Science.gov

Sample records for osmoregulation heme biosynthesis

  1. 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...

  2. Erythroid Heme Biosynthesis and Its Disorders

    PubMed Central

    Meissner, Peter N.

    2013-01-01

    Heme, which is composed of iron and the small organic molecule protoporphyrin, is an essential component of hemoglobin as well as a variety of physiologically important hemoproteins. During erythropoiesis, heme synthesis is induced before, and is essential for, globin synthesis. Although all cells possess the ability to synthesize heme, there are distinct differences between regulation of the pathway in developing erythroid cells and all other types of cells. Disorders that compromise the ability of the developing red cell to synthesize heme can have profound medical implications. The biosynthetic pathway for heme and key regulatory features are reviewed herein, along with specific human genetic disorders that arise from defective heme synthesis such as X-linked sideroblastic anemia and erythropoietic protoporphyria. PMID:23471474

  3. 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

  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. Structure and function of enzymes in heme biosynthesis

    PubMed Central

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

    2010-01-01

    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. PMID:20506125

  6. The CcmC:Heme:CcmE Complex in Heme Trafficking and Cytochrome c Biosynthesis

    PubMed Central

    Richard-Fogal, Cynthia; Kranz, Robert G.

    2012-01-01

    A superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the WWD domain) in an external loop at the inner membrane surface. The three major members of this family (CcmC, CcmF, and CcsBA) are each involved in cytochrome c biosynthesis, yet the function of the WWD domain is unknown. It has been hypothesized that the WWD domain binds heme to present it to an acceptor protein (apoCcmE for CcmC or apocytochrome c for CcmF and CcsBA) such that the heme vinyl group(s) covalently attaches to the acceptors. Alternative proposals suggest that the WWD domain interacts directly with the acceptor protein (e.g., apoCcmE for CcmC). Here, it is shown that CcmC is only trapped with heme when its cognate acceptor protein CcmE is present. It is demonstrated that CcmE only interacts stably with CcmC when heme is present; thus, specific residues in each protein provide sites of interaction with heme to form this very stable complex. For the first time, evidence that the external WWD domain of CcmC interacts directly with heme is presented. Single and multiple substitutions of completely conserved residues in the WWD domain of CcmC alter the spectral properties of heme in the stable CcmC:heme:CcmE complexes. Moreover, some mutations reduce the binding of heme up to 100%. It is likely that endogenously synthesized heme enters the external WWD domain of CcmC either via a channel within this six-transmembrane-spanning protein or from the membrane. The data suggest that a specific heme channel (i.e., heme binding site within membrane spanning helices) is not present in CcmC, in contrast to the CcsBA protein. We discuss the likelihood that it is not important to protect the heme via trafficking in CcmC whereas it is critical in CcsBA. PMID:20599545

  7. Use of heme reporters for studies of cytochrome biosynthesis and heme transport.

    PubMed Central

    Goldman, B S; Gabbert, K K; Kranz, R G

    1996-01-01

    Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes. The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm. To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E. coli. By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant. Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm. Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells. Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space. Such an imbalance could lead to defects in the synthesis of heme-liganded proteins. The heme reporters were also used to analyze strains of E. coli with a defect in genes encoding homologs of a different ABC transporter (helABC). The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R. G. Kranz, J. Bacteriol. 171:456-464, 1989; D. L. Beckman, D. R. Trawick, and R. G. Kranz, Genes Dev. 6:268-283, 1992). This locus was shown to be essential in E. coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis. Consistent with these and previous results, it

  8. Localization of heme biosynthesis pathway enzymes in Plasmodium falciparum.

    PubMed

    Rao, Aditya; Yeleswarapu, Sri Jyothsna; Srinivasan, Rajgopal; Bulusu, Gopalakrishnan

    2008-12-01

    Protein trafficking in the malarial parasite Plasmodium falciparum is dictated by a complex life-cycle that involves a variety of intra-cellular and host cell destinations, such as the mitochondrion, apicoplast, rhoptries and micronemes. Of these, the apicoplast and mitochondrion are believed to account for more than 10% of this traffic. Studies have shown that mechanisms for mitochondrion and apicoplast targeting are distinct, despite their close physical proximity. The heme biosynthesis pathway spans both these organelles, making trafficking studies crucial for the spatial demarcation of the constituent interactions. This minireview highlights the challenges in identifying the possible sub-cellular destinations of the heme pathway enzymes using gleanings from literature survey as well as focussed bioinformatic analysis. PMID:19239121

  9. Sampangine Inhibits Heme Biosynthesis in both Yeast and Human ▿ †

    PubMed Central

    Huang, Zhiwei; Chen, Kaifu; Xu, Tao; Zhang, Jianhuai; Li, Yongxiang; Li, Wei; Agarwal, Ameeta K.; Clark, Alice M.; Phillips, John D.; Pan, Xuewen

    2011-01-01

    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 primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism—by hyperactivating uroporhyrinogen III synthase—to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain. PMID:21908598

  10. 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. PMID:26627829

  11. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis

    PubMed Central

    Kardon, Julia R.; Yien, Yvette Y.; Huston, Nicholas C.; Branco, Diana S.; Hildick-Smith, Gordon J.; Rhee, Kyu Y.; Paw, Barry H.; Baker, Tania A.

    2015-01-01

    SUMMARY The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is five-fold reduced in yeast lacking mtClpX activity, and total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX therefore is a widely conserved stimulator of an essential biosynthetic pathway, and employs a previously unrecognized mechanism for AAA+ unfoldases. PMID:25957689

  12. Uridine Affects Liver Protein Glycosylation, Insulin Signaling, and Heme Biosynthesis

    PubMed Central

    Urasaki, Yasuyo; Pizzorno, Giuseppe; Le, Thuc T.

    2014-01-01

    Purines and pyrimidines are complementary bases of the genetic code. The roles of purines and their derivatives in cellular signal transduction and energy metabolism are well-known. In contrast, the roles of pyrimidines and their derivatives in cellular function remain poorly understood. In this study, the roles of uridine, a pyrimidine nucleoside, in liver metabolism are examined in mice. We report that short-term uridine administration in C57BL/6J mice increases liver protein glycosylation profiles, reduces phosphorylation level of insulin signaling proteins, and activates the HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. Short-term uridine administration is also associated with reduced liver hemin level and reduced ability for insulin-stimulated blood glucose removal during an insulin tolerance test. Some of the short-term effects of exogenous uridine in C57BL/6J mice are conserved in transgenic UPase1−/− mice with long-term elevation of endogenous uridine level. UPase1−/− mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. UPase1−/− mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other short-term effects of exogenous uridine in C57BL/6J mice are not conserved in UPase1−/− mice. UPase1−/− mice exhibit normal phosphorylation level of liver insulin signaling proteins and increased liver hemin concentration compared to untreated control C57BL/6J mice. Contrasting short-term and long-term consequences of uridine on liver metabolism suggest that uridine exerts transient effects and elicits adaptive responses. Taken together, our data support potential roles of pyrimidines and their derivatives in the regulation of liver metabolism. PMID:24918436

  13. 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

  14. 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. PMID:24043703

  15. 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

  16. Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin

    PubMed Central

    Dailey, Harry A.; Gerdes, Svetlana; Dailey, Tamara A.; Burch, Joseph S.; Phillips, John D.

    2015-01-01

    It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme. This pathway is specified by three genes named hemY, hemH, and hemQ. The analysis of 982 representative prokaryotic genomes is consistent with this pathway being the most ancient heme synthesis pathway in the Eubacteria. Our results identifying a previously unknown branch of tetrapyrrole synthesis support a significant shift from current models for the evolution of bacterial heme and chlorophyll synthesis. Because some organisms that possess this coproporphyrin-dependent branch are major causes of human disease, HemQ is a novel pharmacological target of significant therapeutic relevance, particularly given high rates of antimicrobial resistance among these pathogens. PMID:25646457

  17. Molecular Evolution of Multiple-Level Control of Heme Biosynthesis Pathway in Animal Kingdom

    PubMed Central

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5′ untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway. PMID:24489775

  18. Effect of occupational lead exposure on lymphocyte enzymes involved in heme biosynthesis

    SciTech Connect

    Rossi, E.; Costin, K.A.; Garcia-Webb, P. )

    1990-11-01

    Lead exposure is a well-known cause of increases in urinary coproporphyrin and erythrocyte zinc-protoporphyrin, so these compounds are often used to monitor occupational lead exposure. The increased concentrations are usually assumed to result from lead inhibition of two of the mitochondrial enzymes of heme biosynthesis, coproporphyrinogen oxidase and ferrochelatase. We studied 88 subjects in whom the degree of occupational lead exposure was established by measuring erythrocyte lead and protoporphyrin. Assay of lymphocyte coproporphyrinogen oxidase and ferrochelatase activities showed that these enzymes were unaffected by lead exposure, as was a related enzyme, lymphocyte NADH-ferricyanide reductase. We propose alternative explanations for the increased concentrations of coproporphyrin and zinc-protoporphyrin seen in lead exposure.

  19. Complex formation between heme oxygenase and phytochrome during biosynthesis in Pseudomonas syringae pv. tomato.

    PubMed

    Shah, Rashmi; Schwach, Julia; Frankenberg-Dinkel, Nicole; Gärtner, Wolfgang

    2012-06-01

    The plant pathogen Pseudomonas syringae pv. tomato carries two genes encoding bacterial phytochromes. Sequence motifs identify both proteins (PstBphP1 and PstBphP2, respectively) as biliverdin IXα (BV)-binding phytochromes. PstbphP1 is arranged in an operon with a heme oxygenase (PstBphO)-encoding gene (PstbphO), whereas PstbphP2 is flanked downstream by a gene encoding a CheY-type response regulator. Expression of the heme oxygenase PstBphO yielded a green protein (λ(max) = 650 nm), indicative for bound BV. Heterologous expression of PstbphP1 and PstbphP2 and in vitro assembly with BV IXα yielded the apoproteins for both phytochromes, but only in the case of PstBphP1 a light-inducible chromoprotein. Attempts to express the endogenous heme oxygenase BphO and either of the two phytochromes from two plasmids yielded only holo-PstBphP1. Relatively small amounts of soluble holo-PstBphP2 were just obtained upon co-expression with BphO from P. aeruginosa. Expression of the operon containing PstbphO:PstbphP1 led to an improved yield and better photoreactivity for PstBphP1, whereas an identical construct, exchanging PstbphP1 for PstbphP2 (PstbphO:PstbphP2), again yielded only minute amounts of chromophore-loaded BphP2-holoprotein. The improved yield for PstBphP1 from the PstbphO:PstbphP1 operon expression is apparently caused by complex formation between both proteins during biosynthesis as affinity chromatography of either protein using two different tags always co-purified the reaction partner. These results support the importance of protein-protein interactions during tetrapyrrole metabolism and phytochrome assembly. PMID:22415794

  20. 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

  1. 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.

  2. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

    PubMed

    Connor, Katherine L; Colabroy, Keri L; Gerratana, Barbara

    2011-10-18

    We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases. PMID:21919439

  3. 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

  4. Heme transport and erythropoiesis

    PubMed Central

    Yuan, Xiaojing; Fleming, Mark D.; Hamza, Iqbal

    2013-01-01

    In humans, systemic heme homeostasis is achieved via coordinated regulation of heme synthesis, transport and degradation. Although the heme biosynthesis and degradation pathways have been well characterized, the pathways for heme trafficking and incorporation into hemoproteins remains poorly understood. In the past few years, researchers have exploited genetic, cellular and biochemical tools, to identify heme transporters and, in the process, reveal unexpected functions for this elusive group of proteins. However, given the complexity of heme trafficking pathways, current knowledge of heme transporters is fragmented and sometimes contradictory. This review seeks to focus on recent studies on heme transporters with specific emphasis on their functions during erythropoiesis. PMID:23415705

  5. 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

  6. Metals control activity and expression of the heme biosynthesis enzyme delta-aminolevulinic acid dehydratase in Bradyrhizobium japonicum.

    PubMed Central

    Chauhan, S; Titus, D E; O'Brian, M R

    1997-01-01

    The heme biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) requires magnesium or zinc for activity, depending on the organism, and the heme moiety contains iron. Thus, metals are important for heme formation in at least two different ways. Bradyrhizobium japonicum ALAD* is an engineered derivative of wild-type ALAD that requires Zn2+ for activity rather than Mg2+ (S. Chauhan and M. R. O'Brian, J. Biol. Chem. 270:19823-19827, 1995). The pH optimum for ALAD* activity was over 3.5 units lower than for that of the wild-type enzyme, and ALAD* activity was inhibited by lead and cadmium, as reported for the zinc-containing dehydratases of animals. In addition, ALAD* was significantly more thermostable than ALAD; the temperature optima are 50 and 37 degrees C, respectively. These observations strongly suggest that the metal contributes to both catalysis and structure, and this conclusion may be extrapolated to ALADs in general. Although iron did not affect the activity of the preformed protein, enzyme assays and immunoblot analysis demonstrated that the iron concentration in which the cells were grown had a strong positive effect on ALAD activity and the protein level. RNase protection analysis showed that the transcript quantity of hemB, the gene encoding ALAD, was iron dependent; thus, iron regulates hemB at the mRNA level. Induction of hemB mRNA in response to iron was rapid, suggesting that the factor(s) needed to mediate iron control was present in iron-limited cells and did not need to be synthesized de novo. ALAD protein levels and enzyme activities were similar in cells of the wild type and a heme-defective strain, indicating that control by iron is not an indirect effect of the cellular heme status. We conclude that the heme biosynthetic pathway is coordinated with cellular iron levels and that this control may prevent the accumulation of toxic porphyrin intermediates. PMID:9287008

  7. 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

  8. 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. PMID:26075523

  9. Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (-)-4'-Methoxycyclopenin Biosynthesis.

    PubMed

    Chang, Wei-Chen; Li, Jikun; Lee, Justin L; Cronican, Andrea A; Guo, Yisong

    2016-08-24

    Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis. PMID:27442345

  10. Interaction of HoloCcmE with CcmF in Heme Trafficking and Cytochrome c Biosynthesis

    PubMed Central

    Francisco, Brian San; Kranz, Robert G.

    2013-01-01

    The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD), and ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is “trapped”, purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence is presented that P-His1 and P-His2 function as heme-binding ligands. These results show that heme in holoCcmE is essential for complex formation with CcmF, and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex (Richard-Fogal and Kranz, JMB 2010), and suggest common mechanistic and structural aspects. PMID:24513106

  11. Heme Metabolism and Erythropoiesis

    PubMed Central

    Chung, Jacky; Chen, Caiyong; Paw, Barry H.

    2014-01-01

    Purpose of review Heme biosynthesis requires a series of enzymatic reactions that take place in the cytosol and the mitochondria as well as the proper inter- and intracellular trafficking of iron. Heme can also be acquired by intestinal absorption and intercellular transport. The purpose of this review is to highlight recent work on heme and iron transport with an emphasis on their relevance in erythropoiesis. Recent findings While the enzymes responsible for heme biosynthesis have been identified, transport mechanisms for iron, heme, or heme synthesis intermediates are only emerging. Recent studies have shed light into how these molecules are transported among various cellular compartments, as well as tissues. Much of this progress can be attributed to the use of model organisms such as S. cerevisiae, C. elegans, D. rerio, and M. musculus. Genetic studies in these models have led to the identification of several new genes involved in heme metabolism. Although our understanding has greatly improved, it is highly likely that other regulators exist and additional work is required to characterize the pathways by which heme and iron are transported within the erythron. Summary The identification of heme and iron transport mechanisms will improve our understanding of blood development and provide new insight into human blood disorders. PMID:22406824

  12. 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.

  13. 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

  14. Gene cluster for dissimilatory nitrite reductase (nir) from Pseudomonas aeruginosa: sequencing and identification of a locus for heme d1 biosynthesis.

    PubMed Central

    Kawasaki, S; Arai, H; Kodama, T; Igarashi, Y

    1997-01-01

    The primary structure of an nir gene cluster necessary for production of active dissimilatory nitrite reductase was determined from Pseudomonas aeruginosa. Seven open reading frames, designated nirDLGHJEN, were identified downstream of the previously reported nirSMCF genes. From nirS through nirN, the stop codon of one gene and the start codon of the next gene were closely linked, suggesting that nirSMCFDLGHJEN are expressed from a promoter which regulates the transcription of nirSM. The amino acid sequences deduced from the nirDLGH genes were homologous to each other. A gene, designated nirJ, which encodes a protein of 387 amino acids, showed partial identity with each of the nirDLGH genes. The nirE gene encodes a protein of 279 amino acids homologous to S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from other bacterial strains. In addition, NirE shows 21.0% identity with NirF in the N-terminal 100-amino-acid residues. A gene, designated nirN, encodes a protein of 493 amino acids with a conserved binding motif for heme c (CXXCH) and a typical N-terminal signal sequence for membrane translocation. The derived NirN protein shows 23.9% identity with nitrite reductase (NirS). Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1. PMID:8982003

  15. 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.

  16. 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

  17. Direct Assay of δ-Aminolevulinic Acid Dehydratase in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry

    PubMed Central

    Choiniere, John R.; Scott, C. Ronald; Gelb, Michael H.; Tureček, František

    2010-01-01

    We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid-liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The Km of ALAD for ALA was measured as 333 μM, and the Vmax was 19.3 μM/hr. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95–99%. The assay shows good reproducibility, low background, requires a simple workup, and uses a commercially available substrate. PMID:20583792

  18. Transmembrane heme delivery systems

    PubMed Central

    Goldman, Barry S.; Beck, David L.; Monika, Elizabeth M.; Kranz, Robert G.

    1998-01-01

    Heme proteins play pivotal roles in a wealth of biological processes. Despite this, the molecular mechanisms by which heme traverses bilayer membranes for use in biosynthetic reactions are unknown. The biosynthesis of c-type cytochromes requires that heme is transported to the bacterial periplasm or mitochondrial intermembrane space where it is covalently ligated to two reduced cysteinyl residues of the apocytochrome. Results herein suggest that a family of integral membrane proteins in prokaryotes, protozoans, and plants act as transmembrane heme delivery systems for the biogenesis of c-type cytochromes. The complete topology of a representative from each of the three subfamilies was experimentally determined. Key histidinyl residues and a conserved tryptophan-rich region (designated the WWD domain) are positioned at the site of cytochrome c assembly for all three subfamilies. These histidinyl residues were shown to be essential for function in one of the subfamilies, an ABC transporter encoded by helABCD. We believe that a directed heme delivery pathway is vital for the synthesis of cytochromes c, whereby heme iron is protected from oxidation via ligation to histidinyl residues within the delivery proteins. PMID:9560218

  19. 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.

  20. A trilogy on. delta. -aminolevulinic acid biosynthesis in plants and algae: I. Glutamate is the sole precursor to protoheme and heme a in maize. II. The UUC glutamate anticodon is a general feature of the tRNA required for ALA biosynthesis. III. Protein and ALA biosynthesis use the same tRNA

    SciTech Connect

    Schneegurt, M.A.

    1989-01-01

    Specifically radiolabeled substrates can be used to determine whether the heme and chlorophyll precursor {delta}-aminolevulinic acid (ALA) is synthesized via the fife-carbon pathway (incorporation from L-1-({sup 14}C)glutamate) or ALA synthase (incorporation from 2-({sup 14}C)glycine). In etiolated maize epicotyl sections, highly purified total cellular protoheme was labeled 29.7 times more effectively by glutamate than by glycine. Mitochondrial heme {alpha} was labeled 4.1 times more effectively by glutamate than by glycine. Cell-free plant and algal preparations require tRNA for the enzymatic conversion of glutamate to ALA. The tRNA required for ALA biosynthesis ahs been shown to contain the UUC glutamate anticodon, as determined by its specific retention through anticodon:anticodon interactions by tRNA{sup Phe(GAA)}-acrylamide. A fraction that was highly enriched in the RNA which supported ALA formation was obtained by affinity chromatography of RNA extracts from Chlorella vulgaris, Euglena garcilis, Cyanidium caldarium, Synechocystis, sp. PCC 6803, pea, and spinach. Other glutamate-accepting RNAs that were not retained by the affinity column were ineffective in supporting ALA formation.

  1. Biology of Heme in Mammalian Erythroid Cells and Related Disorders

    PubMed Central

    Fujiwara, Tohru; Harigae, Hideo

    2015-01-01

    Heme is a prosthetic group comprising ferrous iron (Fe2+) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis. PMID:26557657

  2. Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis

    SciTech Connect

    Zhang,Y.; Kang, S.; Mukherjee, T.; Bale, S.; Crane, B.; Begley, T.; Ealick, S.

    2007-01-01

    The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 {angstrom}. TDO catalyzes the irreversible oxidation of L-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate L-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.

  3. 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.

  4. Control of intracellular heme levels: Heme transporters and Heme oxygenases

    PubMed Central

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

  5. Direct Assay of Enzymes in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry. Uroporphyrinogen Decarboxylase and Coproporphyrinogen III Oxidase

    PubMed Central

    Wang, Yuesong; Gatti, Paula; Sadílek, Martin; Scott, C. Ronald; Tureèek, František; Gelb, Michael H.

    2008-01-01

    We report new assays of enzymes uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen III oxidase (CPO) in the heme biosynthetic pathway. The assays were developed for use in clinical diagnostics of inherited disorders porphyria cutanea tarda and hereditary coproporphyria, respectively. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylation of pentaporphyrinogen I or uroporphyrinogen III catalyzed by UROD and to determine the enzyme activity in human erythrocytes by measuring the production of coproporphyrinogen I or III. The Km value for pentaporphyrinogen I was measured as 0.17 ± 0.03 μM. A mass spectrometric assay was also developed for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes (Km = 0.066 ± 0.009 μM). The assays show good reproducibility, use simple workup by liquid–liquid extraction of enzymatic products, and employ commercially available substrates and internal standards. PMID:18294003

  6. Direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry. Uroporphyrinogen decarboxylase and coproporphyrinogen III oxidase.

    PubMed

    Wang, Yuesong; Gatti, Paula; Sadílek, Martin; Scott, C Ronald; Turecek, Frantisek; Gelb, Michael H

    2008-04-01

    We report new assays of enzymes uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen III oxidase (CPO) in the heme biosynthetic pathway. The assays were developed for use in clinical diagnostics of inherited disorders porphyria cutanea tarda and hereditary coproporphyria, respectively. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylation of pentaporphyrinogen I or uroporphyrinogen III catalyzed by UROD and to determine the enzyme activity in human erythrocytes by measuring the production of coproporphyrinogen I or III. The Km value for pentaporphyrinogen I was measured as 0.17 +/- 0.03 microM. A mass spectrometric assay was also developed for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes (Km = 0.066 +/- 0.009 microM). The assays show good reproducibility, use simple workup by liquid-liquid extraction of enzymatic products, and employ commercially available substrates and internal standards. PMID:18294003

  7. 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.

  8. Identification of the Mitochondrial Heme Metabolism Complex

    PubMed Central

    Medlock, Amy E.; Shiferaw, Mesafint T.; Marcero, Jason R.; Vashisht, Ajay A.; Wohlschlegel, James A.; Phillips, John D.; Dailey, Harry A.

