas, Nino A.; Kobayashi, Koichi; Sato, Yasushi; Mochizuki, Nobuyoshi; Takahashi, Kaori; Tanaka, Ryouichi; Masuda, Tatsuru

    2016-01-01

    Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

  13. Allocation of Heme Is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells.

    PubMed

    Espinas, Nino A; Kobayashi, Koichi; Sato, Yasushi; Mochizuki, Nobuyoshi; Takahashi, Kaori; Tanaka, Ryouichi; Masuda, Tatsuru

    2016-01-01

    Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions. PMID:27630653

  14. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    PubMed

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

  15. Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

    PubMed Central

    Wu, M. L.; Morgan, W. T.

    1995-01-01

    Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7773173

  16. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

    PubMed Central

    Kathiresan, Meena; Martins, Dorival; English, Ann M.

    2014-01-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  17. Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media.

    PubMed Central

    Whitby, P W; Sim, K E; Morton, D J; Patel, J A; Stull, T L

    1997-01-01

    Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme. PMID:9353052

  18. Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon

    PubMed Central

    Ijssennagger, Noortje; Belzer, Clara; Hooiveld, Guido J.; Dekker, Jan; van Mil, Saskia W. C.; Müller, Michael; Kleerebezem, Michiel; van der Meer, Roelof

    2015-01-01

    Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function. PMID:26216954

  19. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    PubMed

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  20. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration. PMID:19560175

  1. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  2. Tetrahydrobiopterin biosynthesis, regeneration and functions.

    PubMed Central

    Thöny, B; Auerbach, G; Blau, N

    2000-01-01

    Tetrahydrobiopterin (BH(4)) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH(4) is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH(4) from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH(4), but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH(4) are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH(4) has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH(4) is a self-protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH(4) deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be

  3. Heme as a danger molecule in pathogen recognition.

    PubMed

    Wegiel, Barbara; Hauser, Carl J; Otterbein, Leo E

    2015-12-01

    Appropriate control of redox mechanisms are critical for and effective innate immune response, which employs multiple cell types, receptors and molecules that recognize danger signals when they reach the host. Recognition of pathogen-associated pattern molecules (PAMPs) is a fundamental host survival mechanism for efficient elimination of invading pathogens and resolution of the infection and inflammation. In addition to PAMPs, eukaryotic cells contain a plethora of intracellular molecules that are normally secured within the confines of the plasma membrane, but if liberated and encountered in the extracellular milieu can provoke rapid cell activation. These are known as Alarmins or Danger-Associated Molecular Patterns (DAMPs) and can be released actively by cells or passively as a result of sterile cellular injury after trauma, ischemia, or toxin-induced cell rupture. Both PAMPs and DAMPs are recognized by a series of cognate receptors that increase the generation of free radicals and activate specific signaling pathways that result in regulation of a variety of stress response, redox sensitive genes. Multiple mediators released, as cells die include, but are not limited to ATP, hydrogen peroxide, heme, formyl peptides, DNA or mitochondria provide the second signal to amplify immune responses. In this review, we will focus on how sterile and infective stimuli activate the stress response gene heme oxygenase-1 (Hmox1, HO-1), a master gene critical to an appropriate host response that is now recognized as one with enormous therapeutic potential. HO-1 gene expression is regulated in large part by redox-sensitive proteins including but not limited to nrf2. Both PAMPs and DAMPs increase the activation of nrf2 and HO-1. Heme is a powerful pro-oxidant and as such should be qualified as a DAMP. With its degradation by HO-1a molecule of carbon monoxide (CO) is generated that in turn serves as a bioactive signaling molecule. PAMPs such as bacterial endotoxin activate HO-1

  4. Heme coordination states of unfolded ferrous cytochrome C.

    PubMed

    Droghetti, Enrica; Oellerich, Silke; Hildebrandt, Peter; Smulevich, Giulietta

    2006-10-15

    The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process. PMID:16877519

  5. Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy

    DOE PAGES

    Levantino, M.; Lemke, H. T.; Schirò, G.; Glownia, M.; Cupane, A.; Cammarata, M.