    2015-01-01

    Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex. PMID:26287972

  9. An osmoregulated dipeptide in stressed Rhizobium meliloti.

    PubMed Central

    Smith, L T; Smith, G M

    1989-01-01

    One common mechanism of cellular adaptation to osmotic stress is the accumulation of organic solutes in the cytosol. We have used natural-abundance 13C nuclear magnetic resonance to identify all organic solutes that accumulate to significant levels in Rhizobium meliloti. Our studies led to the discovery of a new dipeptide, N-acetylglutaminylglutamine amide (NAGGN), which is accumulated during osmotic stress. Only rarely have peptides been shown to function in bacteria, and furthermore, this is the first example of a peptide playing a role in osmoregulation. Evidence for the biological role of NAGGN in osmotic-stress protection is presented. PMID:2768187

  10. 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

  11. [Heme metabolism and oxidative stress].

    PubMed

    Kaliman, P A; Barannik, T B

    2001-01-01

    The role of heme metabolism in oxidative stress development and defense reactions formation in mammals under different stress factors are discussed in the article. Heme metabolism is considered as the totality of synthesis, degradation, transport and exchange processes of exogenous heme and heme liberated from erythrocyte hemoglobin under erythrocyte aging and hemolysis. The literature data presented display normal heme metabolism including mammals heme-binding proteins and intracellular free heme pool and heme metabolism alterations under oxidative stress development. The main attention is focused to the prooxidant action of heme, the interaction of heme transport and lipid exchange, and to the heme metabolism key enzymes (delta-aminolevulinate synthase and heme oxygenase), serum heme-binding protein hemopexin and intracellular heme-binding proteins participating in metabolism adaptation under the action of factors, which cause oxidative stress. PMID:11599427

  12. [Heme metabolism as an integral part of iron homeostasis].

    PubMed

    Lipiński, Paweł; Starzyński, Rafał R; Styś, Agnieszka; Gajowiak, Anna; Staroń, Robert

    2014-01-01

    Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways--heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S])--is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis) as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages. PMID:24864106

  13. CcsBA is a cytochrome c synthetase that also functions in heme transport

    PubMed Central

    Frawley, Elaine R.; Kranz, Robert G.

    2009-01-01

    Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel. PMID:19509336

  14. 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

  15. 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

  16. Osmoregulation capacity in Bulgarian durum wheat

    PubMed Central

    Todorovska, Elena Georgieva; Bozhanova, Violeta; Dechev, Dechko; Valkova, Nelly

    2014-01-01

    The phenotypic variation in osmotic adjustment (OA) capacity of five Bulgarian winter durum wheat genotypes and their progenies was determined using a modified method based on the measurement of seedling growth suppression after three-day exposure to osmotic stress induced by 1 mol/L sucrose. The genetic parameters of the studied trait in a diallel crossing scheme, including the selected genotypes and the microsatellite polymorphism at 43 loci, were determined. The old Bulgarian cultivar Apulicum 233 and all hybrid combinations involving this genotype showed higher OA. In the heritability of osmoregulation ability, the non-additive gene effects (specific combining ability) strongly predominated over the additive ones and had a significant impact on the observed high heterosis effect. Distinct polymorphisms were identified between the studied genotypes. Cluster analysis of the phenotypic data obtained from a multiyear test under water-limited conditions and the molecular data, both based on Euclidean distance, showed similar grouping of the genotypes with specific separation of cultivar Apulicum 233 (high OA) in a single cluster. Principal component analysis revealed not only interrelationships between the important agronomic and morpho-physiological traits in Bulgarian durum wheat under water-limited conditions, but also presence of relations between them and some microsatellite loci located near or within known quantitative trait loci (QTLs) for these traits. Further studies based on segregating population between genotypes with contrasting levels of OA will allow mapping QTLs for phenotypic traits expressed under water deficit and isolation of genes that can be used as potential markers in marker-assisted selection for drought tolerance. PMID:26019562

  17. PHEF HEME performance

    SciTech Connect

    Baich, M.A.

    1992-08-15

    The DWPF Salt Cell Vent Condenser (SCVC) includes a High Efficiency Mist Eliminator (HEME) designed to remove mercury aerosols that may form in the Precipitate Reactor (PR) condenser. The Savannah River Technology Center was requested by DWPF to make a performance assesssment of a prototypic HEME element in the vent system of the Precipitate Hydrolysis Experimental Facility at TNX.[sup a

  18. PHEF HEME performance

    SciTech Connect

    Baich, M.A.

    1992-08-15

    The DWPF Salt Cell Vent Condenser (SCVC) includes a High Efficiency Mist Eliminator (HEME) designed to remove mercury aerosols that may form in the Precipitate Reactor (PR) condenser. The Savannah River Technology Center was requested by DWPF to make a performance assesssment of a prototypic HEME element in the vent system of the Precipitate Hydrolysis Experimental Facility at TNX.

  19. 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...

  20. Heme Deficiency in Alzheimer's Disease: A Possible Connection to Porphyria

    PubMed Central

    Dwyer, Barney E.; Stone, Meghan L.; Zhu, Xiongwei; Perry, George; Smith, Mark A.

    2006-01-01

    Mechanisms that cause Alzheimer's disease (AD), an invariably fatal neurodegenerative disease, are unknown. Important recent data indicate that neuronal heme deficiency may contribute to AD pathogenesis. If true, factors that contribute to the intracellular heme deficiency could potentially alter the course of AD. The porphyrias are metabolic disorders characterized by enzyme deficiencies in the heme biosynthetic pathway. We hypothesize that AD may differ significantly in individuals possessing the genetic trait for an acute hepatic porphyria. We elaborate on this hypothesis and briefly review the characteristics of the acute hepatic porphyrias that may be relevant to AD. We note the proximity of genes encoding enzymes of the heme biosynthesis pathway to genetic loci linked to sporadic, late-onset AD. In addition, we suggest that identification of individuals carrying the genetic trait for acute porphyria may provide a unique resource for investigating AD pathogenesis and inform treatment and management decisions. PMID:17047301

  1. Kinetics of heme interaction with heme-binding proteins: the effect of heme aggregation state.

    PubMed

    Kuzelová, K; Mrhalová, M; Hrkal, Z

    1997-10-20

    The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins. PMID:9367177

  2. 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

  3. Heme, an essential nutrient from dietary proteins, critically impacts diverse physiological and pathological processes.

    PubMed

    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

  4. 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

  5. Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes

    PubMed Central

    Hofbauer, Stefan; Hagmüller, Andreas; Schaffner, Irene; Mlynek, Georg; Krutzler, Michael; Stadlmayr, Gerhard; Pirker, Katharina F.; Obinger, Christian; Daims, Holger; Djinović-Carugo, Kristina; Furtmüller, Paul G.

    2015-01-01

    Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0 Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2 μM (circular dichroism spectroscopy) and 16.8 μM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis. PMID:25602700

  6. Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes.

    PubMed

    Hofbauer, Stefan; Hagmüller, Andreas; Schaffner, Irene; Mlynek, Georg; Krutzler, Michael; Stadlmayr, Gerhard; Pirker, Katharina F; Obinger, Christian; Daims, Holger; Djinović-Carugo, Kristina; Furtmüller, Paul G

    2015-05-15

    Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2μM (circular dichroism spectroscopy) and 16.8μM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis. PMID:25602700

  7. 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

  8. 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

  9. Impaired osmoregulation at high altitude. Studies on Mt Everest.

    PubMed

    Blume, F D; Boyer, S J; Braverman, L E; Cohen, A; Dirkse, J; Mordes, J P

    1984-07-27

    Osmoregulation was studied in 13 mountaineers who had experienced long-term exposure to high altitude on Mt Everest. Serum osmolality rose from 290 +/- 1 mOsm/kg to 295 +/- 2 mOsm/kg at 5,400 m and finally to 302 +/- 4 mOsm/kg at 6,300 m after a mean of 26.5 days above 5,400 m. Despite this degree of osmoconcentration, plasma arginine-vasopressin concentration remained unchanged: 1.1 +/-0.1 microU/mL at sea level, 0.8 +/- 0.1 microU/mL at 5,400 m, and 0.9 +/- 0.1 microU/mL at 6,300 m. Urinary vasopressin excretion was also similar at all three altitudes. We conclude that prolonged exposure to high altitude may result in persistent impairment of osmoregulation, caused in part by an inappropriate arginine-vasopressin response to hyperosmolality. PMID:6429358

  10. The Heme Sensor System of Staphylococcus aureus

    PubMed Central

    Stauff, Devin L.; Skaar, Eric P.

    2016-01-01

    The important human pathogen Staphylococcus aureus is able to satisfy its nutrient iron requirement by acquiring heme from host hemoglobin in the context of infection. However, heme acquisition exposes S. aureus to heme toxicity. In order to detect the presence of toxic levels of exogenous heme, S. aureus is able to sense heme through the heme sensing system (HssRS) two-component system. Upon sensing heme, HssRS directly regulates the expression of the heme-regulated ABC transporter HrtAB, which alleviates heme toxicity. Importantly, the inability to sense or respond to heme alters the virulence of S. aureus, highlighting the importance of heme sensing and detoxification to staphylococcal pathogenesis. Furthermore, potential orthologues of the Hss and Hrt systems are found in many species of Gram-positive bacteria, a possible indication that heme stress is a challenge faced by bacteria whose habitats include host tissues rich in heme. PMID:19494582

  11. 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

  12. How Heme Oxygenase-1 Prevents Heme-Induced Cell Death

    PubMed Central

    Lanceta, Lilibeth; Mattingly, Jacob M.

    2015-01-01

    Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by “loose” (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity–CO and bilirubin–have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity. PMID:26270345

  13. Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein.

    PubMed

    Kobayashi, Kazuo; Nakagaki, Megumi; Ishikawa, Haruto; Iwai, Kazuhiro; O'Brian, Mark R; Ishimori, Koichiro

    2016-07-26

    The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. Irr degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 × 10(4) s(-1). The Cys-29 → Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with kon values of 1.3 × 10(5) and 2.5 × 10(4) M(-1) s(-1), a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process. PMID:27379473

  14. Analysis of Oligomerization Properties of Heme a Synthase Provides Insights into Its Function in Eukaryotes.

    PubMed

    Swenson, Samantha; Cannon, Andrew; Harris, Nicholas J; Taylor, Nicholas G; Fox, Jennifer L; Khalimonchuk, Oleh

    2016-05-01

    Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 exhibits homotypic interactions, forming highly stable complexes dependent upon hydrophobic interactions. This multimerization is evolutionarily conserved and independent of heme levels and heme a synthase catalytic activity. Four conserved histidine residues are demonstrated to be critical for eukaryotic heme a synthase activity and cannot be substituted with other heme-ligating amino acids. The 20-residue linker region connecting the two conserved domains of Cox15 is also important; removal of this linker impairs both Cox15 multimerization and enzymatic activity. Mutations of COX15 causing single amino acid conversions associated with fatal infantile hypertrophic cardiomyopathy and the neurological disorder Leigh syndrome result in impaired stability (S344P) or catalytic function (R217W), and the latter mutation affects oligomeric properties of the enzyme. Structural modeling of Cox15 suggests these two mutations affect protein folding and heme binding, respectively. We conclude that Cox15 multimerization is important for heme a biosynthesis and/or transfer to maturing cytochrome c oxidase. PMID:26940873

  15. 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

  16. The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

    PubMed Central

    Haufschildt, Kristin; Neumann, Alexander; Storbeck, Sonja; Streif, Judith

    2014-01-01

    In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme. PMID:24669201

  17. 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.

  18. 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

  19. 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. PMID:9678505

  20. 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

  1. [Metabolism of heme and hemeproteins and some indices of the antioxidant system in rat erythrocytes and tissues under anemia caused by phenylhydrazine].

    PubMed

    Kaliman, P A; Strel'chenko, K V; Barannik, T V; Nikitchenko, I V; Inshina, N M; Pavychenko, O V; Fylymonenko, V P

    2003-01-01

    The decrease of activity of several antioxidant enzymes in erythrocytes in the first hours after injection of phenylhydrazine to rats (7 mg per 100 g body weight) was found to be accompanied by accumulation of heme-containing compounds in rat serum and appearance of free heme in liver and decrease of cytochrome P450 content. Tissue-specific features of dynamics of activity of enzymes studied and reduced glutathione content were revealed, that might be caused by differences in total and free heme content in these organs. The role of key enzymes of heme biosynthesis and degradation in adaptation of metabolism under phenylhydrazine action is discussed. PMID:12945117

  2. 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…

  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. Differential effects of metalloporphyrins on messenger RNA levels of delta-aminolevulinate synthase and heme oxygenase. Studies in cultured chick embryo liver cells.

    PubMed Central

    Cable, E E; Pepe, J A; Karamitsios, N C; Lambrecht, R W; Bonkovsky, H L

    1994-01-01

    The acute porphyrias in relapse are commonly treated with intravenous heme infusion to decrease the activity of delta-aminolevulinic acid synthase, normally the rate-controlling enzyme in heme biosynthesis. The biochemical effects of heme treatment are short-lived, probably due in part to heme-mediated induction of heme oxygenase, the rate-controlling enzyme for heme degradation. In this work, selected nonheme metalloporphyrins were screened for their ability to reduce delta-aminolevulinic acid synthase mRNA and induce heme oxygenase mRNA in chick embryo liver cell cultures. Of the metalloporphyrins tested, only zinc-mesoporphyrin reduced delta-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. The combination of zinc-mesoporphyrin and heme, at nanomolar concentrations, decreased delta-aminolevulinic acid synthase mRNA in a dose-dependent manner. The combination of zinc-mesoporphyrin (50 nM) and heme (200 nM) decreased the half-life of the mRNA for delta-aminolevulinic acid synthase from 5.2 to 2.5 h, while a similar decrease was produced by heme (10 microM) alone (2.2 h). The ability of zinc-mesoporphyrin to supplement the reduction of delta-aminolevulinic acid synthase mRNA by heme, in a process similar to that observed with heme alone, provides a rationale for further investigation of this compound for eventual use as a supplement to heme therapy of the acute porphyrias and perhaps other conditions in which heme may be of benefit. Images PMID:8040318

  5. Intracellular redistribution of heme in rat liver under oxidative stress: the role of heme synthesis.

    PubMed

    Kaliman, Pavel A; Barannik, Tatyana; Strel'chenko, Ekaterina; Inshina, Natalya; Sokol, Oxana

    2005-01-01

    Heme distribution in subcellular fractions of rat liver was studied first hours under the action of several agents causing oxidative stress in vivo. Total and post-mitochondrial heme content in liver was found to depend on both the level of hemolysis products in blood and agent's capacity to modify heme and hemoproteins. The increase of activity of 5-aminolevulinate synthase (ALAS) and/or heme accumulation in mitochondria was accompanied by increase of tryptophan-2,3-dioxygenase (TDO) heme saturation. Membrane stabilisation by tocopherol or prevention of early ALAS induction by cycloheximide prevented both mitochondrial heme accumulation and increase of TDO heme saturation. Modification of heme fully prevented the alterations of total heme content even under severe hemolysis as well as the increase of TDO heme saturation if no increase of heme synthesis occurred. Thus heme synthesis can greatly contribute to heme intracellular redistribution under oxidative stress. PMID:15763493

  6. 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

  7. A water-specific aquaporin involved in aphid osmoregulation.

    PubMed

    Shakesby, A J; Wallace, I S; Isaacs, H V; Pritchard, J; Roberts, D M; Douglas, A E

    2009-01-01

    The osmotic pressure of plant phloem sap is generally higher than that of insect body fluids. Water cycling from the distal to proximal regions of the gut is believed to contribute to the osmoregulation of aphids and other phloem-feeding insects, with the high flux of water mediated by a membrane-associated aquaporin. A putative aquaporin referred to as ApAQP1 was identified by RT-PCR of RNA isolated from the guts of pea aphids Acyrthosiphon pisum. The ApAQP1 protein has a predicted molecular mass 28.94kDa. Molecular modeling suggests that ApAQP1 has the general aquaporin topology and possesses the conserved pore properties of water-specific aquaporins. When expressed in Xenopus oocytes, ApAQP1 showed the hallmarks of aquaporin-mediated water transport, including an 18-fold increase in the osmotic water permeability of the oolemma, a reduced activation energy, and inhibition of elevated water transport activity by Hg ions. The ApAQP1 transcript was localised to the stomach and distal intestine, and RNAi-mediated knockdown of its expression resulted in elevated osmotic pressure of the haemolymph. Taken together, these data suggest that ApAQP1 contributes to the molecular basis of water cycling in the aphid gut. PMID:18983920

  8. [Osmoregulation--an important parameter of bacterial growth].

    PubMed

    Sochocka, Marta; Boratyński, Janusz

    2011-01-01

    Environmental conditions such as temperature, pH, radiation and osmotic pressure are important factors limiting the growth and multiplication of bacteria. Regular structure and metabolism of bacterial cells are maintained through a stable arrangement of the water-electrolyte system, regulated by osmosis. The rapid changes caused by osmotic shock (dehydration, rehydration) might lead to modifications of the phospholipid structure of the cell membrane and even cell death. Advances disturbing the osmosis, which are a natural part of living cells, may appear for example in colloid systems. The biological identification of the osmotic pressure is connected with an increase or decrease in the environmental osmotic strength of microorganisms' habitat. Cells exposed to osmotic stress, such as an increase in osmotic pressure, initiate mechanisms of active coping with the adverse consequences of its effects. Osmoregulatory processes are designed to maintain cell turgor, hence ensuring proper conditions for bacterial growth. Osmoregulation, which consists of maintaining fluid and electrolyte balance of cells, raising concerns accumulation of specific compatible solutes (osmolytes). Osmolytes are small, soluble organic molecules with a positive influence on membrane stabilization and proteins, without disrupting cellular functions. Storage of compatible solutes takes place by synthesis or by downregulation from the medium by means of special transport systems, activated by mechanical stimuli. Knowledge of the impact of osmotic pressure on microbial cells and the regulation of its activity led to the appropriate use of bacteria in various branches of the biotechnology industry. PMID:22173436

  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. Heme Mobilization in Animals: A Metallolipid's Journey.

    PubMed

    Reddi, Amit R; Hamza, Iqbal

    2016-06-21

    Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient. PMID:27254265

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

    PubMed

    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

  12. 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

  13. 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

  14. Auxin biosynthesis.

    PubMed

    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

  15. 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

  16. 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. PMID:25773436

  17. 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.

  18. 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

  19. Oxytetracycline Biosynthesis*

    PubMed Central

    Pickens, Lauren B.; Tang, Yi

    2010-01-01

    Oxytetracycline (OTC) is a broad-spectrum antibiotic that acts by inhibiting protein synthesis in bacteria. It is an important member of the bacterial aromatic polyketide family, which is a structurally diverse class of natural products. OTC is synthesized by a type II polyketide synthase that generates the poly-β-ketone backbone through successive decarboxylative condensation of malonyl-CoA extender units, followed by modifications by cyclases, oxygenases, transferases, and additional tailoring enzymes. Genetic and biochemical studies have illuminated most of the steps involved in the biosynthesis of OTC, which is detailed here as a representative case study in type II polyketide biosynthesis. PMID:20522541

  20. 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. PMID:16143108

  1. Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

    PubMed Central

    Takayama, Shin-ichi J.; Ukpabi, Georgia; Murphy, Michael E. P.; Mauk, A. Grant

    2011-01-01

    IsdI, a heme-degrading protein from Staphylococcus aureus, binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in 1H-NMR spectra of the protein argues that the catalytic formation of β- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations. PMID:21788475

  2. Chloride cells and the hormonal control of teleost fish osmoregulation.

    PubMed

    Foskett, J K; Bern, H A; Machen, T E; Conner, M

    1983-09-01

    Teleost fish osmoregulation is largely the result of integrated transport activities of the gill, gut and renal system. The basic 'epithelial fabric' in each of these tissues is adapted to provide the appropriate transport mechanisms depending upon whether the fish is in fresh water or sea water. Net NaCl transport by the branchial epithelium reverses direction when euryhaline species migrate between the two media, providing a useful focus in experiments designed to elucidate mechanisms of differentiation and integration of transport function. Isolated opercular membranes and skins from certain seawater-adapted species are good models to study branchial salt extrusion mechanisms. These heterogeneous tissues generate short-circuit currents equal to net chloride secretion. The vibrating probe technique has allowed localization of all current and almost all conductance to the apical crypt of chloride cells. Area-specific surface current and conductance of chloride cells are 18 mA cm-2 and 580 mS cm-2 (1.7 omega cm2), ranking them as one of the most actively transporting and conductive cells known. There is no net sodium transport under short-circuit conditions but the chloride secretion process is sodium-dependent and ouabain and 'loop'-diuretic sensitive. Sodium fluxes through chloride cells are large (PNa = 5.2 X 10(-4) cms-1) nd appear passive and rate-limited by a single barrier. A link may exist between the active transport and leak pathways since sodium fluxes always account for 50% of chloride cell conductance. The sodium pathway is probably the chloride cell-accessory cell tight junction, although this is still unresolved. Chloride secretion can be rapidly modulated by several hormones, including catecholamines, somatostatin, glucagon, vasoactive intestinal polypeptide and urotensins I and II. Regulation by these hormones may be by rapid alterations of cellular cAMP levels. Differentiation of chloride cells and chloride secretion may be controlled by cortisol

  3. RESPIRATION AND OSMOREGULATION OF THE ESTUARINE CRAB RHITHROPANOPEUS HARRISII (GOULD): EFFECTS OF THE HERBICIDE ALACHLOR

    EPA Science Inventory

    The effects of a sudden decrease in salinity and exposure to sublethal concentrations of the herbicide, Alachlor, on osmoregulation and respiration of the crab, Rhithropanopeus harrisii, were studied. 2. Crabs were hyperosmotic regulators at salinities below 24 ppt and became hyp...

  4. 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...

  5. A role for heme in Alzheimer's disease: heme binds amyloid beta and has altered metabolism.

    PubMed

    Atamna, Hani; Frey, William H

    2004-07-27

    Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P = 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in heme-a/heme-b ratio (P < 0.001). Moreover, the strong Pearson correlation between heme-a and heme-b measured in control individuals (r(2) = 0.66, P < 0.002, n = 11) was abolished in AD subjects (r(2) = 0.076, P = 0.39, n = 12). The level of ferrochelatase (which makes heme-b in the mitochondrial matrix) in AD subjects was 4.2 times (P < 0.04) that in nondemented controls, suggesting up-regulated heme synthesis. To look for a possible connection between these observations and established mechanisms in AD pathology, we examined possible interactions between amyloid beta (A beta) and heme. A beta((1-40)) and A beta((1-42)) induced a redshift of 15-20 nm in the spectrum of heme-b and heme-a, suggesting that heme binds A beta, likely to one or more of the histidine residues. Lastly, in a tissue culture model, we found that clioquinol, a metal chelator in clinical trials for AD therapy, decreased intracellular heme. In light of these observations, we have proposed a model of AD pathobiology in which intracellular A beta complexes with free heme, thereby decreasing its bioavailability (e.g., heme-a) and resulting in functional heme deficiency. The model integrates disparate observations, including A beta, mitochondrial dysfunction, cholesterol, and the proposed efficacy of clioquinol. PMID:15263070

  6. 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

  7. MicroRNA‐210 Decreases heme Levels by Targeting Ferrochelatase in Cardiomyocytes

    PubMed Central

    Qiao, Aijun; Khechaduri, Arineh; Kannan Mutharasan, R.; Wu, Rongxue; Nagpal, Varun; Ardehali, Hossein

    2013-01-01

    Background MicroRNA‐210 (miR‐210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron‐sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster synthesis. However, it is not known how miR‐210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function. Methods and Results To screen for micro‐ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR‐210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia‐inducible factor (HIF) pathway. Furthermore, miR‐210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme‐containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR‐210 binding sites in the 3′ untranslated region (UTR) of FECH reversed the miR‐210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR‐210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3′ UTR does not alter miR‐210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR‐210 knockdown, suggesting that the effects of miR‐210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia. Conclusions Our results identify a role for miR‐210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions. PMID:23608607

  8. Nitrosylation of rabbit ferrous heme-hemopexin.

    PubMed

    Fasano, Mauro; Bocedi, Alessio; Mattu, Marco; Coletta, Massimo; Ascenzi, Paolo

    2004-10-01

    Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4+/-0.2)x10(-7) M, at pH 7.0 and 10.0 degrees C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by epsilon=146 mM(-1) cm(-1) at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by epsilon=74 mM(-1) cm(-1) at 387 nm. The p K(a) value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8+/-0.1 in the absence of sodium chloride and 4.3+/-0.1 in the presence of 1.5x10(-1) M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems. PMID:15378409

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

    PubMed Central

    Pang, Wei; Coghill, George M.

    2015-01-01

    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. PMID:25864377

  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. PMID:17766221

  11. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  12. The role of osmoregulation in the pathophysiology and management of severe ovarian hyperstimulation syndrome.

    PubMed

    Evbuomwan, Isaac

    2013-09-01

    Severe ovarian hyperstimulation syndrome (OHSS), with an incidence of 1-2% of superovulation cycles, remains one of the most important complications of gonadotrophin use in assisted reproductive technologies because of its associated morbidity and rarely, mortality. Despite the wealth of scientific and clinical interest that this iatrogenic complication has generated, its pathophysiology is still not adequately elucidated and its management has thus remained empirical. Disorders of salt and water balance are two very important features that have been reported during severe OHSS. Some of the clinical and biochemical changes resulting from this disorder of salt and water balance are similar to those previously reported in pregnancy and liver cirrhosis. The pathophysiology of these clinical changes has been explained in part in pregnancy and liver cirrhosis by changes in osmoregulation function. It is this similarity in the clinical and biochemical changes in OHSS, pregnancy and liver cirrhosis that has prompted the investigation of the role of osmoregulation function in the pathophysiology of OHSS. The current article has been written to provide further details in support of recent excellent articles and guidelines, highlighting the physiological basis and rationale governing some aspects of, and the role of osmoregulation in the management of the OHSS syndrome. PMID:24047195

  13. 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

  14. Geometric and electronic structures of the His–Fe(IV)=O and His–Fe(IV)–Tyr hemes of MauG

    PubMed Central

    Jensen, Lyndal M. R.; Meharenna, Yergalem T.; Davidson, Victor L.; Poulos, Thomas L.; Hedman, Britt

    2012-01-01

    Biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor activates the enzyme methylamine dehydrogenase. The diheme enzyme MauG catalyzes O-atom insertion and cross-linking of two Trp residues to complete TTQ synthesis. Solution optical and Mössbauer spectroscopic studies have indicated that the reactive form of MauG during turnover is an unusual bisFe(IV) intermediate, which has been formulated as a His-ligated ferryl heme [Fe(IV)=O] (heme A), and an Fe(IV) heme with an atypical His/Tyr ligation (heme B). In this study, Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure studies have been combined with density functional theory (DFT) and time-dependent DFT methods to solve the geometric and electronic structures of each heme site in the MauG bisFe(IV) redox state. The ferryl heme site (heme A) is compared with the well-characterized compound I intermediate of cytochrome c peroxidase. Heme B is unprecedented in biology, and is shown to have a six-coordinate, S = 1 environment, with a short (1.85-Å) Fe–O(Tyr) bond. Experimentally calibrated DFT calculations are used to reveal a strong covalent interaction between the Fe and the O(Tyr) ligand of heme B in the high-valence form. A large change in the Fe–O(Tyr) bond distance on going from Fe(II) (2.02 Å) to Fe(III) (1.89 Å) to Fe(IV) (1.85 Å) signifies increasing localization of spin density on the tyrosinate ligand upon sequential oxidation of heme B to Fe(IV). As such, O(Tyr) plays an active role in attaining and stabilizing the MauG bisFe(IV) redox state. PMID:23053529

  15. 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

  16. Characterization of the heme synthesis enzyme coproporphyrinogen oxidase (CPO) in zebrafish erythrogenesis.

    PubMed

    Hanaoka, Ryuki; Katayama, Shiori; Dawid, Igor B; Kawahara, Atsuo

    2006-03-01

    Hemoglobin consists of heme and globin proteins and is essential for oxygen transport in all vertebrates. Although biochemical features of heme synthesis enzymes have been well characterized, the function of these enzymes in early embryogenesis is not fully understood. We found that the sixth heme synthesis enzyme, coproporphyrinogen oxidase (CPO), is predominantly expressed in the intermediate cell mass (ICM) that is a major site of zebrafish primitive hematopoiesis. Knockdown of zebrafish CPO using anti-sense morpholinos (CPO-MO) leads to a significant suppression of hemoglobin production without apparent reduction of blood cells. Injection of human CPO RNA, but not a mutant CPO RNA that is similar to a mutant responsible for a hereditary coproporphyria (HCP), restores hemoglobin production in the CPO-MO-injected embryos. Furthermore, expression of CPO in the ICM is severely suppressed in both vlad tepes/gata1 mutants and in biklf-MO-injected embryos. In contrast, over-expression of biklf and gata1 significantly induces ectopic CPO expression. The function of CPO in heme biosynthesis is apparently conserved between zebrafish and human, suggesting that CPO-MO-injected zebrafish embryos might be a useful in vivo assay system to measure the biological activity of human CPO mutations. PMID:16483317

  17. 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.

  18. Heme sensing in Bacillus thuringiensis: a supplementary HssRS-regulated heme resistance system.

    PubMed

    Schmidt, Rachel M; Carter, Micaela M; Chu, Michelle L; Latario, Casey J; Stadler, Sarah K; Stauff, Devin L

    2016-05-01

    Several Gram-positive pathogens scavenge host-derived heme to satisfy their nutritional iron requirement. However, heme is a toxic molecule capable of damaging the bacterial cell. Gram-positive pathogens within the phylum Firmicutes overcome heme toxicity by sensing heme through HssRS, a two-component system that regulates the heme detoxification transporter HrtAB. Here we show that heme sensing by HssRS and heme detoxification by HrtAB occur in the insect pathogen Bacillus thuringiensis We find that in B. thuringiensis, HssRS directly regulates an operon, hrmXY, encoding hypothetical membrane proteins that are not found in other Firmicutes with characterized HssRS and HrtAB systems. This novel HssRS-regulated operon or its orthologs BMB171_c3178 and BMB171_c3330 are required for maximal heme resistance. Furthermore, the activity of HrmXY is not dependent on expression of HrtAB. These results suggest that B. thuringiensis senses heme through HssRS and induces expression of separate membrane-localized systems capable of overcoming different aspects of heme toxicity. PMID:27030728

  19. 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

  20. 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

  1. Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

    PubMed

    Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

    2009-01-01

    Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders. PMID:19046352

  2. HEME-HEME COMUNICATION DURING THE ALKALINE INDUCED STRUCTURAL TRANSITION IN CYTOCROME C OXIDASE

    PubMed Central

    Ji, Hong; Rousseau, Denis L.; Yeh, Syun-Ru

    2009-01-01

    Alkaline induced conformational changes at pH 12.0 in the oxidized as well as the reduced state of cytochrome c oxidase have been systematically studied with time-resolved optical absorption and resonance Raman spectroscopies. In the reduced state, the heme a3 first converts from the native five-coordinate configuration to a six-coordinate bis-histidine intermediate as a result of the coordination of one of the CuB ligands, H290 or H291, to the heme iron. The coordination state change in the heme a3 causes the alteration in the microenvironment of the formyl group of the heme a3 and the disruption of the H-bond between R38 and the formyl group of the heme a. This structural transition, which occurs within 1 minute following the initiation of the pH jump, is followed by a slower reaction, in which Schiff base linkages are formed between the formyl groups of the two hemes and their nearby amino acid residues, presumably R38 and R302 for the heme a and a3, respectively. In the oxidized enzyme, a similar Schiff base modification on heme a and a3 was observed but it is triggered by the coordination of the H290 or H291 to heme a3 followed by the breakage of the native proximal H378-iron and H376-iron bonds in heme a and a3, respectively. In both oxidation states, the synchronous formation of the Schiff base linkages in heme a and a3 relies on the structural communication between the two hemes via the H-bonding network involving R438 and R439 and the propionate groups of the two hemes as well as the helix X housing the two proximal ligands, H378 and H376, of the hemes. The heme-heme communication mechanism revealed in this work may be important in controlling the coupling of the oxygen and redox chemistry in the heme sites to proton pumping during the enzymatic turnover of CcO. PMID:18187199

  3. 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.

  4. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    SciTech Connect

    Wojtowicz, Halina; Wojaczynski, Jacek; Olczak, Mariusz; Kroliczewski, Jaroslaw; Latos-Grazynski, Lechoslaw; Olczak, Teresa

    2009-05-29

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  5. 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

  6. Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

    PubMed

    Neya, Saburo; Nagai, Masako; Nagatomo, Shigenori; Hoshino, Tyuji; Yoneda, Tomoki; Kawaguchi, Akira T

    2016-05-01

    Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson

  7. Spectroscopic investigations of heme proteins

    NASA Astrophysics Data System (ADS)

    Ogilvie, Jennifer Pauline

    Using several novel spectroscopic techniques, we investigate the dynamics of heme proteins over the full range of time scales relevant to their function. With ˜10 femtosecond time resolution we use ultrafast pump-probe spectroscopy to gain insight into the earliest dynamics initiated by the photodissociation of the carbon monoxide ligand from myoglobin. Coherent oscillations that are driven by the bond-breaking event reveal several vibrational modes of the heme that provide the driving force for the initial motions along the pathway to protein function. Much later along this pathway we address the question of ligand escape from myoglobin. With this purpose we develop heterodyne-detected diffractive-optics-based phase-grating spectroscopy, which provides more than 2 orders of magnitude increase in sensitivity for the measurement of volume changes and energetics. The improved sensitivity allows us to directly observe the ligand escape, which occurs via a number of discrete routes through the protein. Following the escape process, we observe the full cycle of dynamics that is complete when the carbon monoxide ligand rebinds to the protein. Using a resonant probe we re-examine the dynamics of ligand escape from myoglobin using transient absorption and transient-grating spectroscopy. This study confirms the findings of the previous off-resonant work, and allows us to explore the relationship between the observables in the phase-grating experiment and other resonant spectroscopies. The various dynamical processes of myoglobin provide a basis for understanding the structure/function relationship at the single protein level. This lays the foundation for a description of protein-protein interactions such as cooperativity in hemoglobin.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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. PMID:26872994

  14. 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...

  15. 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

  16. Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions.

    PubMed

    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

    2015-02-13

    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

  17. Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS).

    PubMed

    Du, Yongming; Liu, Gefei; Yan, Yinxia; Huang, Dongyang; Luo, Wenhong; Martinkova, Marketa; Man, Petr; Shimizu, Toru

    2013-10-01

    The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species. PMID:23736976

  18. Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2.

    PubMed

    Alam, Jawed; Killeen, Erin; Gong, Pengfei; Naquin, Ryan; Hu, Bin; Stewart, Daniel; Ingelfinger, Julie R; Nath, Karl A

    2003-04-01

    The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene. PMID:12453873

  19. One ring to rule them all: Trafficking of heme and heme synthesis intermediates in the metazoans

    PubMed Central

    Hamza, Iqbal; Dailey, Harry A.

    2012-01-01

    The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. PMID:22575458

  20. 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.

  1. Heme synthesis in normal mouse liver and mouse liver tumors

    SciTech Connect

    Stout, D.L.; Becker, F.F. )

    1990-04-15

    Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors {sup 55}FeCl3 and (2-{sup 14}C)glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated (2-14C)glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool.

  2. 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.

  3. 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

  4. Heme Synthesis by Plastid Ferrochelatase I Regulates Nuclear Gene Expression in Plants

    PubMed Central

    Woodson, Jesse D.; Perez-Ruiz, Juan M.; Chory, Joanne

    2016-01-01

    Summary Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling [1, 2]. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF [3, 4]. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1Dmutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development. PMID:21565502

  5. 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. PMID:25575902

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Ultrafast dynamics of ligands within heme proteins.

    PubMed

    Vos, Marten H

    2008-01-01

    Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes. PMID:17996720

  13. Control of heme metabolism with synthetic metalloporphyrins.

    PubMed Central

    Kappas, A; Drummond, G S

    1986-01-01

    Studies with synthetic metal-porphyrin complexes in which the central iron atom of heme is replaced by other elements indicate that those heme analogues that cannot be enzymatically degraded to bile pigments possess novel biological properties that may have considerable clinical as well as experimental value. Such studies have revealed the important role that the central metal atom plays in determining the physiological and pharmacological properties of metal-porphyrin complexes; and they have demonstrated that the form in which animals and humans are exposed to trace metals, i.e., inorganic, organified, porphyrin-chelated, etc., can be of great importance in determining the biological responses that such elements elicit, especially with respect to actions on heme synthesis and degradation and cytochrome P-450 formation and function. Study of the biological properties of synthetic metalloporphyrins represents a potentially fruitful area of research and the results may have significant value for basic as well as clinical disciplines. PMID:3511095

  14. Heme compounds in dinosaur trabecular bone

    PubMed Central

    Schweitzer, Mary H.; Marshall, Mark; Carron, Keith; Bohle, D. Scott; Busse, Scott C.; Arnold, Ernst V.; Barnard, Darlene; Horner, J. R.; Starkey, Jean R.

    1997-01-01

    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. PMID:9177210

  15. 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. PMID:9177210

  16. Regulation of heme iron absorption by young children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heme iron is an important source of dietary iron for children. Little is known of its absorption as only radio-isotopically labeled heme iron has been available to date. We have recently developed a method of intrinsically labeling bovine heme iron in vivo with the stable isotope iron-58. Our object...

  17. Structures of prostacyclin synthase and its complexes with substrate analog and inhibitor reveal a ligand-specific heme conformation change.

    PubMed

    Li, Yi-Ching; Chiang, Chia-Wang; Yeh, Hui-Chun; Hsu, Pei-Yung; Whitby, Frank G; Wang, Lee-Ho; Chan, Nei-Li

    2008-02-01

    Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels. PMID:18032380

  18. 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

  19. Characterization of Heme-DNA Complexes Composed of Some Chemically Modified Hemes and Parallel G-Quadruplex DNAs.

    PubMed

    Yamamoto, Yasuhiko; Kinoshita, Masashi; Katahira, Yuya; Shimizu, Haruna; Di, Yue; Shibata, Tomokazu; Tai, Hulin; Suzuki, Akihiro; Neya, Saburo

    2015-12-15

    Heme {Fe(II)- or Fe(III)-protoporphyrin IX complex [heme(Fe(2+)) or heme(Fe(3+)), respectively]} binds selectively to the 3'-terminal G-quartet of a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The binding affinities of some chemically modified hemes(Fe(3+)) for DNA and the structures of complexes between the modified hemes(Fe(2+)) and DNA, with carbon monoxide (CO) coordinated to the heme Fe atom on the side of the heme opposite the G6 G-quartet, have been characterized to elucidate the interaction between the heme and G-quartet in the complexes through analysis of the effects of the heme modification on the structural properties of the complex. The study revealed that the binding affinities and structures of the complexes were barely affected by the heme modification performed in the study. Such plasticity in the binding of heme to the G-quartet is useful for the versatile design of the complex through heme chemical modification and DNA sequence alteration. Furthermore, exchangeable proton signals exhibiting two-proton intensity were observed at approximately -3.5 ppm in the (1)H nuclear magnetic resonance (NMR) spectra of the CO adducts of the complexes. Through analysis of the NMR results, together with theoretical consideration, we concluded that the heme(Fe(2+)) axial ligand trans to CO in the complex is a water molecule (H2O). Identification of the Fe-bound H2O accommodated between the heme and G-quartet planes in the complex provides new insights into the structure-function relationship of the complex. PMID:26595799

  20. 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

  1. 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

  2. Is the calcium transporter a potential candidate for heme transport?

    PubMed

    Latunde-Dada, Gladys O

    2016-06-01

    Heme is of significant importance in iron nutrition and in systemic iron metabolism. The crux of the matter is that while much is known about non-heme metabolism, the vectorial import of exogenous porphyrin macromolecules into the enterocyte and possibly into blood circulation is still speculative. The inhibitory effect of calcium on heme iron absorption has been previously reported in the literature. This paper postulates that the gastrointestinal Ca transporter, TRPV6, might be a putative transporter of heme and might account for reduced heme absorption in the presence of Ca. The hypothesis needs to be investigated in vitro and in vivo with targeted TRPV6 deletion models to explore the nature of the competitive inhibition of heme uptake by Ca. Studies are required to characterize fully this function in the gut and in systemic metabolism. If the hypothesis is proven, modulators of TRPV6 expression could have clinical implications in the management of heme-induced disorders. PMID:27142151

  3. 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

  4. Functional Importance of Tyrosine 294 and the Catalytic Selectivity for the Bis-Fe(IV) State of MauG Revealed by Replacement of This Axial Heme Ligand with Histidine

    SciTech Connect

    Tarboush, Nafez Abu; Jensen, Lyndal M.R.; Feng, Manliang; Tachikawa, Hiroyasu; Wilmot, Carrie M.; Davidson, Victor L.

    2010-12-07

    The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 {angstrom} separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)=O/{pi} radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)=O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.

  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. Potential role of mitochondrial superoxide decreasing ferrochelatase and heme in coronary artery soluble guanylate cyclase depletion by angiotensin II.

    PubMed

    Patel, Dhara; Alhawaj, Raed; Kelly, Melissa R; Accarino, John J O; Lakhkar, Anand; Gupte, Sachin A; Sun, Dong; Wolin, Michael S

    2016-06-01

    Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 μM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide. PMID:27037373

  7. 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

  8. 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.

  9. Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability

    PubMed Central

    Kořený, Luděk; Sobotka, Roman; Kovářová, Julie; Gnipová, Anna; Flegontov, Pavel; Horváth, Anton; Oborník, Miroslav; Ayala, Francisco J.; Lukeš, Julius

    2012-01-01

    Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme. PMID:22355128

  10. Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability.

    PubMed

    Kořený, Luděk; Sobotka, Roman; Kovářová, Julie; Gnipová, Anna; Flegontov, Pavel; Horváth, Anton; Oborník, Miroslav; Ayala, Francisco J; Lukeš, Julius

    2012-03-01

    Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme. PMID:22355128

  11. 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

  12. Thyroid hormone regulation of heme oxidation in the liver.

    PubMed Central

    Smith, T J; Drummond, G S; Kourides, I A; Kappas, A

    1982-01-01

    The effects of 3,5,3'-triiodothyronine (T3) on heme oxygenase (EC 1.14.99.3) activity and cytochrome P-450 content in liver were examined in thyroidectomized rats. T3, when administered for 5 days at a dose of 6 micrograms/100 g of body weight, stimulated basal heme oxygenase activity approximately equal to 2-fold compared to diluent-treated animals. The induction of heme oxygenase by cobalt heme also was enhanced approximately equal to 3-fold in T3-treated animals. T3 treatment lowered cytochrome P-450 content by approximately equal to 50% and potentiated the depletion of this heme protein after cobalt heme administration. Reverse T3 had no effect either on cytochrome P-450 content or on heme oxygenase activity in liver. The time course of response to a single dose of T3 (50 micrograms/100 g of body weight) revealed that both basal and cobalt heme-induced heme oxygenase activity peaked at 48 hr and that cytochrome P-450 content declined to approximately equal to 40% of controls at 96 hr. Examination of microsomal proteins by polyacrylamide gel electrophoresis after T3 treatment disclosed that major bands in the Mr approximately equal to 50,000-55,000 region were diminished. The administration of T3 together with SKF-525A, a compound known to complex with the heme prosthetic group of cytochrome P-450, resulted in partial preservation of these proteins. These data indicate that thyroid hormone can regulate heme oxygenase activity and concomitantly can lower cytochrome P-450 content in liver. The hormone also can act in a synergistic fashion to enhance the response of hepatic heme oxygenase to a chemical inducer of the enzyme. Thyroid status thus may be a potentially significant determinant of the rate of heme oxidation in the liver. Images PMID:6961431

  13. 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.

  14. 21 CFR 862.1410 - Iron (non-heme) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  15. 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...

  16. 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...

  17. 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...

  18. 21 CFR 862.1410 - Iron (non-heme) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  19. Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

    PubMed Central

    Docampo, Roberto; Ulrich, Paul; Moreno, Silvia N. J.

    2010-01-01

    Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca2+-ATPase, V-H+-ATPase, H+-PPase), exchangers (Na+/H+, Ca2+/H+), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes. PMID:20124344

  20. 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

  1. Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes.

    PubMed

    Docampo, Roberto; Ulrich, Paul; Moreno, Silvia N J

    2010-03-12

    Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca(2+)-ATPase, V-H(+)-ATPase, H(+)-PPase), exchangers (Na(+)/H(+), Ca(2+)/H(+)), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes. PMID:20124344

  2. 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.

  3. 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

  4. 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.

  5. 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

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

    PubMed

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

    2016-01-01

    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

  7. 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.

  8. 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.

  9. Heme Catabolism by Heme Oxygenase-1 Confers Host Resistance to Mycobacterium Infection

    PubMed Central

    Silva-Gomes, Sandro; Appelberg, Rui; Larsen, Rasmus; Soares, Miguel Parreira

    2013-01-01

    Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (Mϕ) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1−/−) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1+/+) controls. Furthermore, Hmox1−/− mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1−/− versus Hmox1+/+ SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected Mϕ, an effect mimicked by exogenous heme administration to M. avium-infected wild-type Mϕ in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in Mϕ, contributing critically to host resistance to Mycobacterium infection. PMID:23630967

  10. Identification of essential histidine residues involved in heme binding and Hemozoin formation in heme detoxification protein from Plasmodium falciparum.

    PubMed

    Nakatani, Keisuke; Ishikawa, Haruto; Aono, Shigetoshi; Mizutani, Yasuhisa

    2014-01-01

    Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum. PMID:25138161

  11. Targeting heme oxygenase after intracerebral hemorrhage

    PubMed Central

    Chen-Roetling, Jing; Lu, Xiangping; Regan, Raymond F.