    2015-07-01

    We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (~70 fs) relaxation preceding a slower (~400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

  6. The magnesium chelation step in chlorophyll biosynthesis

    SciTech Connect

    Weinstein, J.D.

    1991-01-01

    The biogenesis of energy transducing membranes requires the coordinate synthesis of prosthetic groups, proteins and lipids. Two of the major prosthetic groups, chlorophyll and heme, share a common biosynthetic pathway that diverges at the point of metal insertion into protoporphyrin IX. Insertion of iron leads to heme, while insertion of magnesium leads to chlorophyll. The Mg-chelatase from intact cucumber chloroplasts has been characterized with regard to substrate specificity, regulation, ATP requirement, and a requirement for intact chloroplasts. Mg-chelatase was isolated from maize, barley and peas and characterized in order to circumvent the intact chloroplast requirement of cucumber Mg-chelatase. Pea Mg-chelatase activity is higher than cucumber Mg-chelatase activity, and lacks the requirement for intact chloroplasts. Studies on isolated pea Mg-chelatase have shown more cofactors are required for the reaction than are seen with ferrochelatase, indicating a greater opportunity for regulatory control of this pathway. Two of the cofactors are proteins, and there appears to be a requirement for a protease-sensitive component which is outside the outer envelope. We are developing a continuous spectrophotometric assay for Mg-chelatase activity, and an assay for free heme which has shown heme efflux from intact chloroplasts. 18 refs. (MHB)

  7. Influence of some growth regulators and cations on inhibition of chlorophyll biosynthesis by lead in maize

    SciTech Connect

    Sinha, S.K. ); Srivastava, H.S. ); Tripathi, R.D. )

    1993-08-01

    Phytotoxic effects of Pb pollution are well established. In order to analyse the physiological basis of toxic symptoms and of reduced plant productivity, its effect on chlorophyll content has been examined in some plants. Thus, a decrease in total chlorophyll content during Pb supply has been observed in oats, mung beam, pea, etc. The activity of delta aminolevulinic acid dehydratase, an important enzyme in the biosynthesis of heme pigments, is inhibited by Pb in mung bean and several other species. This observation may perhaps indicate that a reduction in chlorophyll content in the presence of lead is due to an inhibition of pigment synthesis. The effect of Pb on greening maize leaf segments in the presence of various precursors of chlorophyll has been studied in the present investigation to evaluate this hypothesis. The effect of some growth regulators and cations, which could otherwise modify chlorophyll biosynthesis, has been examined to see whether the toxic effects of Pb on photosynthetic pigments could also be modified by these effectors. 16 refs., 4 tabs.

  8. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

    PubMed

    DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

    2016-03-01

    The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

  9. Role of rice heme oxygenase in lateral root formation

    PubMed Central

    Huei Kao, Ching

    2013-01-01

    Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. In rice, exposure to auxin, methyl jasmonate (MJ), apocynin, and CoCl2 has been shown to increase LR formation. This review provides evidence showing a close link between rice heme oxygenase (HO) and LR formation. The effect of auxin and MJ is nitric oxide (NO) dependent, whereas that of apocynin requires H2O2. The effect of CoCl2 on the LR formation could be by some other pathway unrelated to NO and H2O2. This review also highlights future lines of research that should increase our knowledge of HO-involved LR formation in rice. PMID:23887491