    2015-01-01

    Intracerebral hemorrhage (ICH) is the primary event in approximately 10% of strokes, and has higher rates of morbidity and mortality than ischemic stroke. Experimental evidence suggests that the toxicity of hemoglobin and its degradation products contributes to secondary injury that may be amenable to therapeutic intervention. Hemin, the oxidized form of heme, accumulates in intracranial hematomas to cytotoxic levels. The rate limiting step of its breakdown is catalyzed by the heme oxygenase (HO) enzymes, which consist of inducible HO-1 and constitutively-expressed HO-2. The effect of these enzymes on perihematomal injury and neurological outcome has been investigated in ICH models using both genetic and pharmacological approaches to alter their expression, with variable results reported. These findings are summarized and reconciled in this review; therapeutic strategies that may optimize HO expression and activity after ICH are described. PMID:25642455

  12. Heme oxygenase-1 system and gastrointestinal tumors

    PubMed Central

    Zhu, Xiao; Fan, Wen-Guo; Li, Dong-Pei; Lin, Marie CM; Kung, Hsiangfu

    2010-01-01

    Heme oxygenase-1 (HO-1) system catabolizes heme into three products: carbon monoxide, biliverdin/bilirubin and free iron. It is involved in many physiological and pathophysiological processes. A great deal of data has demonstrated the roles of HO-1 in the formation, growth and metastasis of tumors. The interest in this system by investigators involved in gastrointestinal tumors is fairly recent, and few papers on HO-1 have touched upon this subject. This review focuses on the current understanding of the physiological significance of HO-1 induction and its possible roles in the gastrointestinal tumors studied to date. The implications for possible therapeutic manipulation of HO-1 in gastrointestinal tumors are also discussed. PMID:20518085

  13. Unusual effects of crowders on heme retention in myoglobin.

    PubMed

    Kundu, Jayanta; Kar, Uddipan; Gautam, Saurabh; Karmakar, Sandip; Chowdhury, Pramit K

    2015-12-21

    Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll-based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu. PMID:26606908

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine.

    PubMed

    Hussy, N; Brès, V; Rochette, M; Duvoid, A; Alonso, G; Dayanithi, G; Moos, F C

    2001-09-15

    Osmotic regulation of supraoptic nucleus (SON) neuron activity depends in part on activation of neuronal glycine receptors (GlyRs), most probably by taurine released from adjacent astrocytes. In the neurohypophysis in which the axons of SON neurons terminate, taurine is also concentrated in and osmo-dependently released by pituicytes, the specialized glial cells ensheathing nerve terminals. We now show that taurine release from isolated neurohypophyses is enhanced by hypo-osmotic and decreased by hyper-osmotic stimulation. The high osmosensitivity is shown by the significant increase on only 3.3% reduction in osmolarity. Inhibition of taurine release by 5-nitro-2-(3-phenylpropylamino)benzoic acid, niflumic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid suggests the involvement of volume-sensitive anion channels. On purified neurohypophysial nerve endings, activation of strychnine-sensitive GlyRs by taurine or glycine primarily inhibits the high K(+)-induced rise in [Ca(2+)](i) and subsequent release of vasopressin. Expression of GlyRs in vasopressin and oxytocin terminals is confirmed by immunohistochemistry. Their implication in the osmoregulation of neurohormone secretion was assessed on isolated whole neurohypophyses. A 6.6% hypo-osmotic stimulus reduces by half the depolarization-evoked vasopressin secretion, an inhibition totally prevented by strychnine. Most importantly, depletion of taurine by a taurine transport inhibitor also abolishes the osmo-dependent inhibition of vasopressin release. Therefore, in the neurohypophysis, an osmoregulatory system involving pituicytes, taurine, and GlyRs is operating to control Ca(2+) influx in and neurohormone release from nerve terminals. This elucidates the functional role of glial taurine in the neurohypophysis, reveals the expression of GlyRs on axon terminals, and further defines the role of glial cells in the regulation of neuroendocrine function. PMID:11549721

  2. 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. PMID:25590591

  3. A perspective on the role of natriuretic peptides in amphibian osmoregulation.

    PubMed

    Donald, John A; Trajanovska, Sofie

    2006-05-15

    The natriuretic peptide (NP) system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. In amphibians, the potential role(s) of NPs is complicated by the range of osmoregulatory strategies found in amphibians, and the different tissues that participate in osmoregulation. Atrial NP, brain NP, and C-type NP have been isolated or cloned from a number of species, which has enabled physiological studies to be performed with homologous peptides. In addition, three types of NP receptors have been cloned and partially characterised. Natriuretic peptides are always potent vasodilators in amphibian blood vessels, and ANP has been shown to increase the permeability of the microcirculation. In the perfused kidney, ANP causes vasodilation, diuresis and natriuresis that are caused by an increased GFR rather than effects in the renal tubules. These data are supported by the presence of ANP receptors only on the glomeruli and renal blood vessels. In the bladder and skin, the function of NPs is enigmatic because physiological analysis of the effects of ANP on bladder and skin function has yielded conflicting data with no clear role for NPs being revealed. Overall, NPs often have no direct effect, but in some studies they have been shown to inhibit the function of AVT. In addition, there is evidence that ANP can inhibit salt retention in amphibians since it can inhibit the ability of adrenocorticotrophic hormone or angiotensin II to stimulate corticosteroid secretion. It is proposed that an important role for cardiac NPs could be in the control of hypervolaemia during periods of rapid rehydration, which occurs in terrestrial amphibians. PMID:16343494

  4. An Acanthamoeba castellanii metacaspase associates with the contractile vacuole and functions in osmoregulation.

    PubMed

    Saheb, Entsar; Trzyna, Wendy; Bush, John

    2013-03-01

    Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection. PMID:23274641

  5. Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation 1

    PubMed Central

    Laliberté, Gilles; Hellebust, Johan A.

    1989-01-01

    Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments. PMID:16667157

  6. 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

  7. 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

  8. Keratan Sulfate Biosynthesis

    PubMed Central

    Funderburgh, James L.

    2010-01-01

    Summary Keratan sulfate was originally identified as the major glycosaminoglycan of cornea but is now known to modify at least a dozen different proteins in a wide variety of tissues. Despite a large body of research documenting keratan sulfate structure, and an increasing interest in the biological functions of keratan sulfate, until recently little was known of the specific enzymes involved in keratan sulfate biosynthesis or of the molecular mechanisms that control keratan sulfate expression. In the last 2 years, however, marked progress has been achieved in identification of genes involved in keratan sulfate biosynthesis and in development of experimental conditions to study keratan sulfate secretion and control in vitro. This review summarizes current understanding of keratan sulfate structure and recent developments in understanding keratan sulfate biosynthesis. PMID:12512857

  9. 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.

  10. 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

  11. 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

  12. Mammalian cardiolipin biosynthesis.

    PubMed

    Mejia, Edgard M; Nguyen, Hieu; Hatch, Grant M

    2014-04-01

    Cardiolipin is a major phospholipid in mitochondria and is involved in the generation of cellular energy in the form of ATP. In mammalian and eukaryotic cells it is synthesized via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol phosphate pathway. This brief review will describe some of the more recent studies on mammalian cardiolipin biosynthesis and provide an overview of regulation of cardiolipin biosynthesis. In addition, the important role that this key phospholipid plays in disease processes including heart failure, diabetes, thyroid hormone disease and the genetic disease Barth Syndrome will be discussed. PMID:24144810

  13. Placental heme receptor LRP1 correlates with the heme exporter FLVCR1 and neonatal iron status.

    PubMed

    Cao, Chang; Pressman, Eva K; Cooper, Elizabeth M; Guillet, Ronnie; Westerman, Mark; O'Brien, Kimberly O

    2014-09-01

    LDL receptor-related protein 1 (LRP1) is a transmembrane receptor highly expressed in human placenta. It was recently found to be the receptor for heme and its plasma-binding protein hemopexin (Hx) and is integral to systemic heme clearance. Little is known about systemic concentrations of Hx during pregnancy and whether maternal Hx and placental LRP1 contributes to fetal iron (Fe) homeostasis during pregnancy. We hypothesized that placental LRP1 would be upregulated in maternal/neonatal Fe insufficiency and would be related to maternal circulating Hx. Placental LRP1 expression was assessed in 57 pregnant adolescents (14-18 years) in relationship with maternal and cord blood Fe status indicators (hemoglobin (Hb), serum ferritin, transferrin receptor), the Fe regulatory hormone hepcidin and serum Hx. Hx at mid-gestation correlated positively with Hb at mid-gestation (r=0.35, P=0.02) and Hx at delivery correlated positively with cord hepcidin (r=0.37, P=0.005). Placental LRP1 protein expression was significantly higher in women who exhibited greater decreases in serum Hx from mid-gestation to term (r=0.28, P=0.04). Significant associations were also found between placental LRP1 protein with cord hepcidin (r=-0.29, P=0.03) and placental heme exporter feline leukemia virus C receptor 1 (r=0.34, P=0.03). Our data are consistent with a role for placental heme Fe utilization in supporting fetal Fe demands. PMID:24947444

  14. 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

  15. Chlorophyll Deficiency in the Maize elongated mesocotyl2 Mutant Is Caused by a Defective Heme Oxygenase and Delaying Grana Stacking

    PubMed Central

    Shi, Dianyi; Zheng, Xu; Li, Liang; Lin, Wanhuang; Xie, Wenjun; Yang, Jianping; Chen, Shaojiang; Jin, Weiwei

    2013-01-01

    Background Etiolated seedlings initiate grana stacking and chlorophyll biosynthesis in parallel with the first exposure to light, during which phytochromes play an important role. Functional phytochromes are biosynthesized separately for two components. One phytochrome is biosynthesized for apoprotein and the other is biosynthesized for the chromophore that includes heme oxygenase (HO). Methodology/Principal Finding We isolated a ho1 homolog by map-based cloning of a maize elongated mesocotyl2 (elm2) mutant. cDNA sequencing of the ho1 homolog in elm2 revealed a 31 bp deletion. De-etiolation responses to red and far-red light were disrupted in elm2 seedlings, with a pronounced elongation of the mesocotyl. The endogenous HO activity in the elm2 mutant decreased remarkably. Transgenic complementation further confirmed the dysfunction in the maize ho1 gene. Moreover, non-appressed thylakoids were specifically stacked at the seedling stage in the elm2 mutant. Conclusion The 31 bp deletion in the ho1 gene resulted in a decrease in endogenous HO activity and disrupted the de-etiolation responses to red and far-red light. The specific stacking of non-appressed thylakoids suggested that the chlorophyll biosynthesis regulated by HO1 is achieved by coordinating the heme level with the regulation of grana stacking. PMID:24244620

  16. 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

  17. Relationship between natural and heme-mediated antibody polyreactivity.

    PubMed

    Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D

    2016-03-25

    Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. PMID:26926563

  18. 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...

  19. 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

  20. The heme oxygenases: important regulators of pregnancy and preeclampsia

    PubMed Central

    George, Eric M.; Warrington, Junie P.; Spradley, Frank T.; Palei, Ana C.

    2014-01-01

    The heme oxygenase system has long been believed to act largely as a housekeeping unit, converting prooxidant free heme from heme protein degradation into the benign bilirubin for conjugation and safe excretion. In recent decades, however, heme oxygenases have emerged as important regulators of cardiovascular function, largely through the production of their biologically active metabolites: carbon monoxide, bilirubin, and elemental iron. Even more recently, a number of separate lines of evidence have demonstrated an important role for the heme oxygenases in the establishment and maintenance of pregnancy. Early preclinical and clinical studies have associated defects in the heme oxygenase with the obstetrical complication preeclampsia, as well as failure to establish adequate placental blood flow, an underlying mechanism of the disorder. Several recent preclinical studies have suggested, however, that the heme oxygenase system could serve as a valuable therapeutic tool for the management of preeclampsia, which currently has few pharmacological options. This review will summarize the role of heme oxygenases in pregnancy and highlight their potential in advancing the management of patients with preeclampsia. PMID:24898840

  1. 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

  2. Extracellular Heme Uptake and the Challenges of Bacterial Cell Membranes

    PubMed Central

    Smith, Aaron D.; Wilks, Angela

    2013-01-01

    In bacteria, the fine balance of maintaining adequate iron levels while preventing the deleterious effects of excess iron has led to the evolution of sophisticated cellular mechanisms to obtain, store, and regulate iron. Iron uptake provides a significant challenge given its limited bioavailability and need to be transported across the bacterial cell wall and membranes. Pathogenic bacteria have circumvented the iron-availability issue by utilizing the hosts' heme-containing proteins as a source of iron. Once internalized, iron is liberated from the porphyrin enzymatically for cellular processes within the bacterial cell. Heme, a lipophilic and toxic molecule, poses a significant challenge in terms of transport given its chemical reactivity. As such, pathogenic bacteria have evolved sophisticated membrane transporters to coordinate, sequester, and transport heme. Recent advances in the biochemical and structural characterization of the membrane-bound heme transport proteins are discussed in the context of ligand coordination, protein–protein interaction, and heme transfer. PMID:23046657

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

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

    PubMed

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

    2015-10-01

    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. PMID:26374626

  10. Biosynthesis of Polyisoprenoids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The invention is a process for synthesis of a polymer with the same chemical structure as Natural Rubber (NR) obtained from Hevea brasiliensis and other plant species. The research collaborators recently proposed that NR biosynthesis proceeds via a carbocationic polymerization. Based on this theory...

  11. Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. OBJECTIVE: Our objectives were to produce heme iron enriched in (58)Fe and to use it to study the absorption of heme iron and the effect of iron and ...

  12. 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. PMID:19135260

  13. 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

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

    PubMed

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

    2012-02-14

    Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O(2) transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O(2) and facilitate its diffusion to the N(2)-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 Lba(m), Lbc(m), and Lbd(m), 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 O(2) binding. PMID:22308405

  15. 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

  16. 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

  17. Heme Trafficking and Modifications during System I Cytochrome c Biogenesis: Insights from Heme Redox Potentials of Ccm Proteins.

    PubMed

    Sutherland, Molly C; Rankin, Joel A; Kranz, Robert G

    2016-06-01

    Cytochromes c require covalent attachment of heme via two thioether bonds at conserved CXXCH motifs, a process accomplished in prokaryotes by eight integral membrane proteins (CcmABCDEFGH), termed System I. Heme is trafficked from inside the cell to outside (via CcmABCD) and chaperoned (holoCcmE) to the cytochrome c synthetase (CcmF/H). Purification of key System I pathway intermediates allowed the determination of heme redox potentials. The data support a model whereby heme is oxidized to form holoCcmE and subsequently reduced by CcmF/H for thioether formation, with Fe(2+) being required for attachment to CXXCH. Results provide insight into mechanisms for the oxidation and reduction of heme in vivo. PMID:27198710

  18. Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash-waters and colonization in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host-microbe interactions. Here we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and b...

  19. Bioinspired heme, heme/nonheme diiron, heme/copper, and inorganic NOx chemistry: *NO((g)) oxidation, peroxynitrite-metal chemistry, and *NO((g)) reductive coupling.

    PubMed

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

    2010-07-19

    The focus of this Forum Article 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 (NODs) and (ii) reductive coupling of two molecules of *NO((g)) to give N(2)O(g). In the former case, NODs are described, and the highlighting of possible peroxynitrite/heme intermediates and the consequences of this are given by a 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 O(2)(g), leading to peroxynitrite species, are given. The coverage of biological reductive coupling of *NO((g)) deals with bacterial nitric oxide reductases (NORs) with heme/nonheme diiron active sites and on heme/copper oxidases such as cytochrome c oxidase, which can mediate the same chemistry. Recently designed protein and synthetic model compounds (heme/nonheme/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, that 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

  20. 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/.

  1. Increase on the Initial Soluble Heme Levels in Acidic Conditions Is an Important Mechanism for Spontaneous Heme Crystallization In Vitro

    PubMed Central

    Egan, Timothy J.; Wright, David W.; Oliveira, Marcus F.

    2010-01-01

    Background Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. Methodology/Principal Findings We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. Conclusions The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels. PMID:20856937

  2. 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. PMID:26938274

  3. The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change.

    PubMed

    Pan, Jie; Zhang, Xiaoxue; Yuan, Hong; Xu, Qiming; Zhang, Huijuan; Zhou, Yajun; Huang, Zhong-Xian; Tan, Xiangshi

    2016-05-01

    Heme oxidation and loss of soluble guanylate cyclase (sGC) is thought to be an important contributor to the development of cardiovascular diseases. Nevertheless, it remains unknown why the heme loses readily in oxidized sGC. In the current study, the conformational change of sGC upon heme oxidation by ODQ was studied based on the fluorescence resonance energy transfer (FRET) between the heme and a fluorophore fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of sGC β1. This study provides an opportunity to monitor the domain movement of sGC relative to the heme. The results indicated that heme oxidation by ODQ in truncated sCC induced the heme-associated αF helix moving away from the heme, the Per/Arnt/Sim domain (PAS) domain moving closer to the heme, but led the helical domain going further from the heme. We proposed that the synergistic effect of these conformational changes of the discrete region upon heme oxidation forces the heme pocket open, and subsequent heme loss readily. Furthermore, the kinetic studies suggested that the heme oxidation was a fast process and the conformational change was a relatively slow process. The kinetics of heme loss from oxidized sGC was monitored by a new method based on the heme group de-quenching the fluorescence of FlAsH-EDT2. PMID:26876536

  4. 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

  5. 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

  6. 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

  7. Analysis of Heme Biosynthetic Pathways in a Recombinant Escherichia coli.

    PubMed

    Pranawidjaja, Stephanie; Choi, Su-In; Lay, Bibiana W; Kim, Pil

    2015-06-01

    Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. colisynthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results. PMID:25537720

  8. 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.

  9. The free heme concentration in healthy human erythrocytes

    PubMed Central

    Aich, Anupam; Freundlich, Melissa; Vekilov, Peter G.

    2016-01-01

    Heme, the prosthetic group of hemoglobin, may be released from its host due to an intrinsic instability of hemoglobin and accumulate in the erythrocytes. Free heme is in the form of hematin (Fe3+ protoporphyrin IX OH) and follows several pathways of biochemical toxicity to tissues, cells, and organelles since it catalyzes the production of reactive oxygen species. To determine concentration of soluble free heme in human erythrocytes, we develop a new method. We lyse the red blood cells and isolate free heme from hemoglobin by dialysis. We use the heme to reconstitute horseradish peroxidase (HRP) from an excess of the apoenzyme and determine the HRP reaction rate from the evolution of the emitted luminescence. We find that in a population of five healthy adults the average free heme concentration in the erythrocytes is 21 ± 2 μM, ca. 100× higher than previously determined. Tests suggest that the lower previous value was due to the use of elevated concentrations of NaCl, which drive hematin precipitation and re-association with apoglobin. We show that the found hematin concentration is significantly higher than estimates based on equilibrium release and the known hematin dimerization. The factors that lead to enhanced heme release remain an open question. PMID:26460266

  10. 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

  11. 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. PMID:19337276

  12. 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

  13. Spectroscopic Studies Reveal That the Heme Regulatory Motifs of Heme Oxygenase-2 Are Dynamically Disordered and Exhibit Redox-Dependent Interaction with Heme

    PubMed Central

    2015-01-01

    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 with a second bound heme

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

    DOE PAGESBeta

    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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Artemisinin effectiveness in erythrocyte is reduced by heme and heme-containing proteins

    PubMed Central

    Ponmee, Napawan; Chuchue, Tatsanee; Wilairat, Prapon; Yuthavong, Yongyuth; Kamchonwongpaisan, Sumalee

    2007-01-01

    Artemisinin loses its antimalarial activity on prolonged exposure to erythrocytes, especially α-thalassemic erythrocytes. In this report, we show that the major artemisinin-inactivating factor in cytosol of normal erythrocytes was heat-labile but a heat-stable factor from α-thalassemic cells also played a significant role in reducing artemisinin effectiveness, which was shown to be heme released from hemoglobin (Hb). Studies of fractionated lysate from genetically normal erythrocytes revealed that the protein fraction with molecular weight greater than 100 kDa was capable of reducing artemisinin effectiveness more readily than lower molecular weight fraction. Catalase and Hb A, but not selenoprotein glutathione peroxidase, were capable of reducing artemisinin effectiveness. Hemin (ferriprotoporphyrin IX) also reduced artemisinin effectiveness in a concentration- and time-dependent manner. It is concluded that heme and heme-containing proteins in erythrocyte are largely responsible for reducing artemisinin effectiveness and may contribute to resistance of P. falciparum infecting α-thalassemic erythrocytes observed in vitro. PMID:17498668

  20. Reaction of ferric heme proteins with nitrite and sulfite

    SciTech Connect

    Young, L.J.; Siegel, L.M.

    1988-04-19

    Optical and EPR spectroscopy of ferric heme proteins of the porphyrin, oxyporphyrin, and isobacteriochlorin classes has indicated that nitrite reacts with these proteins at the heme iron. Sulfite has been conclusively proven to react only with proteins containing the isobacteriochlorin macrocycle. Quantitative EPR spectroscopy of these nitrite and sulfite adducts showed that most contained a substantial quantity of undetectable heme. It is suggested that protein-induced autoreduction of nitrite (but not sulfite) and a strained and/or uniaxial g-tensor are the principal ways by which the silent state is produced.

  1. Structural Basis for the Transcriptional Regulation of Heme Homeostasis in Lactococcus lactis*

    PubMed Central

    Sawai, Hitomi; Yamanaka, Masaru; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Aono, Shigetoshi

    2012-01-01

    Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtR·DNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 Å, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-π interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the α4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the α4 helix upon heme sensing. PMID:22798069

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

    PubMed Central

    Martin, Emil; Lee, Yu-Chen; Murad, Ferid

    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. PMID:11687640

  3. 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.

  4. Ultrafast transient absorption measurements of heme proteins

    NASA Astrophysics Data System (ADS)

    Ye, Xiong; Demidov, Andrey; Wang, Wei; Christian, James; Champion, Paul

    1998-03-01

    Transient absorption spectra reveal the dynamics and intermediate states of the heme active site after ligand photodissociation, which helps clarify the physical process of ligand dissociation and geminate recombination. To measure the transient absorption spectra, we apply a femtosecond pump-probe technique with frequency resolved detection using a multichannel diode array. The femtosecond pulse output from a regenerative laser amplifier system is split in two; one beam pumps the optical parametric amplifier to produce a tunable wavelength pump pulse, the other beam generates a white light continuum that is varied in time with respect to pump pulse and probe the transient absorbance of the sample. We make a comparative study of myoglobin with different ligands, mutants and pH conditions.

  5. Introduction of a covalent histidine–heme linkage in a hemoglobin: A promising tool for heme protein engineering1

    PubMed Central

    Rice, Selena L.; Preimesberger, Matthew R.; Johnson, Eric A.; Lecomte, Juliette T. J.; Jenkins, T. C.

    2014-01-01

    The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine–heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (> 90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl in a 60% yield, indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His–heme modification will be applicable to the design of proteins with a non-dissociable heme group. PMID:25304367

  6. 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.

  7. Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

    PubMed Central

    Vallelian, F; Deuel, J W; Opitz, L; Schaer, C A; Puglia, M; Lönn, M; Engelsberger, W; Schauer, S; Karnaukhova, E; Spahn, D R; Stocker, R; Buehler, P W; Schaer, D J

    2015-01-01

    Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death. PMID:25301065

  8. 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. PMID:24782769

  9. 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

  10. Ligand preference and orientation in b- and c-type heme-binding proteins

    PubMed Central

    Fufezan, Christian; Zhang, Jun; Gunner, M. R.

    2009-01-01

    Hemes are often incorporated into designed proteins. The importance of the heme ligand type and its orientation is still a matter of debate. Here, heme ligands and ligand orientation were investigated using a nonredundant (87 structures) and a redundant (1503 structures) set of structures to compare and contrast design features of natural b- and c-type heme-binding proteins. Histidine is the most common ligand. Marked differences in ligation motifs between b- and c-type hemes are higher occurrence of His-Met in c-type heme binding motifs (16.4% vs. 1.4%) and higher occurrence of exchangeable, small molecules in b-type heme binding motifs (67.6% vs. 9.9%). Histidine ligands that are part of the c-type CXXCH heme-binding motif show a distinct asymmetric distribution of orientation. They tend to point between either the heme propionates or between the NA and NB heme nitrogens. Molecular mechanics calculations show that this asymmetry is due to the bonded constraints of the covalent attachment between the heme and the protein. In contrast, the orientations of b-type hemes histidine ligands are found evenly distributed with no preference. Observed histidine heme ligand orientations show no dominating influence of electrostatic interactions between the heme propionates and the ligands. Furthermore, ligands in bis-His hemes are found more frequently perpendicular rather than parallel to each other. These correlations support energetic constraints on ligands that can be used in designing proteins. PMID:18491383

  11. 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

  12. Carbon monoxide-driven reduction of ferric heme and heme proteins.

    PubMed

    Bickar, D; Bonaventura, C; Bonaventura, J

    1984-09-10

    Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to

  13. 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

  14. Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.

    PubMed

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

    2008-01-01

    The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix. PMID:18078349

  15. Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

    PubMed

    Taylor, J R; Mager, E M; Grosell, M

    2010-02-01

    Although endogenous CO2 hydration and serosal HCO3- are both known to contribute to the high rates of intestinal HCO3- secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3- secretion is accomplished has received little attention. Isolated intestine HCO3- secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3- concentration and sensitive to serosal DIDS. Elevated mucosal Cl- concentration had the unexpected effect of reducing HCO3- secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3- secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3- in the physiological range (Km=10.2 mmol l(-1)), indicating a potential to efficiently fine-tune systemic acid-base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3- cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3- (Km=8.5 mmol l(-1)). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3- rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3- secretion rates of the marine fish intestine. PMID:20086131

  16. 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.

  17. Heme: Modulator of Plasma Systems in Hemolytic Diseases.

    PubMed

    Roumenina, Lubka T; Rayes, Julie; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D

    2016-03-01

    Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal. PMID:26875449

  18. 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

  19. Heme and blood-feeding parasites: friends or foes?

    PubMed Central

    2010-01-01

    Hemoparasites, like malaria and schistosomes, are constantly faced with the challenges of storing and detoxifying large quantities of heme, released from their catabolism of host erythrocytes. Heme is an essential prosthetic group that forms the reactive core of numerous hemoproteins with diverse biological functions. However, due to its reactive nature, it is also a potentially toxic molecule. Thus, the acquisition and detoxification of heme is likely to be paramount for the survival and establishment of parasitism. Understanding the underlying mechanism involved in this interaction could possibly provide potential novel targets for drug and vaccine development, and disease treatment. However, there remains a wide gap in our understanding of these mechanisms. This review summarizes the biological importance of heme for hemoparasite, and the adaptations utilized in its sequestration and detoxification. PMID:21087517

  20. Primary processes in heme-based sensor proteins.

    PubMed

    Liebl, Ursula; Lambry, Jean-Christophe; Vos, Marten H

    2013-09-01

    A wide and still rapidly increasing range of heme-based sensor proteins has been discovered over the last two decades. At the molecular level, these proteins function as bistable switches in which the catalytic activity of an enzymatic domain is altered mostly by binding or dissociation of small gaseous ligands (O2, NO or CO) to the heme in a sensor domain. The initial "signal" at the heme level is subsequently transmitted within the protein to the catalytic site, ultimately leading to adapted expression levels of specific proteins. Making use of the photolability of the heme-ligand bond that mimics thermal dissociation, early processes in this intra-protein signaling pathway can be followed using ultrafast optical spectroscopic techniques; they also occur on timescales accessible to molecular dynamics simulations. Experimental studies performed over the last decade on proteins including the sensors FixL (O2), CooA (CO) and soluble guanylate cyclase (NO) are reviewed with an emphasis on emerging general mechanisms. After heme-ligand bond breaking, the ligand can escape from the heme pocket and eventually from the protein, or rebind directly to the heme. Remarkably, in all sensor proteins the rebinding, specifically of the sensed ligand, is highly efficient. This "ligand trap" property possibly provides means to smoothen the effects of fast environmental fluctuations on the switching frequency. For 6-coordinate proteins, where exchange between an internal heme-bound residue and external gaseous ligands occurs, the study of early processes starting from the unliganded form indicates that mobility of the internal ligand may facilitate signal transfer. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. PMID:23485911

  1. Hal Is a Bacillus anthracis heme acquisition protein.

    PubMed

    Balderas, Miriam A; Nobles, Christopher L; Honsa, Erin S; Alicki, Embriette R; Maresso, Anthony W

    2012-10-01

    The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

  2. Heme-based Sensing by the Mammalian Circadian Protein, CLOCK

    PubMed Central

    Lukat-Rodgers, Gudrun S.; Correia, Cristina; Botuyan, Maria Victoria; Mer, Georges; Rodgers, Kenton R.

    2010-01-01

    Heme is emerging as a key player in the synchrony of circadian-coupled transcriptional regulation. Current evidence suggests that levels of circadian-linked transcription are regulated in response to both the availability of intracellular heme and by heme-based sensing of carbon monoxide and possibly nitric oxide. The protein CLOCK is central to the regulation and maintenance of circadian rhythms in mammals. CLOCK comprises two PAS domains, each with a heme binding site. Our studies focus on the functionality of the Murine CLOCK PAS–A domain (residues 103-265). We show that CLOCK PAS–A binds Fe(III) protoporhyrin IX to form a complex with 1:1 stoichiometry. Optical absorbance and resonance Raman studies reveal that the heme of ferric CLOCK PAS–A is a six-coordinate, low spin complex whose resonance Raman signature is insensitive to pH over the range of protein stability. Ferrous CLOCK PAS–A is a mixture of five-coordinate, high spin and six-coordinate, low spin complexes. Ferrous CLOCK PAS–A forms complexes with CO and NO. Ferric CLOCK PAS–A undergoes reductive nitrosylation in the presence of NO to generate a CLOCK PAS–A–NO, which is a pentacoordinate {FeNO}7 complex. Formation of the highly stable {FeNO}7 heme complex from either ferrous or ferric heme makes possible the binding of NO at very low concentration, a characteristic of NO sensors. Comparison of the spectroscopic properties and CO binding kinetics of CLOCK PAS–A with other CO sensor proteins reveals that CLOCK PAS–A exhibits chemical properties consistent with a heme-based gas sensor protein. PMID:20666392

  3. 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

  4. 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. PMID:19694439

  5. HmuS and HmuQ of Ensifer/Sinorhizobium meliloti degrade heme in vitro and participate in heme metabolism in vivo.

    PubMed

    Amarelle, Vanesa; Rosconi, Federico; Lázaro-Martínez, Juan Manuel; Buldain, Graciela; Noya, Francisco; O'Brian, Mark R; Fabiano, Elena

    2016-04-01

    Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo. PMID:26906560

  6. A heme fusion tag for protein affinity purification and quantification

    PubMed Central

    Asher, Wesley B; Bren, Kara L

    2010-01-01

    We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an l-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination. PMID:20665691

  7. Carnitine biosynthesis in mammals.

    PubMed Central

    Vaz, Frédéric M; Wanders, Ronald J A

    2002-01-01

    Carnitine is indispensable for energy metabolism, since it enables activated fatty acids to enter the mitochondria, where they are broken down via beta-oxidation. Carnitine is probably present in all animal species, and in numerous micro-organisms and plants. In mammals, carnitine homoeostasis is maintained by endogenous synthesis, absorption from dietary sources and efficient tubular reabsorption by the kidney. This review aims to cover the current knowledge of the enzymological, molecular, metabolic and regulatory aspects of mammalian carnitine biosynthesis, with an emphasis on the human and rat. PMID:11802770

  8. Binding and storage of heme by vitellin from the cattle tick, Boophilus microplus.

    PubMed

    Logullo, C; Moraes, J; Dansa-Petretski, M; Vaz, I S; Masuda, A; Sorgine, M H F; Braz, G R; Masuda, H; Oliveira, P L

    2002-12-01

    We have previously shown (, Curr. Biol. 9, 703-706) that the cattle tick Boophilus microplus does not synthesize heme, relying solely on the recovery of the heme from the diet to make all its hemeproteins. Here we present evidence that Vitellin (VN(1)), the main tick yolk protein, is a reservoir of heme for embryo development. VN was isolated from eggs at different days throughout embryogenesis. Immediately after oviposition, Boophilus VN contains approximately one mol of heme/mol of protein. During embryo development about one third of egg VN is degraded. The remaining VN molecules bind part of the heme released. These results suggest that VN functions as a heme reservoir, binding any free heme that exceeds the amount needed for development. In vitro measurement of the binding of heme to VN showed that each VN molecule binds up to 31 heme molecules. The association of heme with VN strongly inhibits heme-induced lipid peroxidation, suggesting that binding of heme is an important antioxidant mechanism to protect embryo cells from oxidative damage. This mechanism allows this hematophagous arthropod to safely store heme obtained from a blood meal inside their eggs for future use. Taken together our data suggest that, besides its known roles, VN also plays additional functions as a heme deposit and an antioxidant protective molecule. PMID:12429132

  9. 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

  10. Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

    PubMed Central

    Davison, Jack; al Fahad, Ahmed; Cai, Menghao; Song, Zhongshu; Yehia, Samar Y.; Lazarus, Colin M.; Bailey, Andrew M.; Simpson, Thomas J.; Cox, Russell J.

    2012-01-01

    A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus (Penicillium stipitatum) and investigated by targeted gene knockout. A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group. The tropA gene was characterized by heterologous expression in Aspergillus oryzae, whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro. Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group. Comparison of the T. stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone. PMID:22508998

  11. Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury.

    PubMed

    Origassa, Clarice Silvia Taemi; Câmara, Niels Olsen Saraiva

    2013-10-27

    The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury. PMID:24179613

  12. Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin

    PubMed Central

    Müllebner, Andrea; Moldzio, Rudolf; Redl, Heinz; Kozlov, Andrey V.; Duvigneau, J. Catharina

    2015-01-01

    Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have

  13. 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. PMID:25795718

  14. Terpene Biosynthesis: Modularity Rules

    PubMed Central

    Oldfield, Eric; Lin, Fu-Yang

    2013-01-01

    Terpenes are the largest class of small molecule natural products on Earth, and the most abundant by mass. Here, we summarize recent developments in elucidating the structure and function of the proteins involved in their biosynthesis. There are 6 main building blocks or modules (α,β,γ,δ,ε and ζ) that make up the structures of these enzymes: the αα and αδ head-to-tail trans-prenyl transferases that produce trans-isoprenoid diphosphates from C5 precursors; the ε head-to-head prenyl transferases that convert these diphosphates into the tri-and tetra-terpene precursors of sterols, hopanoids and carotenoids; the βγ di- and tri-terpene synthases; the ζ head-to-tail cis-prenyl transferases that produce the cis-isoprenoid diphosphates involved in bacterial cell wall biosynthesis, and finally the α, αβ and αβγ terpene synthases that produce plant terpenes, with many of these modular enzymes having originated from ancestral α and β domain proteins. We also review progress in determining the structure and function of the two 4Fe-4S reductases involved in formation of the C5 diphosphates in many bacteria, where again, highly modular structures are found. PMID:22105807

  15. Taxol biosynthesis: an update.

    PubMed

    Hezari, M; Croteau, R

    1997-08-01

    The novel diterpenoid taxol (paclitaxel) is now well-established as a potent chemotherapeutic agent. Total synthesis of the drug is not commercially feasible and, in the foreseeable future, the supply of taxol and its synthetically useful progenitors must rely on biological methods of production. The first three steps of taxol biosynthesis have been defined and the responsible enzymes described. These are the cyclization of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, the cytochrome P450-catalyzed hydroxylation of this olefin to taxa-4(20), 11(12)-dien-5 alpha-ol, and the acetyl CoA-dependent conversion of the alcohol to the corresponding acetate ester. Demonstration of these early steps of taxol biosynthesis suggests that the complete pathway can be defined by a systematic, stepwise approach at the cell-free enzyme level. When combined with in vivo studies to determine contribution to pathway flux, slow steps can be targeted for gene isolation and subsequent overexpression in Taxus to improve the yield of taxol and related compounds. PMID:9270370

  16. 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

  17. Heme-nitrosyls: electronic structure implications for function in biology.

    PubMed

    Hunt, Andrew P; Lehnert, Nicolai

    2015-07-21

    The question of why mammalian systems use nitric oxide (NO), a potentially hazardous and toxic diatomic, as a signaling molecule to mediate important functions such as vasodilation (blood pressure control) and nerve signal transduction initially perplexed researchers when this discovery was made in the 1980s. Through extensive research over the past two decades, it is now well rationalized why NO is used in vivo for these signaling functions, and that heme proteins play a dominant role in NO signaling in mammals. Key insight into the properties of heme-nitrosyl complexes that make heme proteins so well poised to take full advantage of the unique properties of NO has come from in-depth structural, spectroscopic, and theoretical studies on ferrous and ferric heme-nitrosyls. This Account highlights recent findings that have led to greater understanding of the electronic structures of heme-nitrosyls, and the contributions that model complex studies have made to elucidate Fe-NO bonding are highlighted. These results are then discussed in the context of the biological functions of heme-nitrosyls, in particular in soluble guanylate cyclase (sGC; NO signaling), nitrophorins (NO transport), and NO-producing enzymes. Central to this Account is the thermodynamic σ-trans effect of NO, and how this relates to the activation of the universal mammalian NO sensor sGC, which uses a ferrous heme as the high affinity "NO detection unit". It is shown via detailed spectroscopic and computational studies that the strong and very covalent Fe(II)-NO σ-bond is at the heart of the strong thermodynamic σ-trans effect of NO, which greatly weakens the proximal Fe-NHis (or Fe-SCys) bond in six-coordinate ferrous heme-nitrosyls. In sGC, this causes the dissociation of the proximally bound histidine ligand upon NO binding to the ferrous heme, inducing a significant conformational change that activates the sGC catalytic domain for the production of cGMP. This, in turn, leads to vasodilation and

  18. 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. PMID:26827851

  19. 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

  20. Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV.

    PubMed

    Lambry, Jean-Christophe; Stranava, Martin; Lobato, Laura; Martinkova, Marketa; Shimizu, Toru; Liebl, Ursula; Vos, Marten H

    2016-01-01

    An important question for the functioning of heme proteins is whether different ligands present within the protein moiety can readily exchange with heme-bound ligands. Studying the dynamics of the heme domain of the Escherichia coli sensor protein YddV upon dissociation of NO from the ferric heme by ultrafast spectroscopy, we demonstrate that when the hydrophobic leucine residue in the distal heme pocket is mutated to glycine, in a substantial fraction of the protein water replaces NO as an internal ligand in as fast as ∼4 ps. This process, which is near-barrierless and occurs orders of magnitude faster than the corresponding process in myoglobin, corresponds to a ligand swap of NO with a water molecule present in the heme pocket, as corroborated by molecular dynamics simulations. Our findings provide important new insight into ligand exchange in heme proteins that functionally interact with different external ligands. PMID:26651267

  1. Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry

    SciTech Connect

    Yang, Feng; Bogdanov, Bogdan; Strittmatter, Eric F.; Vilkov, Andrey N.; Gritsenko, Marina A.; Shi, Liang; Elias, Dwayne A.; Ni, Shuisong; Romine, Margaret F.; Pasa-Tolic, Liljiana; Lipton, Mary S.; Smith, Richard D.

    2005-05-01

    We describe methods for mass spectrometric identification of heme-containing peptides from digests of c-type cytochromes that contain the CXXCH(X = any amino acid) sequence motif. Analysis of purified standard heme-containing peptides showed that the charged heme group was present both before and after peptide fragmentation in the gas phase. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference (DAA=1) for both 2+ and 3+ peptide charge states and the extent of heme loss during peptide fragmentation was affected by both sequence and charge. A modified search strategy was evaluated with tryptic digests of one known and two unknown cytochromes from Shewanella oneidenis, demonstrating that this approach can be generally applied for identification of c-type heme-containing peptides from complex samples.

  2. A Ferric-Peroxo Intermediate in the Oxidation of Heme by IsdI.

    PubMed

    Takayama, Shin-Ichi J; Loutet, Slade A; Mauk, A Grant; Murphy, Michael E P

    2015-04-28

    The canonical heme oxygenases (HOs) catalyze heme oxidation via a heme-bound hydroperoxo intermediate that is stabilized by a water cluster at the active site of the enzyme. In contrast, the hydrophobic active site of IsdI, a heme-degrading enzyme from Staphylococcus aureus, lacks a water cluster and is expected to oxidize heme by an alternative mechanism. Reaction of the IsdI-heme complex with either H2O2 or m-chloroperoxybenzoic acid fails to produce a specific oxidized heme iron intermediate, suggesting that ferric-hydroperoxo or ferryl derivatives of IsdI are not involved in the catalytic mechanism of this enzyme. IsdI lacks a proton-donating group in the distal heme pocket, so the possible involvement of a ferric-peroxo intermediate has been evaluated. Density functional theory (DFT) calculations indicate that heme oxidation involving a ferric-peroxo intermediate is energetically accessible, whereas the energy barrier for a reaction involving a ferric-hydroperoxo intermediate is too great in the absence of a proton donor. We propose that IsdI catalyzes heme oxidation through nucleophilic attack by the heme-bound peroxo species. This proposal is consistent with our previous demonstration by nuclear magnetic resonance spectroscopy that heme ruffling increases the susceptibility of the meso-carbon of heme to nucleophilic attack. PMID:25853501

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

    PubMed

    Cunnington, Aubrey J; de Souza, J Brian; Walther, Michael; Riley, Eleanor M

    2012-01-01

    In sub-Saharan Africa, invasive nontyphoid Salmonella (NTS) infection 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 Salmonella enterica serovar Typhimurium 12023 (Salmonella typhimurium) causes acute, fatal bacteremia with high bacterial load, features reproduced by phenylhydrazine-induced 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

  4. 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

  5. Thermodynamic investigation into the mechanisms of proton-coupled electron transfer events in heme protein maquettes.

    PubMed

    Reddi, Amit R; Reedy, Charles J; Mui, Steven; Gibney, Brian R

    2007-01-01

    To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[delta7-His]2, are four-alpha-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Delta7-His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [delta7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[delta7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, <3.0 kcal/mol. The 1H+/1e- redox-Bohr effect of heme b-[H10A24]2 is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H+/1e- redox-Bohr effect observed for heme b-[delta7-His]2 is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These

  6. Biosynthesis of cylindrospermopsin.

    PubMed

    Burgoyne, D L; Hemscheidt, T K; Moore, R E; Runnegar, M T

    2000-01-14

    Studies on the biosynthesis of cylindrospermopsin (1), a potent hepatotoxin associated with the cyanobacterium Cylindrospermopsis raciborskii, indicate that 1 is an acetogenin with guanidinoacetic acid serving as the starter unit of the polyketide chain. Feeding experiments show that C14 and C15 of 1 are derived from C1 and C2 of glycine, respectively, and C4 through C13 arise from five contiguous acetate units attached head to tail. The methyl carbon on C13 originates from the C(1) pool. The starter unit, established by the incorporation of [guanidino-(13)C,alpha-(15)N]-guanidinoacetic acid into N16 and C17 of 1, does not appear to be formed from glycine by known amidination pathways. The origin of the NH-CO-NH segment in the uracil ring is also unknown. PMID:10813909

  7. Biosynthesis of plant sulfolipids

    SciTech Connect

    Kleppinger-Sparace, K.; Mudd, J.B.; Sparace, S. )

    1989-04-01

    The complete biosynthesis of sulfoquinovosyldiacylglycerol (SQDG) remains undetermined although dark synthesis of SQDG by chloroplasts supplied with AP{sup 35}S, PAP{sup 35}S or {sup 35}SO{sub 4} plus ATP suggests the sulfur moiety arises from either APS or sulfite (1). Sulfate incorporation into sulfolipids in isolated chloroplasts and in intact roots is reported here and compared to lipids labelled by {sup 14}C-acetate or {sup 14}C-glycerol. Several unknown {sup 35}S-labelled chloroform-soluble compounds were isolated from sterile roots. These {sup 35}S-labelled compounds differ from those of the chloroplast, identified as elemental sulfur forms. Identification of the unknown root compounds is in progress. Unlike chloroplast, isolated root plastids do not synthesis SQDG from sulfate plus ATP suggesting a requirement for an activated form of sulfate, such as APS or PAPS.

  8. Designer microbes for biosynthesis

    PubMed Central

    Quin, Maureen B.; Schmidt-Dannert, Claudia

    2014-01-01

    Microbes have long been adapted for the biosynthetic production of useful compounds. There is increasing demand for the rapid and cheap microbial production of diverse molecules in an industrial setting. Microbes can now be designed and engineered for a particular biosynthetic purpose, thanks to recent developments in genome sequencing, metabolic engineering, and synthetic biology. Advanced tools exist for the genetic manipulation of microbes to create novel metabolic circuits, making new products accessible. Metabolic processes can be optimized to increase yield and balance pathway flux. Progress is being made towards the design and creation of fully synthetic microbes for biosynthetic purposes. Together, these emerging technologies will facilitate the production of designer microbes for biosynthesis. PMID:24646570

  9. The nature of heme/iron-induced protein tyrosine nitration

    PubMed Central

    Bian, Ka; Gao, Zhonghong; Weisbrodt, Norman; Murad, Ferid

    2003-01-01

    Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H2O2 to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO2-Tyr formation. The data from our study indicate that: (i) heme/iron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H2O2, and NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document}; (ii) H2O2 plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H2O2, and the NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage

  10. Coupling Heme and Iron Metabolism via Ferritin H Chain

    PubMed Central

    2014-01-01

    Abstract Significance: Inflammation and immunity can be associated with varying degrees of heme release from hemoproteins, eventually leading to cellular and tissue iron (Fe) overload, oxidative stress, and tissue damage. Presumably, these deleterious effects contribute to the pathogenesis of systemic infections. Recent Advances: Heme release from hemoglobin sensitizes parenchyma cells to undergo programmed cell death in response to proinflammatory cytokines, such as tumor necrosis factor. This cytotoxic effect is driven by a mechanism involving intracellular accumulation of free radicals, which sustain the activation of the c-Jun N-terminal kinase (JNK) signaling transduction pathway. While heme catabolism by heme oxygenase-1 (HO-1) prevents programmed cell death, this cytoprotective effect requires the co-expression of ferritin H (heart/heavy) chain (FTH), which controls the pro-oxidant effect of labile Fe released from the protoporphyrin IX ring of heme. This antioxidant effect of FTH restrains JNK activation, whereas JNK activation inhibits FTH expression, a cross talk that controls metabolic adaptation to cellular Fe overload associated with systemic infections. Critical Issues and Future Directions: Identification and characterization of the mechanisms via which FTH provides metabolic adaptation to tissue Fe overload should provide valuable information to our current understanding of the pathogenesis of systemic infections as well as other immune-mediated inflammatory diseases. Antioxid. Redox Signal. 20, 1754–1769. PMID:24124891

  11. [Heme oxygenase activity in rat organs during cadmium chloride administration].