  10. Self-Assembled Monolayers as Templates for Heme Crystallization.

    SciTech Connect

    Wang, Xuefeng; Ingall, Ellery; Lai, Barry; Stack, Andrew G

    2010-01-01

    Homogeneous self-assembled monolayers (SAMs) of alkanethiols (HS(CH{sub 2}){sub n}X) on Au(111) were used as substrates for crystallization of ferriprotoporphyrin IX (heme) in acidic aqueous solution. Different terminal functional groups (X = OH, COOH, NH{sub 2}, CH{sub 3}) were used on the SAMs as models of sites where heme crystallization takes place in blood-feeding organisms. Atomic force microscopy, X-ray diffraction (XRD), and X-ray absorption near edge spectroscopy (XANES) were employed to characterize particle morphology, density, crystallographic orientation, and the coordination environment. It was found that the morphology and extent of growth of particulates were strongly affected by the environment in which they crystallize. As has been previously observed, acicular crystals form in DMSO-methanol solution, whereas irregular aggregates of crystals form in acidic aqueous solution. Here tabular crystals were found to form on -NH{sub 2} and -OH terminated SAMs, whereas inclined crystals formed on -COOH and -CH{sub 3} terminated substrates. Particulate coverage on these SAMs decreased in the order of -NH{sub 2}, -COOH, -CH{sub 3}, and -OH. Chloroquine, a widely used antimalaria drug, slowed particle nucleation rate on the SAMs with varying efficacy but was most efficient on the -COOH SAM. XANES measurements showed that the coordination environment surrounding iron in the particles was found to be the same, regardless of the preparation method and matches existing spectra of hemozoin produced in vivo and synthetic {beta}-hematin. Different crystallographic planes were found to be expressed depending on the identity of the SAM using XRD. The interaction between the terminal functional group of the SAM and the density and orientation of crystals is discussed.

  11. Self-Assembled Monolayers as Templates for Heme Crystallization

    SciTech Connect

    Wang, Xuefeng; Ingall, Ellery; Lai, Barry; Stack, Andrew G; Allard Jr, Lawrence Frederick

    2010-01-01

    Homogeneous self-assembled monolayers (SAMs) of alkanethiols (HS(CH{sub 2}){sub n}X) on Au(111) were used as substrates for crystallization of ferriprotoporphyrin IX (heme) in acidic aqueous solution. Different terminal functional groups (X = OH, COOH, NH{sub 2}, CH{sub 3}) were used on the SAMs as models of sites where heme crystallization takes place in blood-feeding organisms. Atomic force microscopy, X-ray diffraction (XRD), and X-ray absorption near edge spectroscopy (XANES) were employed to characterize particle morphology, density, crystallographic orientation, and the coordination environment. It was found that the morphology and extent of growth of particulates were strongly affected by the environment in which they crystallize. As has been previously observed, acicular crystals form in DMSO-methanol solution, whereas irregular aggregates of crystals form in acidic aqueous solution. Here tabular crystals were found to form on -NH{sub 2} and -OH terminated SAMs, whereas inclined crystals formed on -COOH and -CH{sub 3} terminated substrates. Particulate coverage on these SAMs decreased in the order of -NH{sub 2}, -COOH, -CH{sub 3}, and -OH. Chloroquine, a widely used antimalaria drug, slowed particle nucleation rate on the SAMs with varying efficacy but was most efficient on the -COOH SAM. XANES measurements showed that the coordination environment surrounding iron in the particles was found to be the same, regardless of the preparation method and matches existing spectra of hemozoin produced in vivo and synthetic {beta}-hematin. Different crystallographic planes were found to be expressed depending on the identity of the SAM using XRD. The interaction between the terminal functional group of the SAM and the density and orientation of crystals is discussed.

  12. Aldosterone: from biosynthesis to non-genomic action onto the proteome.

    PubMed

    Zöllner, Susanne; Hwang, Kyung Hoon; Wilzewski, Britta; Carapito, Christine; Leize-Wagner, Emmanuelle; Van Dorsselaer, Alain; Bernhardt, Rita

    2008-10-01

    An increased aldosterone concentration can lead to a progression of heart diseases and to myocardial fibrosis. These fatal processes can be prevented by e.g. inhibiting the mineralocorticoid receptor (MR), which is nowadays part of a commonly applied standard therapy. Moreover, selective inhibition of aldosterone synthase (CYP11B2) is a straightforward goal whereby CYP11B1, a key enzyme in glucocorticoid biosynthesis exhibiting a high structure identity with CYP11B2 should not be inhibited. Therefore, effective test systems have been developed and rather potent and selective CYP11B2 compounds like SIAS-1 have been identified by our group. In addition to finding new inhibitors, we investigated which proteins are directly influenced by aldosterone focussing on non-genomic effects. Schizosaccharomyces pombe was chosen as a model organism, since this yeast does not contain nuclear