    PubMed

    Strel'chenko, E V; Nikitchenko, I V; Kaliman, P A

    2002-01-01

    Heme oxygenase activity, the level of spontaneous and ascorbat-induced LPO in the liver, kidney and spleen homogenates of rats and blood serum absorption spectrum in the Soret region in different periods both after CdCl2 and prior alpha-tocopherol administration were studied. The increase in the hemolysis products content in the serum was observed in 15 min after CdCl2 injection and remained during 24 h. Heme oxygenase activity in the liver and kidney increased after 6 h and stayed at the same level 24 h after CdCl2 administration. The level of spontaneous LPO in the spleen increased after 6 h, and in the liver and kidney the level of spontaneous and ascorbat-induced LPO increased in 24 h after CdCl2 injection. The preliminary alpha-tocopherol administration did not prevent the accumulation of hemolysis products in the serum and the increase of heme oxygenase activity in the liver and kidney caused by CdCl2 administration. However, the increase in the ascorbat-induced LPO in these organs was completely blocked. The role of heme and LPO in the heme oxygenase induction by CdCl2 are discussed. PMID:12916165

  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. Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

    PubMed Central

    Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

    1997-01-01

    One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

  14. Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

    PubMed

    Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

    1997-08-01

    One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

  15. 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...

  16. [Metabolism of heme and hemoproteins in rat liver upon administration of mercuric chloride].

    PubMed

    Kaliman, P A; Nikitchenko, I V; Barannik, T V; Sokol, O A

    1999-01-01

    Rat liver delta-aminolevulinate synthase (delta-ALAS) activity in the early period after mercury chloride administration (0.7 mg per 100 g body weight) was found to be followed by free heme level increase, which was registered by the increase of heme saturation of the heme-binding protein tryptophan-2,3-dioxygenase (T-2,3-DO). delta-ALAS and heme oxygenase activity increase was observed 24 h after action. Microsomal cytochromes P450 and b5 levels decrease. Heme saturation of the T-2,3-DO returned to control level. Heme oxygenase and T-2,3-DO induction promoted hepatocytes free heme level normalization. Heme oxygenase and delta-ALAS induction role in the liver cells defense from the oxidative damage is discussed. PMID:10820853

  17. Heme oxygenase metabolites inhibit tubuloglomerular feedback (TGF).

    PubMed

    Ren, YiLin; D'Ambrosio, Martin A; Wang, Hong; Liu, Ruisheng; Garvin, Jeffrey L; Carretero, Oscar A

    2008-10-01

    Tubuloglomerular feedback (TGF) is the mechanism by which the macula densa (MD) senses increases in luminal NaCl concentration and sends a signal to constrict the afferent arteriole (Af-Art). The kidney expresses constitutively heme oxygenase-2 (HO-2) and low levels of HO-1. HOs release carbon monoxide (CO), biliverdin, and free iron. We hypothesized that renal HOs inhibit TGF via release of CO and biliverdin. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the MD perfusate. When HO activity was inhibited by adding stannous mesoporphyrin (SnMP) to the MD perfusate, the TGF response increased from 2.1+/-0.2 to 4.1+/-0.4 microm (P=0.003, control vs. SnMP, n=7). When a CO-releasing molecule, (CORM-3; 50 microM), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6+/-0.3 to 2.1+/-0.2 microm (P<0.001, control vs. CORM-3, n=12). When CORM-3 at 100 microM was added to the perfusate, it completely blocked the TGF response, from 4.2+/-0.4 to -0.2+/-0.3 microm (P<0.001, control vs. CORM-3, n=6). When biliverdin was added to the perfusate, the TGF response decreased by 79%, from 3.4+/-0.3 to 0.7+/-0.4 microm (P=0.001, control vs. biliverdin, n=6). The effects of SnMP and CORM-3 were not blocked by inhibition of nitric oxide synthase. We concluded that renal HO inhibits TGF probably via release of CO and biliverdin. HO regulation of TGF is a novel mechanism that could lead to a better understanding of the control of renal microcirculation and function. PMID:18715939

  18. 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. PMID:26963649

  19. 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

  20. 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

  1. NMR studies of osmoregulation in methanogenic archaebacteria. [NMR (nuclear magnetic resonance)

    SciTech Connect

    Robertson, D.E.

    1991-01-01

    Methanogens are strict anaerobic archaebacteria whose metabolism centers around the reduction of CO[sub 2] to CH[sub 4]. Their environments are often extreme (high temperatures, high salt, few nutrients, etc.) and they may have evolved unique ways to handle these stresses. It is proposed that methanogenic archaebacteria respond to osmotic stress by accumulating a series of organic solutes. In two strains of marine methanogens, Methanogenium cariaci and Methanococcus thermolithotrophicus, four key organic solutes are observed: L-[alpha]-glutamate, [beta]-glutamate, N[sup e]-acetyl-[beta]-lysine, and glycine betaine. The first three of these are synthesized de novo; glycine betaine is transported into the Mg. cariaci cells from the medium. In the absence of betaine, Mg. cariaci synthesizes N[sup e]-acetyl-[beta]-lysine as the dominant osmolyte. Mc. thermolithotrophicus also synthesizes N[sup e]-acetyl-[beta]-lysine but only at salt concentrations greater than 1 M. In Mc. thermolithotrophicus intracellular potassium ion concentrations, determined by [sup 39]K NMR spectroscopy, are balanced by the total concentration of anionic amino acid species, [alpha]-glutamate and [beta]-glutamate. Turnover of the organic solutes has been monitored using [sup 13]C-pulse/[sup 12]C-chase, and [sup 15]N-pulse/[sup 14]N-chase experiments. The [beta]-amino acids exhibit slower turnover rates compared to L-[alpha]-glutamate or aspartate, consistent with their role as compatible solutes. Biosynthetic information for the [beta]-amino acids was provided by [sup 13]C-label incorporation and steady state labeling experiments. [beta]-glutamate shows a lag in [sup 13]C uptake from [sup 13]CO[sub 2], indicative of its biosynthesis from a precursor not in equilibrium with the soluble L-[alpha]-glutamate pool, probably a macromolecule. A novel biosynthetic pathway is proposed for N[sup e]-acetyl-[beta]-lysine from the diaminopimelate pathway.

  2. 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

  3. 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. PMID:15297453

  4. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... evacuation service (HEMES). 135.271 Section 135.271 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... 24-consecutive hour period of a HEMES assignment, unless an emergency medical evacuation operation...

  5. Over-expression of Multi-heme C-type Cytochromes

    SciTech Connect

    Shi, Liang; Lin, Chiann Tso; Markillie, Lye Meng; Squier, Thomas C.; Hooker, Brian S.

    2005-02-01

    ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

  6. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... evacuation service (HEMES). 135.271 Section 135.271 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... 24-consecutive hour period of a HEMES assignment, unless an emergency medical evacuation operation...

  7. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... evacuation service (HEMES). 135.271 Section 135.271 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... 24-consecutive hour period of a HEMES assignment, unless an emergency medical evacuation operation...

  8. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... evacuation service (HEMES). 135.271 Section 135.271 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... 24-consecutive hour period of a HEMES assignment, unless an emergency medical evacuation operation...

  9. 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

  10. Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production.

    PubMed

    Fortes, Guilherme B; Alves, Leticia S; de Oliveira, Rosane; Dutra, Fabianno F; Rodrigues, Danielle; Fernandez, Patricia L; Souto-Padron, Thais; De Rosa, María José; Kelliher, Michelle; Golenbock, Douglas; Chan, Francis K M; Bozza, Marcelo T

    2012-03-01

    Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4(-/-) or to Myd88(-/-) macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1(-/-)) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS. PMID:22262768

  11. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... evacuation service (HEMES). 135.271 Section 135.271 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... 24-consecutive hour period of a HEMES assignment, unless an emergency medical evacuation operation...

  12. 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...

  13. Crystal structure of the Pseudomonas aeruginosa cytoplasmic heme binding protein, Apo-PhuS.

    PubMed

    Tripathi, Sarvind; O'Neill, Maura J; Wilks, Angela; Poulos, Thomas L

    2013-11-01

    Iron is an essential element to all living organisms and is an important determinant of bacterial virulence. Bacteria have evolved specialized systems to sequester and transport iron from the environment or host. Pseudomonas aeruginosa, an opportunistic pathogen, uses two outer membrane receptor mediated systems (Phu and Has) to utilize host heme as a source of iron. PhuS is a 39 kDa soluble cytoplasmic heme binding protein which interacts and transports heme from the inner membrane heme transporter to the cytoplasm where it is degraded by heme oxygenase thus releasing iron. PhuS is unique among other cytoplasmic heme transporter proteins owing to the presence of three histidines in the heme binding pocket which can potentially serve as heme ligands. Out of the three histidine residues on the heme binding helix, His 209 is conserved among heme trafficking proteins while His 210 and His 212 are unique to PhuS. Here we report the crystal structure of PhuS at 1.98Å resolution which shows a unique heme binding pocket and oligomeric structure compared to other known cytoplasmic heme transporter and accounts for some of the unusual biochemical properties of PhuS. PMID:23973453

  14. 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. PMID:24754676

  15. Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation.

    PubMed

    Maringer, Katherine; Yarbrough, Azure; Sims-Lucas, Sunder; Saheb, Entsar; Jawed, Sanaa; Bush, John

    2016-06-01

    Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80 percent homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium. PMID:27240981

  16. C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain

    PubMed Central

    Soloviev, Alexander; Gallagher, Joseph; Marnef, Aline; Kuwabara, Patricia E.

    2011-01-01

    The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins. PMID:21215260

  17. De novo assembly and transcriptome analysis of osmoregulation in Litopenaeus vannamei under three cultivated conditions with different salinities.

    PubMed

    Zhang, Dan; Wang, Fang; Dong, Shuanglin; Lu, Yunliang

    2016-03-10

    Litopenaeus vannamei, one of the most important euryhaline crustaceans, is cultured in seawater, brackish water, and freshwater worldwide. We performed Illumina RNA sequencing of L. vannamei gills, generating 124,914,870; 119,250,450; and 105,487,350 raw reads from the shrimps cultured in seawater, brackish water, and freshwater, respectively. From these reads, 466,293 transcripts were de novo assembled and annotated. Comparative genomic analysis showed that 1752 genes were significantly differentially expressed in the freshwater group compared with the seawater group, including 1242 upregulated and 510 downregulated genes. In addition, 1246 genes were differentially expressed in the brackish group vs. the seawater water group, including 659 upregulated and 587 downregulated genes. These differentially expressed genes were mainly involved in energy metabolism, substance metabolism, ion transport and signal transduction, and genetic process. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to analyze the functional significance of the differentially expressed genes, included those responding to salinity through diverse biological functions and processes and numerous potential genes associated with the osmotic response. L. vannamei responses to the three cultivated salinities were analyzed using next-generation sequencing. The transcriptional database established from the current research adds to the information available on L. vannamei and the findings expand our knowledge of the molecular basis of osmoregulation mechanisms in this species. PMID:26691500

  18. Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation.

    PubMed

    Grosell, M; Genz, J

    2006-10-01

    The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO3- via apical anion exchange to serve Cl- and water absorption for osmoregulatory purposes. Luminal HCO3- secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25 degrees C under in vivo-like conditions, secretion rates averaged 0.45 micromol x cm(-2) x h(-1), of which 0.25 micromol x cm(-2) x h(-1) can be accounted for by hydration of endogenous CO2 partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO3- and H+ arising from the CO2 hydration reaction is evident from equal rates of luminal HCO3- secretion via anion exchange and basolateral H+ extrusion. When basolateral H+ extrusion is partly inhibited by reduction of serosal pH, luminal HCO3- secretion is reduced. Basolateral H+ secretion occurs in exchange for Na+ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na+-K+-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H+ extrusion, intimately linking osmoregulation and acid-base balance. PMID:16709644

  19. Psychrometric Pressure-Volume Analysis of Osmoregulation in Roots, Shoots, and Whole Sporophytes of Salinized Ceratopteris1

    PubMed Central

    Augé, Robert M.; Hickok, Leslie G.; Stodola, Ann J. W.

    1989-01-01

    Osmotic and turgor adjustment in roots, shoots, and whole sporophytes of a NaCl-tolerant mutant and a NaCl-sensitive wild-type strain of the fern Ceratopteris richardii Brongn. were characterized following exposure to 60 millimolar NaCl, using a psychrometric approach to pressure-volume analysis. Water potential components of whole plants and shoots at full or ambient hydration were similar between strains. Roots of the mutant, however, had osmotic potentials at full turgor and water potentials at zero turgor which were lower (0.32 and 0.46 megapascal, respectively) than those in roots of the wild type after salinization. Although compromised in the absence of NaCl, sporophytes of the mutant strain were larger and much less necrotic in 60 millimolar NaCl after 17 days, compared to the wild type. Root growth in the mutant strain was unaffected by salinization and far exceeded root growth in the wild type. Correlation coefficients of the linear regression and the general consistency and precision of the pressure-volume data demonstrated the feasibility of using PV curves for estimating water potential components of roots. The technique should also provide a means of studying osmoregulation in a variety of other rapidly equilibrating plant tissues. PMID:16667019

  20. 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

  1. BIOSYNTHESIS OF STRESS ETHYLENE IN SOYBEAN SEEDLINGS: SIMILARITIES TO ENDOGENOUS ETHYLENE BIOSYNTHESIS

    EPA Science Inventory

    The similarity of stress ethylene biosynthesis in whole plants to endogenous ethylene biosynthesis was investigated using two inhibitors of ethylene biosynthesis, amino-ethoxyvinylglycine (AVG) and cobalt chloride (Co2+); and the intermediates, methionine, S-adenosylmethionine (S...

  2. Impact of Heme and Heme Degradation Products on Vascular Diameter in Mouse Visual Cortex

    PubMed Central

    Joerk, Alexander; Seidel, Raphael Andreas; Walter, Sebastian Gottfried; Wiegand, Anne; Kahnes, Marcel; Klopfleisch, Maurice; Kirmse, Knut; Pohnert, Georg; Westerhausen, Matthias; Witte, Otto Wilhelm; Holthoff, Knut

    2014-01-01

    Background Delayed cerebral vasospasm is the most common cause of mortality and severe neurological impairment in patients who survive subarachnoid hemorrhage. Despite improvements in the field of diagnostic imaging, options for prevention and medical treatment—primarily with the calcium channel antagonist nimodipine or hemodynamic manipulations—are insufficient. Previous studies have suggested that heme and bilirubin oxidation end products, originating from degraded hemoglobin around ruptured blood vessels, are involved in the development of vasospasm by inhibiting large conductance BKCa potassium channels in vascular smooth muscle cells. In this study, we identify individual heme degradation products regulating arteriolar diameter in dependence of BKCa channel activity. Methods and Results Using differential interference contrast video microscopy in acute brain slices, we determined diameter changes of intracerebral arterioles in mouse visual cortex. In preconstricted vessels, the specific BKCa channel blockers paxilline and iberiotoxin as well as iron‐containing hemin caused vasoconstriction. In addition, the bilirubin oxidation end product Z‐BOX A showed a stronger vasoconstrictive potency than its regio‐isomer Z‐BOX B. Importantly, Z‐BOX A had the same vasoconstrictive effect, independent of its origin from oxidative degradation or chemical synthesis. Finally, in slices of Slo1‐deficient knockout mice, paxilline and Z‐BOX A remained ineffective in changing arteriole diameter. Conclusions We identified individual components of the oxidative bilirubin degradation that led to vasoconstriction of cerebral arterioles. The vasoconstrictive effect of Z‐BOX A and Z‐BOX B was mediated by BKCa channel activity that might represent a signaling pathway in the occurrence of delayed cerebral vasospasm in subarachnoid hemorrhage patients. PMID:25169792

  3. Heme-Biosynthetic Porphobilinogen Deaminase Protects Aspergillus nidulans from Nitrosative Stress

    PubMed Central

    Zhou, Shengmin; Narukami, Toshiaki; Nameki, Misuzu; Ozawa, Tomoko; Kamimura, Yosuke; Hoshino, Takayuki

    2012-01-01

    Microorganisms have developed mechanisms to combat reactive nitrogen species (RNS); however, only a few of the fungal genes involved have been characterized. Here we screened RNS-resistant Aspergillus nidulans strains from fungal transformants obtained by introducing a genomic DNA library constructed in a multicopy vector. We found that the AN0121.3 gene (hemC) encodes a protein similar to the heme biosynthesis enzyme porphobilinogen deaminase (PBG-D) and facilitates RNS-tolerant fungal growth. The overproduction of PBG-D in A. nidulans promoted RNS tolerance, whereas PBG-D repression caused growth that was hypersensitive to RNS. PBG-D levels were comparable to those of cellular protoheme synthesis as well as flavohemoglobin (FHb; encoded by fhbA and fhbB) and nitrite reductase (NiR; encoded by niiA) activities. Both FHb and NiR are hemoproteins that consume nitric oxide and nitrite, respectively, and we found that they are required for maximal growth in the presence of RNS. The transcription of hemC was upregulated by RNS. These results demonstrated that PBG-D is a novel NO-tolerant protein that modulates the reduction of environmental NO and nitrite levels by FHb and NiR. PMID:22038601

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

    PubMed

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

    2016-05-10

    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

  5. 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

  6. Mechanism of inhibition of estrogen biosynthesis by azole fungicides.

    PubMed

    Egbuta, Chinaza; Lo, Jessica; Ghosh, Debashis

    2014-12-01

    Biosynthesis of estrogens from androgens is catalyzed by cytochrome P450 aromatase. Aromatase inhibition by the triazole compounds letrozole (LTZ) and anastrozole is a prevalent therapy for estrogen-dependent postmenopausal breast cancer. Azoles are widely used as agricultural fungicides and antimycotic drugs that target 14α-demethylase. Some were previously shown to inhibit aromatase, thereby raising the possibility of endocrine disruptive effects. However, mechanistic analysis of their inhibition has never been undertaken. We have evaluated the inhibitory effects of 3 common fungicides, bifonazole, imazalil, and flusilazole, in human aromatase purified from placenta and compared them with LTZ, the most potent inhibitor of aromatase. Bifonazole exhibits strong inhibitory effects with an IC50 of 270nM and Ki (Michaeles-Menten inhibition constant) of 68nM, compared with 10nM and 13nM, respectively, for LTZ. The IC50 and Ki are 1100nM and 278nM for imazilil and 3200nM and 547nM for flusilazole, respectively. Analyses of inhibition kinetics suggest that the modes of inhibition by azole fungicides are mixed or competitive, whereas LTZ inhibition could be noncompetitive or mixed. We interpret the inhibition mechanism in the context of the x-ray structure of aromatase-androstenedione complex. Structural data show that aromatase has 3 binding pockets in relation to the heme. The substrate-binding cavity at the heme-distal site closely compliments the structures of the natural substrate, androstenedione, and steroidal aromatase inhibitors. Because the structures of LTZ and the azole fungicides are entirely dissimilar to the androstenedione backbone, the azoles possibly inhibit by binding to a structurally rearranged active site, the 2 other catalytically important sites, or both, in agreement with the kinetics data. PMID:25243857

  7. 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.

  8. Biosynthesis of Dolichyl Phosphate

    PubMed Central

    Hopp, H. Esteban; Daleo, Gustavo R.; Romero, Pedro A.; Lezica, Rafael Pont

    1978-01-01

    This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system. A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate. The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [14C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C90 to C105, C95 and C100 being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed. The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate. PMID:16660269

  9. Reductive nitrosylation and peroxynitrite-mediated oxidation of heme-hemopexin.

    PubMed

    Ascenzi, Paolo; Bocedi, Alessio; Antonini, Giovanni; Bolognesi, Martino; Fasano, Mauro

    2007-01-01

    Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2). PMID:17229156

  10. The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive*

    PubMed Central

    Sabbadin, Federico; Jackson, Rosamond; Haider, Kamran; Tampi, Girish; Turkenburg, Johan P.; Hart, Sam; Bruce, Neil C.; Grogan, Gideon

    2009-01-01

    XplA is a cytochrome P450 of unique structural organization, consisting of a heme- domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P21 to a resolution of 1.9 Å and as a monomer in space group P21212 to a resolution of 1.5 Å, with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogen-bonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 → Ala and Ala-395 → Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors. PMID:19692330

  11. Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the pigA Gene of Pseudomonas aeruginosa

    PubMed Central

    Ratliff, Melanie; Zhu, Wenming; Deshmukh, Rahul; Wilks, Angela; Stojiljkovic, Igor

    2001-01-01

    The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity. PMID:11591684

  12. 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

  13. 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

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

    PubMed Central

    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

  15. 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...

  16. 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

  17. 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)

  18. 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. PMID:26666777

  19. 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 ...

  20. 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

  1. Heme and HO-1 Inhibition of HCV, HBV, and HIV

    PubMed Central

    Schmidt, Warren N.; Mathahs, M. Meleah; Zhu, Zhaowen

    2012-01-01

    Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system inhibit replication of all 3 viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections. PMID:23060790

  2. Mechanism of reaction of chlorite with mammalian heme peroxidases

    PubMed Central

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

    2014-01-01

    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. PMID:24632343

  3. 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.

  4. 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. PMID:26593548

  5. Hemopexin Prevents Endothelial Damage and Liver Congestion in a Mouse Model of Heme Overload

    PubMed Central

    Vinchi, Francesca; Gastaldi, Stefania; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2008-01-01

    Intravascular hemolysis results in the release of massive amounts of hemoglobin and heme into plasma, where they are rapidly bound by haptoglobin and hemopexin, respectively. Data from haptoglobin and hemopexin knockout mice have shown that both proteins protect from renal damage after phenylhydrazine-induced hemolysis, whereas double-mutant mice were especially prone to liver damage. However, the specific role of hemopexin remains elusive because of the difficulty in discriminating between hemoglobin and heme recovery. To study the specific role of hemopexin in intravascular hemolysis, we established a mouse model of heme overload. Under these conditions, both endothelial activation and vascular permeability were significantly higher in hemopexin-null mice compared with wild-type controls. Vascular permeability was particularly altered in the liver, where congestion in the centrolobular area was believed to be associated with oxidative stress and inflammation. Liver damage in hemopexin- null mice may be prevented by induction of heme oxygenase-1 before heme overload. Furthermore, heme-treated hemopexin-null mice exhibited hyperbilirubinemia, prolonged heme oxygenase-1 expression, excessive heme metabolism, and lack of H-ferritin induction in the liver compared with heme-treated wild-type controls. Moreover, these mutant mice metabolize an excess of heme in the kidney. These studies highlight the importance of hemopexin in heme detoxification, thus suggesting that drugs mimicking hemopexin activity might be useful to prevent endothelial damage in patients suffering from hemolytic disorders. PMID:18556779

  6. Influence of heme and vitamin B12 on growth and fermentations of Bacteroides species.

    PubMed

    Chen, M; Wolin, M J

    1981-01-01

    We examined the effects of heme on the growth and fermentations of Bacteroides species. Bacteroides fragilis ATCC 25285 required heme for growth and produced malate and lactate as major products of glucose fermentation when the concentration of heme was 1 ng/ml. With 1 microgram of heme per ml, malate was not formed, lactate production decreased, and succinate and acetate were the major fermentation products. B. eggerthii ATCC 27754 grew without heme, with the production of mainly malate and lactate from glucose. Its fermentation with 1 microgram of heme per ml was similar to that of B. fragilis grown with the same concentration of heme. B. splanchicus VPI 6842 grew without heme, with the production of mainly malate, acetate, and H2 from glucose. With 1 microgram of heme per ml, malate disappeared, H2 decreased significantly, and succinate, acetate, and butyrate were the major products. The addition of vitamin B12 to media containing 1 microgram of heme per ml caused all species to produce propionate at the expense of succinate and, with B. splanchnicus, also at the expense of butyrate. Thus, the concentration of heme and the presence of vitamin B12 significantly influenced the course of glucose fermentation by these bacteria. PMID:7462148

  7. 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

  8. Escherichia coli heme oxygenase modulates host innate immune responses

    PubMed Central

    Maharshak, Nitsan; Ryu, Hyungjin Sally; Fan, Ting-Jia; Onyiah, Joseph C.; Schulz, Stephanie; Otterbein, Sherrie L.; Wong, Ron; Hansen, Jonathan; Otterbein, Leo E; Carroll, Ian; Plevy, Scott E.

    2015-01-01

    Induction of mammalian heme oxygenase-1 and exposure of animals to carbon monoxide ameliorates experimental colitis. When enteric bacteria, including Escherichia coli, are exposed to low iron conditions, they express an heme oxygenase-like enzyme, chuS, and metabolize heme into iron, biliverdin and carbon monoxide. Given the abundance of enteric bacteria residing in the intestinal lumen, we hypothesized that commensal intestinal bacteria may be a significant source of carbon monoxide, with the consequence that enteric bacteria expressing chuS and other heme oxygenase -like molecules suppress inflammatory immune responses through release of carbon monoxide. Carbon monoxide exposed mice have altered enteric bacterial composition and increased E. coli 16S and chuS DNA by real-time PCR. Moreover, severity of experimental colitis correlates with increased E. coli chuS expression in IL-10 deficient mice. To explore functional roles, E. coli were genetically modified to overexpress chuS or the chuS gene was deleted. Co-culture of chuS-overexpressing E. coli with bone marrow derived macrophages results in decreased IL-12 p40 and increased IL-10 secretion compared to wild-type or chuS-deficient E. coli. Mice infected with chuS-overexpressing E. coli have increased levels of hepatic carbon monoxide and decreased serum IL-12 p40 compared to mice infected with chuS-deficient E. coli. Thus, carbon monoxide alters the composition of the commensal intestinal microbiota and expands E. coli populations harboring the chuS gene. These bacteria are capable of attenuating innate immune responses through expression of chuS. Bacterial heme oxygenase -like molecules and bacterial-derived carbon monoxide may represent novel targets for therapeutic intervention in inflammatory conditions. PMID:26146866

  9. 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

  10. 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

  11. 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

  12. Participation of Endomembrane Cation/H+ Exchanger AtCHX20 in Osmoregulation of Guard Cells1[W][OA

    PubMed Central

    Padmanaban, Senthilkumar; Chanroj, Salil; Kwak, June M.; Li, Xiyan; Ward, John M.; Sze, Heven

    2007-01-01

    Guard cell movement is induced by environmental and hormonal signals that cause changes in turgor through changes in uptake or release of solutes and water. Several transporters mediating these fluxes at the plasma membrane have been characterized; however, less is known about transport at endomembranes. CHX20, a member of a poorly understood cation/H+ exchanger gene family in Arabidopsis (Arabidopsis thaliana), is preferentially and highly expressed in guard cells as shown by promoter∷β-glucuronidase activity and by whole-genome microarray. Interestingly, three independent homozygous mutants carrying T-DNA insertions in CHX20 showed 35% reduction in light-induced stomatal opening compared to wild-type plants. To test the biochemical function of CHX20, cDNA was expressed in a yeast (Saccharomyces cerevisiae) mutant that lacks Na+(K+)/H+ antiporters (Δnhx1 Δnha1 Δkha1) and plasma membrane Na+ pumps (Δena1-4). Curiously, CHX20 did not enhance tolerance of mutants to moderate Na+ or high K+ stress. Instead, it restored growth of the mutant on medium with low K+ at slightly alkaline pH, but had no effect on growth at acidic pH. Green fluorescent protein-tagged CHX20 expressed in mesophyll protoplasts was localized mainly to membranes of the endosomal system. Furthermore, light-induced stomatal opening of the Arabidopsis mutants was insensitive to external pH and was impaired at high KCl. The results are consistent with the idea that, in exchanging K+ for H+, CHX20 maintains K+ homeostasis and influences pH under certain conditions. Together, these results provide genetic and biochemical evidence that one CHX protein plays a critical role in osmoregulation through K+ fluxes and possibly pH modulation of an active endomembrane system in guard cells. PMID:17337534

  13. 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

  14. A T67A mutation in the proximal pocket of the high-spin heme of MauG stabilizes formation of a mixed-valent FeII/FeIII state and enhances charge resonance stabilization of the bis-FeIV state.

    PubMed

    Shin, Sooim; Feng, Manliang; Li, Chao; Williamson, Heather R; Choi, Moonsung; Wilmot, Carrie M; Davidson, Victor L

    2015-08-01

    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. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket. PMID:25896561

  15. Ultrafast ligand rebinding in the heme domain of the oxygen sensors FixL and Dos: general regulatory implications for heme-based sensors.

    PubMed

    Liebl, Ursula; Bouzhir-Sima, Latifa; Negrerie, Michel; Martin, Jean-Louis; Vos, Marten H

    2002-10-01

    Heme-based oxygen sensors are part of ligand-specific two-component regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate. To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy to study ligand dynamics in the heme domains of the oxygen sensors FixL from Bradyrhizobium japonicum (FixLH) and Dos from Escherichia coli (DosH). The heme coordination with different ligands and the corresponding ground-state heme spectra of FixLH are similar to myoglobin (Mb). After photodissociation, the excited-state properties and ligand-rebinding kinetics are qualitatively similar for FixLH and Mb for CO and NO as ligands. In contrast to Mb, the transient spectra of FixLH after photodissociation of ligands are distorted compared with the ground-state difference spectra, indicating differences in the heme environment with respect to the unliganded state. This distortion is particularly marked for O(2). Strikingly, heme-O(2) recombination occurs with efficiency unprecedented for heme proteins, in approximately 5 ps for approximately 90% of the dissociated O(2). For DosH-O(2), which shows 60% sequence similarity to FixLH, but where signal detection and transmission presumably are quite different, a similarly fast recombination was found with an even higher yield. Altogether these results indicate that in these sensors the heme pocket acts as a ligand-specific trap. The general implications for the functioning of heme-based ligand sensors are discussed in the light of recent studies on heme-based NO and CO sensors. PMID:12271121

  16. 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.

  17. 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. PMID:25096577

  18. 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

  19. 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

  20. 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

  1. Developing a heme iron database for meats according to meat type, cooking method and doneness level

    PubMed Central

    Cross, Amanda J.; Harnly, James M.; Ferrucci, Leah M.; Risch, Adam; Mayne, Susan T.; Sinha, Rashmi

    2012-01-01

    Background Animal studies have demonstrated that iron may be related to carcinogenesis, and human studies found that heme iron can increase the formation of N-nitroso compounds, which are known carcinogens. Objectives One of the postulated mechanisms linking red meat intake to cancer risk involves iron. Epidemiologic studies attempt to investigate the association between heme iron intake and cancer by applying a standard factor to total iron from meat. However, laboratory studies suggest that heme iron levels in meat vary according to cooking method and doneness level. We measured heme iron in meats cooked by different cooking methods to a range of doneness levels to use in conjunction with a food frequency questionnaire to estimate heme iron intake. Methods Composite meat samples were made to represent each meat type, cooking method and doneness level. Heme iron was measured using atomic absorption spectrometry and inductively coupled plasma-atomic emission spectrometry. Results Steak and hamburgers contained the highest levels of heme iron, pork and chicken thigh meat had slightly lower levels, and chicken breast meat had the lowest. Conclusions Although heme iron levels varied, there was no clear effect of cooking method or doneness level. We outline the methods used to create a heme iron database to be used in conjunction with food frequency questionnaires to estimate heme iron intake in relation to disease outcome. PMID:23459329

  2. 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

  3. 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

  4. 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

  5. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    PubMed

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. PMID:26592143

  6. 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. PMID:12433915

  7. Topologically Conserved Residues Direct Heme Transport in HRG-1-related Proteins*

    PubMed Central

    Yuan, Xiaojing; Protchenko, Olga; Philpott, Caroline C.; Hamza, Iqbal

    2012-01-01

    Caenorhabditis elegans and human HRG-1-related proteins are conserved, membrane-bound permeases that bind and translocate heme in metazoan cells via a currently uncharacterized mechanism. Here, we show that cellular import of heme by HRG-1-related proteins from worms and humans requires strategically located amino acids that are topologically conserved across species. We exploit a heme synthesis-defective Saccharomyces cerevisiae mutant to model the heme auxotrophy of C. elegans and demonstrate that, under heme-deplete conditions, the endosomal CeHRG-1 requires both a specific histidine in the predicted second transmembrane domain (TMD2) and the FARKY motif in the C terminus tail for heme transport. By contrast, the plasma membrane CeHRG-4 transports heme by utilizing a histidine in the exoplasmic (E2) loop and the FARKY motif. Optimal activity under heme-limiting conditions, however, requires histidine in the E2 loop of CeHRG-1 and tyrosine in TMD2 of CeHRG-4. An analogous system exists in humans, because mutation of the synonymous histidine in TMD2 of hHRG-1 eliminates heme transport activity, implying an evolutionary conserved heme transport mechanism that predates vertebrate origins. Our results support a model in which heme is translocated across membranes facilitated by conserved amino acids positioned on the exoplasmic, cytoplasmic, and transmembrane regions of HRG-1-related proteins. These findings may provide a framework for understanding the structural basis of heme transport in eukaryotes and human parasites, which rely on host heme for survival. PMID:22174408

  8. 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

  9. Staphylococcus aureus HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response▿ †

    PubMed Central

    Stauff, Devin L.; Bagaley, Danielle; Torres, Victor J.; Joyce, Rose; Anderson, Kelsi L.; Kuechenmeister, Lisa; Dunman, Paul M.; Skaar, Eric P.

    2008-01-01

    During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn2+/Mg2+-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus ΔhrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme. PMID:18326576

  10. The secondary coordination sphere controlled reactivity of a ferric-superoxo heme: unexpected conversion to a ferric hydroperoxo intermediate by reaction with a high-spin ferrous heme.

    PubMed

    Nagaraju, Perumandla; Ohta, Takehiro; Liu, Jin-Gang; Ogura, Takashi; Naruta, Yoshinori

    2016-06-01

    A bio-inspired heme complex involving both a proton donor and an axial imidazole ligand reduces the activation energy for the formation of a ferric hydroperoxo intermediate. A high-spin ferrous heme is shown to be capable of reducing its superoxy species to generate a ferric hydroperoxo intermediate for the first time. PMID:27105471

  11. REGULATION OF RAT HEPATIC DELTA-AMINOLEVULINIC ACID SYNTHETASE AND HEME OXYGENASE ACTIVITIES: EVIDENCE FOR CONTROL BY HEME AND AGAINST MEDIATION BY PROSTHETIC IRON

    EPA Science Inventory

    The effects of in vivo administration of 6 compounds on the activity of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. The order of decreasing potency in reducing ALA synthetase activity was heme, bilirubin, protoporphyrin IX, bilirubin dimethyl es...

  12. The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis

    PubMed Central

    Shin, Sooim; Feng, Manliang; Chen, Yan; Jensen, Lyndal M. R.; Tachikawa, Hiroyasu; Wilmot, Carrie M.; Liu, Aimin; Davidson, Victor L.

    2010-01-01

    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. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca2+ in proximity to the two hemes [Jensen, L.M.R., Sanishvili, R., Davidson, V.L. & Wilmot, C.M. (2010) Science 327, 1392–1394]. This Ca2+ did not readily dissociate; however after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca2+-dependent changes in heme spin-state and conformation. Addition of H2O2 to the Ca2+-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca2+ to the Ca2+-depleted MauG, full TTQ biosynthesis activity and reactivity towards H2O2 was restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca2+ binding to Ca2+-depleted MauG indicated a two-step mechanism. Ca2+ initially reversibly binds to Ca2+-depleted MauG (Kd = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10−3 s−1) but highly favorable (Keq = 4.2) conformational change, yielding an apparent equilibrium Kd,eq value of 5.3 μM. The circular dichroism spectra of native and Ca2+-depleted MauG were essentially the same, consistent with Ca2+-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases. PMID:21128656

  13. 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 ...

  14. Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi.

    PubMed

    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

    2011-01-01

    Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE 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 labelling 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 PDE 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 signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi. PMID:21166893

  15. Heme as trigger and target for trioxane-containing antimalarial drugs.

    PubMed

    Meunier, Bernard; Robert, Anne

    2010-11-16

    Heme is not only just the binding site responsible for oxygen transport by hemoglobin, but it is also the prosthetic group of many different heme-containing enzymes, such as cytochromes P450, peroxidases, catalase, and several proteins involved in electron transfer. Heme plays a key role in the mechanism of action of many different antimalarial drugs. In degrading the host's hemoglobin, the malaria parasite Plasmodium and several other heme-eating parasites are faced with this redox-active metal complex. Heme is able to induce the toxic reductive cascade of molecular oxygen, which leads to the production of destructive hydroxyl radicals. Plasmodium detoxifies heme by converting it into a redox-inactive iron(III) polymer called hemozoin. Artemisinin, a natural drug containing a biologically important 1,2,4-trioxane structure, is now the first-line treatment for multidrug-resistant malaria. The peroxide moiety in artemisinin reacts in the presence of the flat, achiral iron(II)-heme; the mechanism does not reflect the classical "key and lock" paradigm for drugs. Instead, the reductive activation of the peroxide function generates a short-lived alkoxy radical, which quickly rearranges to a C-centered primary radical. This radical alkylates heme via an intramolecular process to produce covalent heme-drug adducts. The accumulation of non-polymerizable redox-active heme derivatives, a consequence of heme alkylation, is thought to be toxic for the parasite. The alkylation of heme by artemisinin has been demonstrated in malaria-infected mice, indicating that heme is acting as the trigger and target of artemisinin. The alkylation of heme by artemisinin is not limited to this natural compound: the mechanism is invoked for a large number of antimalarial semisynthetic derivatives. Synthetic trioxanes or trioxolanes also alkylate heme, and their alkylation ability correlates well with their antimalarial efficacy. In addition, several reports have demonstrated the cytotoxicity of

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Effects of mutating aromatic surface residues of the heme domain of human sulfite oxidase on its heme midpoint potential, intramolecular electron transfer, and steady-state kinetics.

    PubMed

    Davis, Amanda C; Cornelison, Matthew J; Meyers, Kimberly T; Rajapakshe, Asha; Berry, Robert E; Tollin, Gordon; Enemark, John H

    2013-03-01

    Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential. PMID:22975842

  1. Radical Energies and the Regiochemistry of Addition to Heme Groups. Methylperoxy and Nitrite Radical Additions to the Heme of Horseradish Peroxidase

    PubMed Central

    Wojciechowski, Grzegorz; Ortiz de Montellano, Paul R.

    2008-01-01

    The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons and/or vinyl groups of the electrophilic or radical species generated in the catalytic oxidation of halides, pseudohalides, carboxylic acids, aryl and alkyl hydrazines, and other substrates. The determinants of the regiospecificity of these reactions, however, are unclear. We report here modification of the heme of HRP by autocatalytically generated, low energy NO2• and CH3OO• radicals. The NO2• radical adds regioselectively to the 4- over the 2-vinyl group but does not add to the meso positions. Reaction of HRP with tert-BuOOH does not lead to heme modification, but reaction with the F152M mutant, in which the heme vinyls are more sterically accessible, results in conversion of the heme 2-vinyl into a 1-hydroxy-2-(methylperoxy)-ethyl group [-CH(OH)CH2OOCH3]. [18O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO• radical add to both the δ-meso-carbon and the 2-vinyl group. The regiochemistry of these and the other known additions to the heme indicate that only high-energy radicals (e.g., CH3•) add to the meso-carbon. Less energetic radicals, including NO2• and CH3OO•, add to heme vinyl groups if they are small enough but do not add to the meso-carbons. Electrophilic species such as HOBr, HOCl, and HOSCN add to vinyl groups but do not react with the meso-carbons. This meso- versus vinyl-reactivity paradigm, which appears to be general for autocatalytic additions to heme prosthetic groups, suggests that meso-hydroxylation of the heme by heme oxygenase occurs by a controlled radical reaction rather than by electrophilic addition. PMID:17249668

  2. (-)-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

  3. (-)-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.

  4. 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.

  5. 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

  6. Proton delivery to ferryl heme in a heme peroxidase: enzymatic use of the Grotthuss mechanism.

    PubMed

    Efimov, Igor; Badyal, Sandip K; Metcalfe, Clive L; Macdonald, Isabel; Gumiero, Andrea; Raven, Emma Lloyd; Moody, Peter C E

    2011-10-01

    We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two. PMID:21819069

  7. Sexual dimorphism in renal heme-heme oxygenase system in the streptozotocin diabetic rats.

    PubMed

    Bonacasa, Bárbara; Pérez, Cayetano; Salom, Miguel G; López, Bernardo; Sáez-Belmonte, Fara; Martinez, Pedro; Casas, Teresa; Fenoy, Fráncisco J; Rodriguez, Francisca

    2013-01-01

    Heme Oxygenase (HO) -1 and -2 exert antioxidant, cytoprotective and vascular actions in male diabetic rats. However, there is no information about the expression and functional significance of the renal HO system in diabetic females. The present study tested the hypothesis that the HO system is differentially regulated in the kidney of female Sprague Dawley diabetic rats, protecting it from nitrosative and glomerular functional damage. Two weeks after the administration of streptozotocin (STZ; 65 mg/kg. i.p), males (DM) and females (DF) showed hyperglycemia, polyuria and elevated kidney/body weight ratio, compared to their control males (CM) and females (CF). In conscious animals, creatinine clearance was higher (0.5 ± 00 vs. 0.3 ± 00; ml/min/100g BW; p<0.05) and urinary albumin excretion was lower (0.7 ± 0.3 vs 3.1 ± 0.7; mg/day) in DF compared to DM. Acute administration of a HO inhibitor stannous mesoporphyrin (SnMP 40 mol/kg, i.v.) induced a greater renal vasoconstrictor response in DF than in DM. Western blot analysis of renal tissue revealed higher renal cortex HO-1 protein levels in DF compared to all other groups; by immunohistochemistry this induction of HO-1 in DF was localized in tubular segments and glomeruli. Furthermore, renal cortical concentration of nitrosylated protein was higher in DM than in DF animals and inversely related with HO-1 levels in both renal cortex and medulla. These data demonstrate that the HO-1 protein is induced in females, associated with renal vasodilation, decreased renal nitrosative stress and reduced albuminuria, indicating that the HO system is protecting the kidney from diabetes-induced damage specifically in females. PMID:23092315

  8. A Pivotal Heme-transfer Reaction Intermediate in Cytochrome c Biogenesis*

    PubMed Central

    Mavridou, Despoina A. I.; Stevens, Julie M.; Mönkemeyer, Leonie; Daltrop, Oliver; di Gleria, Katalin; Kessler, Benedikt M.; Ferguson, Stuart J.; Allen, James W. A.

    2012-01-01

    c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway. PMID:22121193

  9. 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.

  10. Calcium ions inhibit reduction of heme a in bovine cytochrome c oxidase.

    PubMed

    Dyuba, Artem V; Vygodina, Tatiana; Azarkina, Natalia; Konstantinov, Alexander A

    2015-12-21

    The effect of Ca(2+) on the rate of heme a reduction by dithionite and hexaammineruthenium (RuAm) was studied in the cyanide-complexed bovine cytochrome oxidase (CcO). The rate of heme a reduction is proportional to RuAm concentration below 300 μM with kv of 0.53×10(6) M(-1) s(-1). Ca(2+) inhibits the rate of heme a reduction by dithionite by ∼25%. As the reaction speeds up with increased concentrations of RuAm, the inhibition by Ca(2+) disappears. The inhibition of heme a reduction may contribute to recently described partial inhibition of CcO by Ca(2+) in the enzymatic assays. The inhibitory effect of Ca(2+) on heme a reduction indicates that ET through heme a may be coupled to proton movement in the exit part of the proton channel H. PMID:26611345

  11. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.

    PubMed Central

    Nath, K A; Balla, G; Vercellotti, G M; Balla, J; Jacob, H S; Levitt, M D; Rosenberg, M E

    1992-01-01

    Heme proteins such as myoglobin or hemoglobin, when released into the extracellular space, can instigate tissue toxicity. Myoglobin is directly implicated in the pathogenesis of renal failure in rhabdomyolysis. In the glycerol model of this syndrome, we demonstrate that the kidney responds to such inordinate amounts of heme proteins by inducing the heme-degradative enzyme, heme oxygenase, as well as increasing the synthesis of ferritin, the major cellular repository for iron. Prior recruitment of this response with a single preinfusion of hemoglobin prevents kidney failure and drastically reduces mortality (from 100% to 14%). Conversely, ablating this response with a competitive inhibitor of heme oxygenase exacerbates kidney dysfunction. We provide the first in vivo evidence that induction of heme oxygenase coupled to ferritin synthesis is a rapid, protective antioxidant response. Our findings suggest a therapeutic strategy for populations at a high risk for rhabdomyolysis. Images PMID:1634613

  12. Inherently distorted heme as a novel tool for myoglobin-based oxygen carrier.

    PubMed

    Neya, Saburo; Kawaguchi, Akira T

    2012-02-01

    The two types of artificial myoglobins (Mbs) containing inherently distorted α-ethyl-2,4-dimethyldeuteroheme and undistorted 2,4-dimethyldeuteroheme were prepared to examine the influence of nonplanar heme deformation on the reactivity of Mb. In ferrous deoxy proteins, the paramagnetic proton nuclear magnetic resonance spectra showed that the deformed heme caused a 3.2-ppm lower-field shift of the proximal histidine signal, indicating a larger iron displacement from the heme plane upon the nonplanar deformation. The Mb with the nonplanar heme exhibited a markedly lower oxygen affinity as compared with the Mb containing the planar heme. The result suggests the utility of heme distortion to design the Mb-based oxygen carrier. PMID:21501194

  13. Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17

    PubMed Central

    Mak, Piotr J.; Gregory, Michael C.; Sligar, Stephen G.; Kincaid, James R.

    2014-01-01

    An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the cortico-steroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17 α -hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17 α -hydroxyprogesterone (17-OH PROG). The hydroxylated products then are either used for production of corticoids or undergo a second CYP17 catalyzed-transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions. PMID:24328388

  14. Photoselection in polarized photolysis experiments on heme proteins.

    PubMed Central

    Ansari, A; Jones, C M; Henry, E R; Hofrichter, J; Eaton, W A

    1993-01-01

    Polarized photolysis experiments have been performed on the carbon monoxide complex of myoglobin to assess the effects of photoselection on the kinetics of ligand rebinding and to investigate the reorientational dynamics of the heme plane. The results are analyzed in terms of the optical theory developed in the preceding paper by Ansari and Szabo. Changes in optical density arising from rotational diffusion of the photoselected population produce large deviations from the true geminate ligand rebinding curves if measurements are made with only a single polarization. The apparent ligand rebinding curves are significantly distorted even at photolysis levels greater than 90%. These deviations are eliminated by obtaining isotropically-averaged optical densities from measurements using both parallel and perpendicular polarizations of the probe pulse. These experiments also yield the optical anisotropy, which gives a novel method for accurately determining the degree of photolysis, as well as important information on the reorientational dynamics of the heme plane. The correlation time for the overall rotational diffusion of the molecule is obtained from the decay of the anisotropy. The anisotropy prior to rotational diffusion is lower than that predicted for a rigidly attached, perfectly circular absorber, corresponding to an apparent order parameter of S = 0.95 +/- 0.02. Polarized absorption data on single crystals suggest that the decreased anisotropy results more from internal motions of the heme plane which take place on time scales shorter than the duration of the laser pulse (10 ns) than from out-of-plane polarized transitions. PMID:8471730

  15. Nitric oxide heme interactions in nitrophorin from Cimex lectularius

    NASA Astrophysics Data System (ADS)

    Christmann, R.; Auerbach, H.; Berry, R. E.; Walker, F. A.; Schünemann, V.

    2016-12-01

    The nitrophorin from the bedbug Cimex lectularius (cNP) is a nitric oxide (NO) carrying protein. Like the nitrophorins (rNPs) from the kissing bug Rhodnius prolixus, cNP forms a stable heme Fe(III)-NO complex, where the NO can be stored reversibly for a long period of time. In both cases, the NPs are found in the salivary glands of blood-sucking bugs. The insects use the nitrophorins to transport the NO to the victim's tissues, resulting in vasodilation and reduced blood coagulation. However, the structure of cNP is significantly different to those of the rNPs from Rhodnius prolixus. Furthermore, the cNP can bind a second NO molecule to the proximal heme cysteine when present at higher concentrations. High field Mössbauer spectroscopy on 57Fe enriched cNP complexed with NO shows reduction of the heme iron and formation of a ferrous nitric oxide (Fe(II)-NO) complex. Density functional theory calculations reproduce the experimental Mössbauer parameters and confirm this observation.

  16. 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.

  17. 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. PMID:25449264

  18. Structure, function, and assembly of heme centers in mitochondrial respiratory complexes.

    PubMed

    Kim, Hyung J; Khalimonchuk, Oleh; Smith, Pamela M; Winge, Dennis R

    2012-09-01

    The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O(2), is mediated by protein-bound redox cofactors. In mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc(1) complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a(3), with Cu(B), form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc(1), whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals. PMID:22554985

  19. 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. PMID:25961377

  20. 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.

  1. A heme-degradation pathway in a blood-sucking insect.

    PubMed

    Paiva-Silva, Gabriela O; Cruz-Oliveira, Christine; Nakayasu, Ernesto S; Maya-Monteiro, Clarissa M; Dunkov, Boris C; Masuda, Hatisaburo; Almeida, Igor C; Oliveira, Pedro L

    2006-05-23

    Hematophagous insects are vectors of diseases that affect hundreds of millions of people worldwide. A common physiological event in the life of these insects is the hydrolysis of host hemoglobin in the digestive tract, leading to a massive release of heme, a known prooxidant molecule. Diverse organisms, from bacteria to plants, express the enzyme heme oxygenase, which catalyzes the oxidative degradation of heme to biliverdin (BV) IX, CO, and iron. Here, we show that the kissing bug Rhodnius prolixus, a vector of Chagas' disease, has a unique heme-degradation pathway wherein heme is first modified by addition of two cysteinylglycine residues before cleavage of the porphyrin ring, followed by trimming of the dipeptides. Furthermore, in contrast to most known heme oxygenases, which generate BV IXalpha, in this insect, the end product of heme detoxification is a dicysteinyl-BV IXgamma. Based on these results, we propose a heme metabolizing pathway that includes the identified intermediates produced during modification and cleavage of the heme porphyrin ring. PMID:16698925

  2. A heme-degradation pathway in a blood-sucking insect

    PubMed Central

    Paiva-Silva, Gabriela O.; Cruz-Oliveira, Christine; Nakayasu, Ernesto S.; Maya-Monteiro, Clarissa M.; Dunkov, Boris C.; Masuda, Hatisaburo; Almeida, Igor C.; Oliveira, Pedro L.

    2006-01-01

    Hematophagous insects are vectors of diseases that affect hundreds of millions of people worldwide. A common physiological event in the life of these insects is the hydrolysis of host hemoglobin in the digestive tract, leading to a massive release of heme, a known prooxidant molecule. Diverse organisms, from bacteria to plants, express the enzyme heme oxygenase, which catalyzes the oxidative degradation of heme to biliverdin (BV) IX, CO, and iron. Here, we show that the kissing bug Rhodnius prolixus, a vector of Chagas' disease, has a unique heme-degradation pathway wherein heme is first modified by addition of two cysteinylglycine residues before cleavage of the porphyrin ring, followed by trimming of the dipeptides. Furthermore, in contrast to most known heme oxygenases, which generate BV IXα, in this insect, the end product of heme detoxification is a dicysteinyl-BV IXγ. Based on these results, we propose a heme metabolizing pathway that includes the identified intermediates produced during modification and cleavage of the heme porphyrin ring. PMID:16698925

  3. 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

  4. 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

  5. Regulating the nitrite reductase activity of myoglobin by redesigning the heme active center.

    PubMed

    Wu, Lei-Bin; Yuan, Hong; Gao, Shu-Qin; You, Yong; Nie, Chang-Ming; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2016-07-01

    Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins. PMID:27108710

  6. 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.

  7. Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis.

    PubMed

    Andrade, Bruno B; Pavan Kumar, Nathella; Amaral, Eduardo P; Riteau, Nicolas; Mayer-Barber, Katrin D; Tosh, Kevin W; Maier, Nolan; Conceição, Elisabete L; Kubler, Andre; Sridhar, Rathinam; Banurekha, Vaithilingam V; Jawahar, Mohideen S; Barbosa, Theolis; Manganiello, Vincent C; Moss, Joel; Fontana, Joseph R; Marciano, Beatriz E; Sampaio, Elizabeth P; Olivier, Kenneth N; Holland, Steven M; Jackson, Sharon H; Moayeri, Mahtab; Leppla, Stephen; Sereti, Irini; Barber, Daniel L; Nutman, Thomas B; Babu, Subash; Sher, Alan

    2015-09-15

    Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients. PMID:26268658

  8. 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

  9. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury. PMID:15474356

  10. Investigating beta-hydroxyenduracididine formation in the biosynthesis of the mannopeptimycins.

    PubMed

    Haltli, Brad; Tan, Ying; Magarvey, Nathan A; Wagenaar, Melissa; Yin, Xihou; Greenstein, Michael; Hucul, John A; Zabriskie, T Mark

    2005-11-01

    The mannopeptimycins (MPPs) are potent glycopeptide antibiotics that contain both D and L forms of the unique, arginine-derived amino acid beta-hydroxyenduracididine (betahEnd). The product of the mppO gene in the MPP biosynthetic cluster resembles several non-heme iron, alpha-ketoglutarate-dependent oxygenases, such as VioC and clavaminate synthase. The role of MppO in betahEnd biosynthesis was confirmed through inactivation of mppO, which yielded a strain that produced dideoxy-MPPs, indicating that mppO is essential for generating the beta-hydroxy functionality for both betahEnd residues. Characterization in vitro of recombinant His6-MppO expressed in E. coli revealed that MppO selectively hydroxylates the beta carbon of free L-enduracididine. PMID:16298295

  11. 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.

  12. From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D12

    PubMed Central

    Pinto, John T.; Cooper, Arthur J. L.

    2014-01-01

    Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism. PMID:24618756

  13. 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.

  14. An Unusual Carbon-Carbon Bond Cleavage Reaction During Phosphinothricin Biosynthesis

    PubMed Central

    Cicchillo, Robert M.; Zhang, Houjin; Blodgett, Joshua A.V.; Whitteck, John T.; Li, Gongyong; Nair, Satish K.; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture1. One such compound, phosphinothricin tripeptide (PTT), contains the unusual amino acid phosphinothricin (PT) attached to two alanine residues (Fig. 1). Synthetic PT (glufosinate) is a component of two top-selling herbicides (Basta® and Liberty®), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during PTT biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP) (Fig. 1)2. Reported here are the in vitro reconstitution of this unprecedented C(sp3)-C(sp3) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-heme iron(II)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalyzed by the 2-His-1-carboxylate mononuclear non-heme iron family of enzymes. PMID:19516340

  15. 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.

  16. 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.

  17. Benzodiazepine biosynthesis in Streptomyces refuineus.

    PubMed

    Hu, Yunfeng; Phelan, Vanessa; Ntai, Ioanna; Farnet, Chris M; Zazopoulos, Emmanuel; Bachmann, Brian O

    2007-06-01

    Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A lambda-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a "dehydroproline acrylamide" moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin. PMID:17584616

  18. 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

  19. Biosynthesis of enediyne antitumor antibiotics.

    PubMed

    Van Lanen, Steven G; Shen, Ben

    2008-01-01

    The enediyne polyketides are secondary metabolites isolated from a variety of Actinomycetes. All members share very potent anticancer and antibiotic activity, and prospects for the clinical application of the enediynes has been validated with the recent marketing of two enediyne derivatives as anticancer agents. The biosynthesis of these compounds is of interest because of the numerous structural features that are unique to the enediyne family. The gene cluster for five enediynes has now been cloned and sequenced, providing the foundation to understand natures' means to biosynthesize such complex, exotic molecules. Presented here is a review of the current progress in delineating the biosynthesis of the enediynes with an emphasis on the model enediyne, C-1027. PMID:18397168

  20. 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.

  1. Biosynthesis of Ochratoxin A1

    PubMed Central

    Searcy, J. W.; Davis, N. D.; Diener, U. L.

    1969-01-01

    Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-14C and sodium acetate-2-14C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation. PMID:5369298

  2. 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

  3. Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase

    SciTech Connect

    Li, Lenong; Chang, Zhenzhan; Pan, Zhiqiang; Fu, Zheng-Qing; Wang, Xiaoqiang

    2009-01-12

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between {beta}3-2 and {beta}3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

  4. 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

  5. Reciprocal Allosteric Modulation of Carbon Monoxide and Warfarin Binding to Ferrous Human Serum Heme-Albumin

    PubMed Central

    Bocedi, Alessio; De Sanctis, Giampiero; Ciaccio, Chiara; Tundo, Grazia R.; Di Masi, Alessandra; Fanali, Gabriella; Nicoletti, Francesco P.; Fasano, Mauro; Smulevich, Giulietta; Ascenzi, Paolo; Coletta, Massimo

    2013-01-01

    Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II). PMID:23555601

  6. Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme

    PubMed Central

    2015-01-01

    C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS–heme–cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical “correction” supports the proposed role of heme binding for the corresponding residues. PMID:25054239

  7. Synthesis and characterization of reduced heme and heme/copper carbonmonoxy species.

    PubMed

    Kretzer, Ryan M; Ghiladi, Reza A; Lebeau, Estelle L; Liang, Hong-Chang; Karlin, Kenneth D

    2003-05-01

    Carbon monoxide readily binds to heme and copper proteins, acting as a competitive inhibitor of dioxygen. As such, CO serves as a probe of protein metal active sites. In our ongoing efforts to mimic the active site of cytochrome c oxidase, reactivity toward carbon monoxide offers a unique opportunity to gain insight into the binding and spectroscopic characteristics of synthetic model compounds. In this paper, we report the synthesis and characterization of CO-adducts of ((5/6)L)Fe(II), [((5/6)L)Fe(II)...Cu(I)](B(C(6)F(5))(4)), and [(TMPA)Cu(I)(CH(3)CN)](B(C(6)F(5))(4)), where TMPA = tris(2-pyridylmethyl)amine and (5/6)L = a tetraarylporphyrinate tethered in either the 5-position ((5)L) or 6-position ((6)L) to a TMPA copper binding moiety. Reaction of ((5/6)L)Fe(II) [in THF (293 K): UV-vis 424 (Soret), 543-544 nm; (1)H NMR delta(pyrrole) 52-59 ppm (4 peaks); (2)H NMR (from ((5)L-d(8))Fe(II)) delta(pyrrole) 53.3, 54.5, 55.8, 56.4 ppm] with CO in solution at RT yielded ((5/6)L)Fe(II)-CO [in THF (293 K): UV-vis 413-414 (Soret), 532-533 nm; IR nu(CO)(Fe) 1976-1978 cm(-1); (1)H NMR delta(pyrrole) 8.8 ppm; (2)H NMR (from ((5)L-d(8))Fe(II)-CO) delta(pyrrole) 8.9 ppm; (13)C NMR delta((CO)Fe) 206.8-207.1 ppm (2 peaks)]. Experiments repeated in acetonitrile, acetone, toluene, and dichloromethane showed similar spectroscopic data. Binding of CO resulted in a change from five-coordinate, high-spin Fe(II) to six-coordinate, low-spin Fe(II), as evidenced by the upfield shift of the pyrrole resonances to the diamagnetic region ((1)H and (2)H NMR spectra). Addition of CO to [((5/6)L)Fe(II)...Cu(I)](B(C(6)F(5))(4)) [in THF (293 K): UV-vis ((6)L only) 424 (Soret), 546 nm; (1)H NMR delta(pyrrole) 54-59 ppm (multiple peaks); (2)H NMR (from [((5)L-d(8))Fe(II).Cu(I)](B(C(6)F(5))(4))) delta(pyrrole) 53.4 ppm (br)] gave the bis-carbonyl adduct [((5/6)L)Fe(II)-CO...Cu(I)-CO](B(C(6)F(5))(4)) [in THF (293 K): UV-vis ((6)L only) 413 (Soret), 532 nm; IR nu(CO)(Fe) 1971-1973 cm(-1), nu

  8. Heme uptake across the outer membrane as revealed by crystal structures of the receptor–hemophore complex

    PubMed Central

    Krieg, Stefanie; Huché, Frédéric; Diederichs, Kay; Izadi-Pruneyre, Nadia; Lecroisey, Anne; Wandersman, Cécile; Delepelaire, Philippe; Welte, Wolfram

    2009-01-01

    Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 Å from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor. PMID:19144921

  9. 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

  10. 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.

  11. Osmoregulation in Rhodobacter sphaeroides.

    PubMed

    Abee, T; Palmen, R; Hellingwerf, K J; Konings, W N

    1990-01-01

    Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems. PMID:2294084

  12. 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

  13. 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.

  14. Heme deficiency of soluble guanylate cyclase induces gastroparesis

    PubMed Central

    COSYNS, S. M. R.; DHAESE, I.; THOONEN, R.; BUYS, E. S.; VRAL, A.; BROUCKAERT, P.; LEFEBVRE, R. A.

    2016-01-01

    Background Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme-free status of sGC, as induced by oxidative stress, was investigated. Methods sGCβ1H105F knock-in (apo-sGC) mice, which express heme-free sGC that has basal activity, but cannot be stimulated by NO, were generated. Key Results Diethylenetriamine NONOate did not increase sGC activity in gastrointestinal tissue of apo-sGC mice. Exogenous NO did not induce relaxation in fundic, jejunal and colonic strips, and pyloric rings of apo-sGC mice. The stomach was enlarged in apo-sGC mice with hypertrophy of the muscularis externa of the fundus and pylorus. In addition, gastric emptying and intestinal transit were delayed and whole-gut transit time was increased in the apo-sGC mice, while distal colonic transit time was maintained. The nitrergic relaxant responses to electrical field stimulation at 1–4 Hz were abolished in fundic and jejunal strips from apo-sGC mice, but in pyloric rings and colonic strips, only the response at 1 Hz was abolished, indicating the contribution of other transmitters than NO. Conclusions & Inferences The results indicate that the gastrointestinal consequences of switching from a native sGC to a heme-free sGC, which cannot be stimulated by NO, are most pronounced at the level of the stomach establishing a pivotal role of the activation of sGC by NO in normal gastric functioning. In addition, delayed intestinal transit was observed, indicating that nitrergic activation of sGC also plays a role in the lower gastrointestinal tract. PMID:23551931

  15. Paradoxical effects of heme arginate on survival of myocutaneous flaps.

    PubMed

    Edmunds, Marie-Claire; Czopek, Alicja; Wigmore, Stephen J; Kluth, David C

    2014-01-01

    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

  16. Engineering the proximal heme cavity of catalase-peroxidase.

    PubMed

    Jakopitsch, Christa; Regelsberger, Günther; Furtmüller, Paul Georg; Rüker, Florian; Peschek, Günter A; Obinger, Christian

    2002-07-25

    Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested. PMID:12121764

  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. PMID:14517400

  18. Effect of salinity on the metabolism and osmoregulation of selected ontogenetic stages of an Amazon population of Macrobrachium amazonicum shrimp (Decapoda, Palaemonidae).

    PubMed

    Mazzarelli, C C M; Santos, M R; Amorim, R V; Augusto, A

    2015-05-01

    Probably as a function of their wide geographical distribution, the different population of Macrobrachium amazonicum shrimp may present distinct physiological, biochemical, reproductive, behavioral, and ecological patterns. These differences are so accentuated that the existence of allopatric speciation has been suggested, although initial studies indicate that the genetic variability of populations happen at an intraspecific level. Among the biological responses described for M. amazonicum populations, those regarding osmoregulation and metabolism play a key role for being related to the occupation of diverse habitats. To this effect, we investigated osmoregulation through the role of free amino acids in cell volume control and metabolism, through oxygen consumption in larvae (zoeae I, II, V and IX) and/or post-larvae of a M. amazonicum population from Amazon, kept in aquaculture fish hatcheries in the state of São Paulo. The results add information regarding the existence of distinct physiological responses among M. amazonicum populations and suggest that possible adjustments to metabolism and to the use of free amino acids as osmolytes of the regulation of the larvae and post-larvae cell volume depend on the appearance of structures responsible for hemolymph osmoregulation like, for example, the gills. In this respect, we verified that zoeae I do not alter their metabolism due to the exposition to fresh or brackish water, but they reduce intracellular concentration of free amino acids when exposed to fresh water, what may suggest the inexistence or inefficient performance of the structures responsible for volume regulation and hemolymph composition. On the other hand, in zoeae II and V exposed to fresh and brackish water, metabolism alterations were not followed by changes in free amino acids concentration. Thus it is possible, as the structures responsible for osmoregulation and ionic regulation become functional, that the role of free amino acids gets

  19. Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM).

    PubMed

    Schubert, Erik; Florin, Nicole; Duthie, Fraser; Henning Brewitz, H; Kühl, Toni; Imhof, Diana; Hagelueken, Gregor; Schiemann, Olav

    2015-07-01

    The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy. PMID:26050879

  20. Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein

    PubMed Central

    Weichsel, Andrzej; Maes, Estelle M.; Andersen, John F.; Valenzuela, Jesus G.; Shokhireva, Tatjana Kh.; Walker, F. Ann; Montfort, William R.

    2005-01-01

    Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-Å resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding. PMID:15637157

  1. 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.

  2. 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...

  3. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages.

    PubMed

    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

  4. 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

  5. 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

  6. Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins

    PubMed Central

    Zhong, Fangfang; Lisi, George P.; Collins, Daniel P.; Dawson, John H.; Pletneva, Ekaterina V.

    2014-01-01

    Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates. PMID:24398520

  7. Ultrafast Pump-Probe Experimental Studies of Heme Proteins

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Demidov, Andrey; Ye, Xiong; Christian, James F.; Champion, Paul M.

    1998-03-01

    Femtosecond time-resovled spectroscopy has been applied to explore coherent dynamics of cytochrome c and myoglobin with different diatomic ligands (NO, O2, CO) using amplified 50fs laser pulses which are generated by a regenerative amplifier and optical parametric amplifier laser system at a repetition rate of 250KHz. Signals associated with ``field driven'' coherence and ``reaction driven'' coherence are studied using both single-color and two-color pump-probe schemes. The combination of the two schemes helps us to further understand electronic relaxation, ligand photodissociation and protein relaxation processes in heme proteins.

  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. 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.

  11. Urea-based osmoregulation in the developing embryo of oviparous cartilaginous fish (Callorhinchus milii): contribution of the extraembryonic yolk sac during the early developmental period.

    PubMed

    Takagi, Wataru; Kajimura, Makiko; Tanaka, Hironori; Hasegawa, Kumi; Bell, Justin D; Toop, Tes; Donald, John A; Hyodo, Susumu

    2014-04-15

    Marine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in the extra-embryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in the YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish. PMID:24363418

  12. Systematic Tuning of Heme Redox Potentials and Its Effects on O2 Reduction Rates in a Designed Oxidase in Myoglobin

    PubMed Central

    2015-01-01

    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. 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

  14. 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

  15. /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.

  16. 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.

  17. An iron-carboxylate bond links the heme units of malaria pigment.

    PubMed Central

    Slater, A F; Swiggard, W J; Orton, B R; Flitter, W D; Goldberg, D E; Cerami, A; Henderson, G B

    1991-01-01

    The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme. Images PMID:1988933

  18. A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence

    PubMed Central

    Torres, Victor J.; Stauff, Devin L.; Pishchany, Gleb; Bezbradica, Jelena S.; Gordy, Laura E.; Iturregui, Juan; Anderson, Kelsi L.; Dunman, Paul M.; Joyce, Sebastian; Skaar, Eric P.

    2007-01-01

    SUMMARY Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality worldwide, exists as a harmless commensal organism in approximately 25% of the human population. Identifying the molecular machinery that is activated upon infection is central to understanding staphylococcal pathogenesis. We describe here the Heme-Sensor System (HssRS) that responds to heme exposure and activates expression of the Heme Regulated Transporter (HrtAB). The coordinated activities of HssRS and HrtAB maintain intracellular heme homeostasis and modulate S. aureus virulence. Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. Genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory