Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

PubMed Central

Birke, Jakob; Röther, Wolf

2015-01-01

Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in LcpK30. Heme was identified as a cofactor in purified LcpK30 by (i) detection of characteristic α-, β-, and γ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetone-extractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe3+ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. LcpK30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms. PMID:25819959

Human hemoglobin structural and functional alterations and heme degradation upon interaction with benzene: A spectroscopic study.

PubMed

Hosseinzadeh, Reza; Moosavi-Movahedi, Ali Akbar

2016-03-15

Here, the effect of benzene on hemoglobin structure, stability and heme prosthetic group integrity was studied by different methods. These included UV-vis absorption spectrophotometry, normal and synchronous fluorescence techniques, and differential scanning calorimetry (DSC). Our results indicated that benzene has high hemolytic potential even at low concentrations. The UV-vis spectroscopic results demonstrated that benzene altered both the globin chain and the heme prosthetic group of hemoglobin increasing met- and deoxy-Hb, while decreasing oxy-Hb. However, with increasing benzene the concentration of all species decreased due to heme destruction. The spectrophotometric results show that benzene has a high potential for penetrating the hydrophobic pocket of hemoglobin. These results were consistent with the molecular docking simulation results of benzene-hHb. Aggregation and thermal denaturation studies show that the increased benzene concentration induced hemoglobin aggregation with a decrease in stability, which is consistent with the DSC results. Conventional fluorescence spectroscopy revealed that the heme degradation species were produced in the presence of benzene. The results of constant wavelength synchronous fluorescence spectroscopy (CWSFS) indicated that at least five heme-degraded species were produced. Together, our results indicated that benzene has adverse effects on hemoglobin structure and function, and heme degradation. Copyright © 2015 Elsevier B.V. All rights reserved.

Impact of higher-order heme degradation products on hepatic function and hemodynamics.

PubMed

Seidel, Raphael A; Claudel, Thierry; Schleser, Franziska A; Ojha, Navin K; Westerhausen, Matthias; Nietzsche, Sandor; Sponholz, Christoph; Cuperus, Frans; Coldewey, Sina M; Heinemann, Stefan H; Pohnert, Georg; Trauner, Michael; Bauer, Michael

2017-08-01

Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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,more » 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.« less

SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation

PubMed Central

Mohanty, Joy G.; Nagababu, Enika; Friedman, Jeffrey S.; Rifkind, Joseph M.

2013-01-01

Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress. PMID:23142655

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates.

PubMed

Chen, Qiuying; Hirsch, Rhoda Elison

2006-03-01

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.

Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the hemO Gene

PubMed Central

Zhu, Wenming; Hunt, Desiree J.; Richardson, Anthony R.; Stojiljkovic, Igor

2000-01-01

Heme compounds are an important source of iron for neisseriae. We have identified a neisserial gene, hemO, that is essential for heme, hemoglobin (Hb), and haptoglobin-Hb utilization. The hemO gene is located 178 bp upstream of the hmbR Hb receptor gene in Neisseria meningitidis isolates. The product of the hemO gene is homologous to enzymes that degrade heme; 21% of its amino acid residues are identical, and 44% are similar, to those of the human heme oxygenase-1. DNA sequences homologous to hemO were ubiquitous in commensal and pathogenic neisseriae. HemO genetic knockout strains of Neisseria gonorrhoeae and N. meningitidis were unable to use any heme source, while the assimilation of transferrin-iron and iron-citrate complexes was unaffected. A phenotypic characterization of a conditional hemO mutant, constructed by inserting an isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated promoter upstream of the ribosomal binding site of hemO, confirmed the indispensability of the HemO protein in heme utilization. The expression of HemO also protected N. meningitidis cells against heme toxicity. hemO mutants were still able to transport heme into the cell, since both heme and Hb could complement an N. meningitidis hemA hemO double mutant for growth. The expression of the HmbR receptor was reduced significantly by the inactivation of the hemO gene, suggesting that hemO and hmbR are transcriptionally linked. The expression of the unlinked Hb receptor, HpuAB, was not altered. Comparison of the polypeptide patterns of the wild type and the hemO mutant led to detection of six protein spots with an altered expression pattern, suggesting a more general role of HemO in the regulation of gene expression in Neisseriae. PMID:10629191

Photochemical organic oxidations and dechlorinations with a mu-oxo bridged heme/non-heme diiron complex.

PubMed

Wasser, Ian M; Fry, H Christopher; Hoertz, Paul G; Meyer, Gerald J; Karlin, Kenneth D

2004-12-27

Steady state and laser flash photolysis studies of the heme/non-heme mu-oxo diiron complex [((6)L)Fe(III)-O-Fe(III)-Cl](+) (1) have been undertaken. The anaerobic photolysis of benzene solutions of 1 did not result in the buildup of any photoproduct. However, the addition of excess triphenylphosphine resulted in the quantitative photoreduction of 1 to [((6)L)Fe(II)...Fe(II)-Cl](+) (2), with concomitant production by oxo-transfer of 1 equiv of triphenylphosphine oxide. Under aerobic conditions and excess triphenylphosphine, the reaction produces multiple turnovers (approximately 28) before the diiron complex is degraded. The anaerobic photolysis of tetrahydrofuran (THF) or toluene solutions of 1 likewise results in the buildup of 2. The oxidation products from these reactions included gamma-butyrolactone (approximately 15%) for the reaction in THF and benzaldehyde (approximately 23%) from the reaction in toluene. In either case, the O-atom which is incorporated into the carbonyl product is derived from dioxygen present under workup or under aerobic photolysis conditions. Transient absorption measurements of low-temperature THF solutions of 1 revealed the presence of an (P)Fe(II)-like [P = tetraaryl porphyrinate dianion] species suggesting that the reactive species is a formal (heme)Fe(II)/Fe(IV)=O(non-heme) pair. The non-heme Fe(IV)=O is thus most likely responsible for C-H bond cleavage and subsequent radical chemistry. The photolysis of 1 in chlorobenzene or 1,2-dichlorobenzene resulted in C-Cl cleavage reactions and the formation of [[((6)L)Fe(III)-Cl...Fe(III)-Cl](2)O](2+) (3), with chloride ligands that are derived from solvent dehalogenation chemistry. The resulting organic products are biphenyl trichlorides or biphenyl monochlorides, derived from dichlorobenzene and chlorobenzene, respectively. Similarly, product 3 is obtained by the photolysis of benzene-benzyl chloride solutions of 1; the organic product is benzaldehyde (approximately 70%). A brief discussion of the dehalogenation chemistry, along with relevant environmental perspectives, is included.

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

PubMed

Huber, Warren J; Backes, Wayne L

2007-10-30

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

The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi

PubMed Central

Paes, Marcia Cristina; Cosentino-Gomes, Daniela; de Souza, Cíntia Fernandes; Nogueira, Natália Pereira de Almeida; Meyer-Fernandes, José Roberto

2011-01-01

Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS) which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology. PMID:22007287

Enhancement of nitrite on heme-induced oxidative reactions: A potential toxicological implication.

PubMed

Lu, Naihao; Chen, Wei; Zhu, Jingjie; Peng, Yi-Yuan

2012-02-01

Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Preferential role of iron in heme degradation of hemoglobin upon gamma irradiation.

PubMed

Rafiei, Javad; Yavari, Kamal; Moosavi-Movahedi, Ali A

2017-10-01

It is usually believed that γ-ray interaction with biomolecules is intermediately performed by reactive oxygen species (ROS) produced from radiolysis of water. Hemoglobin (Hb) as one of the most abundant biomolecule in blood and well-studied endogenously affected by ROS, was a good candidate for study. Adult human Hb was extracted and irradiated using four distinct 20, 60, 90 and 170Gy doses from Co-60 γ-ray source. UV-vis, fluorescence and FT-IR spectroscopies were used to study the whole conformational changes and partial degradation of heme. Hb species calculated using Benesch equations indicated that the concentration of oxy-Hb was decreased from 9.97μM to 6.56μM, while the total metastable met and deoxy-Hb concentration were just increased 2.39μM and about 8.4% of total heme was diminished. Heme degradation was studied using fluorescence spectra at two 321 and 460nm excitation wavelengths as fully and partially degradation of heme respectively. Inverse behavior of these two fluorescence spectra suggested a new mechanism of heme degradation in which γ-ray preferably absorbed by heme without any intermediary effects of water. It was confirmed by FT-IR spectra at 900-1000cm -1 where the FeN and NH of porphyrin indicate their own stretching vibrational bands. Thermal stability justified that the gamma radiation induced the conformational changes of Hb which is appeared during thermal unfolding. First derivative of thermal spectra indicated that the Tm of 170Gy dose irradiated sample is 2°C lowered and total concentration of Hb was decreased 14%. Copyright © 2017 Elsevier B.V. All rights reserved.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4).

PubMed

Bertling, Anne; Brodde, Martin F; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C; Kelsch, Reinhard; Kehrel, Beate E

2017-09-01

Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress.

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...

Elevated carboxyhemoglobin in a marine mammal, the northern elephant seal

PubMed Central

Tift, Michael S.; Ponganis, Paul J.; Crocker, Daniel E.

2014-01-01

Low concentrations of endogenous carbon monoxide (CO), generated primarily through degradation of heme from heme-proteins, have been shown to maintain physiological function of organs and to exert cytoprotective effects. However, high concentrations of carboxyhemoglobin (COHb), formed by CO binding to hemoglobin, potentially prevent adequate O2 delivery to tissues by lowering arterial O2 content. Elevated heme-protein concentrations, as found in marine mammals, are likely associated with greater heme degradation, more endogenous CO production and, consequently, elevated COHb concentrations. Therefore, we measured COHb in elephant seals, a species with large blood volumes and elevated hemoglobin and myoglobin concentrations. The levels of COHb were positively related to the total hemoglobin concentration. The maximum COHb value was 10.4% of total hemoglobin concentration. The mean (±s.e.m.) value in adult seals was 8.7±0.3% (N=6), while juveniles and pups (with lower heme-protein contents) had lower mean COHb values of 7.6±0.2% and 7.1±0.3%, respectively (N=9 and N=9, respectively). Serial samples over several hours revealed little to no fluctuation in COHb values. This consistent elevation in COHb suggests that the magnitude and/or rate of heme-protein turnover is much higher than in terrestrial mammals. The maximum COHb values from this study decrease total body O2 stores by 7%, thereby reducing the calculated aerobic dive limit for this species. However, the constant presence of elevated CO in blood may also protect against potential ischemia–reperfusion injury associated with the extreme breath-holds of elephant seals. We suggest the elephant seal represents an ideal model for understanding the potential cytoprotective effects, mechanisms of action and evolutionary adaptation associated with chronically elevated concentrations of endogenously produced CO. PMID:24829326

Elevated carboxyhemoglobin in a marine mammal, the northern elephant seal.

PubMed

Tift, Michael S; Ponganis, Paul J; Crocker, Daniel E

2014-05-15

Low concentrations of endogenous carbon monoxide (CO), generated primarily through degradation of heme from heme-proteins, have been shown to maintain physiological function of organs and to exert cytoprotective effects. However, high concentrations of carboxyhemoglobin (COHb), formed by CO binding to hemoglobin, potentially prevent adequate O2 delivery to tissues by lowering arterial O2 content. Elevated heme-protein concentrations, as found in marine mammals, are likely associated with greater heme degradation, more endogenous CO production and, consequently, elevated COHb concentrations. Therefore, we measured COHb in elephant seals, a species with large blood volumes and elevated hemoglobin and myoglobin concentrations. The levels of COHb were positively related to the total hemoglobin concentration. The maximum COHb value was 10.4% of total hemoglobin concentration. The mean (± s.e.m.) value in adult seals was 8.7 ± 0.3% (N=6), while juveniles and pups (with lower heme-protein contents) had lower mean COHb values of 7.6 ± 0.2% and 7.1 ± 0.3%, respectively (N=9 and N=9, respectively). Serial samples over several hours revealed little to no fluctuation in COHb values. This consistent elevation in COHb suggests that the magnitude and/or rate of heme-protein turnover is much higher than in terrestrial mammals. The maximum COHb values from this study decrease total body O2 stores by 7%, thereby reducing the calculated aerobic dive limit for this species. However, the constant presence of elevated CO in blood may also protect against potential ischemia-reperfusion injury associated with the extreme breath-holds of elephant seals. We suggest the elephant seal represents an ideal model for understanding the potential cytoprotective effects, mechanisms of action and evolutionary adaptation associated with chronically elevated concentrations of endogenously produced CO. © 2014. Published by The Company of Biologists Ltd.

Rapid, convenient method for screening imidazole-containing compounds for heme oxygenase inhibition.

PubMed

Vlahakis, Jason Z; Rahman, Mona N; Roman, Gheorghe; Jia, Zongchao; Nakatsu, Kanji; Szarek, Walter A

2011-01-01

Sensitive assays for measuring heme oxygenase activity have been based on the gas-chromatographic detection of carbon monoxide using elaborate, expensive equipment. The present study describes a rapid and convenient method for screening imidazole-containing candidates for inhibitory activity against heme oxygenase using a plate reader, based on the spectroscopic evaluation of heme degradation. A PowerWave XS plate reader was used to monitor the absorbance (as a function of time) of heme bound to purified truncated human heme oxygenase-1 (hHO-1) in the individual wells of a standard 96-well plate (with or without the addition of a test compound). The degradation of heme by heme oxygenase-1 was initiated using l-ascorbic acid, and the collected relevant absorbance data were analyzed by three different methods to calculate the percent control activity occurring in wells containing test compounds relative to that occurring in control wells with no test compound present. In the cases of wells containing inhibitory compounds, significant shifts in λ(max) from 404 to near 412 nm were observed as well as a decrease in the rate of heme degradation relative to that of the control. Each of the three methods of data processing (overall percent drop in absorbance over 1.5h, initial rate of reaction determined over the first 5 min, and estimated pseudo first-order reaction rate constant determined over 1.5h) gave similar and reproducible results for percent control activity. The fastest and easiest method of data analysis was determined to be that using initial rates, involving data acquisition for only 5 min once reactions have been initiated using l-ascorbic acid. The results of the study demonstrate that this simple assay based on the spectroscopic detection of heme represents a rapid, convenient method to determine the relative inhibitory activity of candidate compounds, and is useful in quickly screening a series or library of compounds for heme oxygenase inhibition. Copyright © 2010 Elsevier Inc. All rights reserved.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4)

PubMed Central

Bertling, Anne; Brodde, Martin F.; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C.; Kelsch, Reinhard; Kehrel, Beate E.

2017-01-01

Background Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Methods Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Results Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Conclusion Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress. PMID:29070980

Enhanced Heme Function and Mitochondrial Respiration Promote the Progression of Lung Cancer Cells

PubMed Central

Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li

2013-01-01

Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer biology and therapeutics. PMID:23704904

[Heme-iron in the human body].

PubMed

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

2007-09-09

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.

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

Characterisation of Anopheles gambiae heme oxygenase and metalloporphyrin feeding suggests a potential role in reproduction.

PubMed

Spencer, Christopher S; Yunta, Cristina; de Lima, Glauber Pacelli Gomes; Hemmings, Kay; Lian, Lu-Yun; Lycett, Gareth; Paine, Mark J I

2018-05-03

The mosquito Anopheles gambiae is the principal vector for malaria in sub-Saharan Africa. The ability of A. gambiae to transmit malaria is strictly related to blood feeding and digestion, which releases nutrients for oogenesis, as well as substantial amounts of highly toxic free heme. Heme degradation by heme oxygenase (HO) is a common protective mechanism, and a gene for HO exists in the An. gambiae genome HO (AgHO), although it has yet to be functionally examined. Here, we have cloned and expressed An. gambiae HO (AgHO) in E. coli. Purified recombinant AgHO bound hemin stoichiometrically to form a hemin-enzyme complex similar to other HOs, with a K D of 3.9 ± 0.6 μM; comparable to mammalian and bacterial HOs, but 7-fold lower than that of Drosophila melanogaster HO. AgHO also degraded hemin to biliverdin and released CO and iron in the presence of NADPH cytochrome P450 oxidoreductase (CPR). Optimal AgHO activity was observed at 27.5 °C and pH 7.5. To investigate effects of AgHO inhibition, adult female A. gambiae were fed heme analogues Sn- and Zn-protoporphyrins (SnPP and ZnPP), known to inhibit HO. These led to a dose dependent decrease in oviposition. Cu-protoporphyrin (CuPP), which does not inhibit HO had no effect. These results demonstrate that AgHO is a catalytically active HO and that it may play a key role in egg production in mosquitoes. It also presents a potential target for the development of compounds aimed at sterilising mosquitoes for vector control. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of pH on Structural Changes in Perch Hemoglobin that Can Alter Redox Stability and Heme Affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richards, Mark P.; Aranda, IV, Roman; He, Cai

2010-01-07

pH can be manipulated to alter the oxidative stability of fish-based foods during storage. X-ray diffraction was used to investigate the ability of reduced pH to cause structural changes in fish hemoglobins that lead to enhanced oxidative degradation. Decreasing pH from 8.0 to 6.3 and 5.7 created a large channel for solvent entry into the heme crevice of perch hemoglobin beta chains. The proton-induced opening of this channel occurred between site CD3 and the heme-6-propionate. Solvent entry into the heme crevice can enhance metHb formation and hemin loss, processes that accelerate lipid oxidation. Reduced pH also decreased the distance betweenmore » Ile at E11 in one of the alpha chains and the ligand above the heme iron atom. This sterically displaces O{sub 2} and protonated O{sub 2} which increases metHb formation. These studies demonstrate that pH reduction causes structural changes in perch hemoglobin which increase oxidative degradation of the heme pigment.« less

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family

PubMed Central

Vasina, Daria V.; Moiseenko, Konstantin V.; Fedorova, Tatiana V.; Tyazhelova, Tatiana V.

2017-01-01

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle. PMID:28301519

Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

PubMed Central

Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

2013-01-01

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

Protein aggregation as a cellular response to oxidative stress induced by heme and iron

PubMed Central

Vasconcellos, Luiz R. C.; Dutra, Fabianno F.; Siqueira, Mariana S.; Paula-Neto, Heitor A.; Dahan, Jennifer; Kiarely, Ellen; Carneiro, Leticia A. M.; Bozza, Marcelo T.; Travassos, Leonardo H.

2016-01-01

Hemolytic diseases include a variety of conditions with diverse etiologies in which red blood cells are destroyed and large amounts of hemeproteins are released. Heme has been described as a potent proinflammatory molecule that is able to induce multiple innate immune responses, such as those triggered by TLR4 and the NLRP3 inflammasome, as well as necroptosis in macrophages. The mechanisms by which eukaryotic cells respond to the toxic effects induced by heme to maintain homeostasis are not fully understood, however. Here we describe a previously uncharacterized cellular response induced by heme: the formation of p62/SQTM1 aggregates containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). This action is part of a response driven by the transcription factor NRF2 to the excessive generation of reactive oxygen species induced by heme that results in the expression of genes involved in antioxidant responses, including p62/SQTM1. Furthermore, we show that heme degradation by HO-1 is required for ALIS formation, and that the free iron released on heme degradation is necessary and sufficient to induce ALIS. Moreover, ferritin, a key protein in iron metabolism, prevents excessive ALIS formation. Finally, in vivo, hemolysis promotes an increase in ALIS formation in target tissues. Our data unravel a poorly understood aspect of the cellular responses induced by heme that can be explored to better understand the effects of free heme and free iron during hemolytic diseases such as sickle cell disease, dengue fever, malaria, and sepsis. PMID:27821769

Insights on how the Mycobacterium tuberculosis heme uptake pathway can be used as a drug target

PubMed Central

Owens, Cedric P; Chim, Nicholas; Goulding, Celia W

2013-01-01

Mycobacterium tuberculosis (Mtb) acquires non-heme iron through salicylate-derived siderophores termed mycobactins whereas heme iron is obtained through a cascade of heme uptake proteins. Three proteins are proposed to mediate Mtb heme iron uptake, a secreted heme transporter (Rv0203), and MmpL3 and MmpL11, which are potential transmembrane heme transfer proteins. Furthermore, MhuD, a cytoplasmic heme-degrading enzyme, has been identified. Rv0203, MmpL3 and MmpL11 are mycobacteria-specific proteins, making them excellent drug targets. Importantly, MmpL3, a necessary protein, has also been implicated in trehalose monomycolate export. Recent drug-discovery efforts revealed that MmpL3 is the target of several compounds with antimycobacterial activity. Inhibition of the Mtb heme uptake pathway has yet to be explored. We propose that inhibitor design could focus on heme analogs, with the goal of blocking specific steps of this pathway. In addition, heme uptake could be hijacked as a method of importing drugs into the mycobacterial cytosol. PMID:23919550

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.

Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.

PubMed

Kumar, Devesh; de Visser, Samuël P; Shaik, Sason

2005-06-08

The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.

CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages.

PubMed

Srisook, Klaokwan; Han, Shan-Shu; Choi, Hyung-Sim; Li, Mei-Hua; Ueda, Hideo; Kim, Chaekyun; Cha, Young-Nam

2006-01-12

Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.

Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

PubMed

Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

2018-05-15

Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

PubMed

Vukomanovic, Dragic; Rahman, Mona N; Jia, Zongchao; Nakatsu, Kanji

2017-01-01

Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thein, S.L.; Hesketh, C.; Wood, W.G.

Analysis of the molecular basis of dominantly inherited {beta}-thalassemia in four families has revealed different mutations involving exon 3 of the {beta}-globin gene. It is suggested that the phenotypic difference between this condition and the more common recessive forms of {beta}-thalassemia lies mainly in the length and stability of the abnormal translation products that are synthesized and, in particular, whether they are capable of binding heme and producing aggregations that are relatively resistant to proteolytic degradation.

Pro-stimulatory role of methemoglobin in inflammation through hemin oxidation and polymerization.

PubMed

Deshmukh, Rohitas; Trivedi, Vishal

2013-02-01

Inflammation or vascular occlusion by parasitized red blood cell contributes to the pathogenesis of cerebral malaria. The current study aimed to characterize the role of major pro-oxidant factor methemoglobin present in the malaria culture supernatant contributing in inflammation during malaria. Heme and heme polymer stimulate macrophage to secrete large amount of reactive oxygen species into the external micro-environment. The addition of methemoglobin along with heme or heme polymer amplifies production of ROS from macrophages several folds. Methemoglobin mediated stimulatory effect is not due to release of iron, enhanced production of H2O2 or mutual interaction of reaction components. Spectroscopic studies show that methemoglobin accepts heme as a substrate and oxidizes it through a single electron transfer mechanism. Heme oxidation product is a heme polymer with similar chemical and structural properties to synthetic β-hematin. Phenyl N-t-butylnitrone inhibits heme polymerization (IC50=30 nM) and indicates the absolute necessity of heme oxidation and heme free radical generation for heme polymerization. Methemoglobin produced heme polymer is a potent pro-inflammatory factor to release ROS into external microenvironment. Interestingly, methemoglobin not only produces pro-inflammatory heme polymer, but it also amplifies the potential of heme or preformed heme polymer (haemozoin or β-hematin) to produce several folds high ROS production from macrophages. This study illustrates the pro-inflammatory effect of methemoglobin, the underlying novel mechanism by which this occurs and a possible clinical intervention. Based on the results, we recommend methemoglobin directed peroxidase inhibitors as an adjuvant therapy during malaria.

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae*

PubMed Central

Lechardeur, Delphine; Fernandez, Annabelle; Robert, Bruno; Gaudu, Philippe; Trieu-Cuot, Patrick; Lamberet, Gilles; Gruss, Alexandra

2010-01-01

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein. PMID:20332091

An Integrated Clinico-transcriptomic Approach Identifies a Central Role of the Heme Degradation Pathway for Septic Complications after Trauma.

PubMed

Rittirsch, Daniel; Schoenborn, Veit; Lindig, Sandro; Wanner, Elisabeth; Sprengel, Kai; Günkel, Sebastian; Blaess, Markus; Schaarschmidt, Barbara; Sailer, Patricia; Märsmann, Sonja; Simmen, Hans-Peter; Cinelli, Paolo; Bauer, Michael; Claus, Ralf A; Wanner, Guido A

2016-12-01

The present study was aimed to identify mechanisms linked to complicated courses and adverse events after severe trauma by a systems biology approach. In severe trauma, overwhelming systemic inflammation can result in additional damage and the development of complications, including sepsis. In a prospective, longitudinal single-center study, RNA samples from circulating leukocytes from patients with multiple injury (injury severity score ≥17 points; n = 81) were analyzed for dynamic changes in gene expression over a period of 21 days by whole-genome screening (discovery set; n = 10 patients; 90 samples) and quantitative RT-PCR (validation set; n = 71 patients, 517 samples). Multivariate correlational analysis of transcripts and clinical parameters was used to identify mechanisms related to sepsis. Transcriptome profiling of the discovery set revealed the strongest changes between patients with either systemic inflammation or sepsis in gene expression of the heme degradation pathway. Using quantitative RT-PCR analyses (validation set), the key components haptoglobin (HP), cluster of differentiation (CD) 163, heme oxygenase-1 (HMOX1), and biliverdin reductase A (BLVRA) showed robust changes following trauma. Upregulation of HP was associated with the severity of systemic inflammation and the development of sepsis. Patients who received allogeneic blood transfusions had a higher incidence of nosocomial infections and sepsis, and the amount of blood transfusion as source of free heme correlated with the expression pattern of HP. These findings indicate that the heme degradation pathway is associated with increased susceptibility to septic complications after trauma, which is indicated by HP expression in particular.

Spectroscopic characterization of a truncated hemoglobin from the nitrogen-fixing bacterium Herbaspirillum seropedicae.

PubMed

Razzera, Guilherme; Vernal, Javier; Baruh, Debora; Serpa, Viviane I; Tavares, Carolina; Lara, Flávio; Souza, Emanuel M; Pedrosa, Fábio O; Almeida, Fábio C L; Terenzi, Hernán; Valente, Ana Paula

2008-09-01

The Herbaspirillum seropedicae genome sequence encodes a truncated hemoglobin typical of group II (Hs-trHb1) members of this family. We show that His-tagged recombinant Hs-trHb1 is monomeric in solution, and its optical spectrum resembles those of previously reported globins. NMR analysis allowed us to assign heme substituents. All data suggest that Hs-trHb1 undergoes a transition from an aquomet form in the ferric state to a hexacoordinate low-spin form in the ferrous state. The close positions of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in the distal pocket place them as candidates for heme coordination and ligand regulation. Peroxide degradation kinetics suggests an easy access to the heme pocket, as the protein offered no protection against peroxide degradation when compared with free heme. The high solvent exposure of the heme may be due to the presence of a flexible loop in the access pocket, as suggested by a structural model obtained by using homologous globins as templates. The truncated hemoglobin described here has unique features among truncated hemoglobins and may function in the facilitation of O(2) transfer and scavenging, playing an important role in the nitrogen-fixation mechanism.

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

PubMed Central

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.

2012-01-01

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

Insights into lignin degradation and its potential industrial applications.

PubMed

Abdel-Hamid, Ahmed M; Solbiati, Jose O; Cann, Isaac K O

2013-01-01

Lignocellulose is an abundant biomass that provides an alternative source for the production of renewable fuels and chemicals. The depolymerization of the carbohydrate polymers in lignocellulosic biomass is hindered by lignin, which is recalcitrant to chemical and biological degradation due to its complex chemical structure and linkage heterogeneity. The role of fungi in delignification due to the production of extracellular oxidative enzymes has been studied more extensively than that of bacteria. The two major groups of enzymes that are involved in lignin degradation are heme peroxidases and laccases. Lignin-degrading peroxidases include lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP). LiP, MnP, and VP are class II extracellular fungal peroxidases that belong to the plant and microbial peroxidases superfamily. LiPs are strong oxidants with high-redox potential that oxidize the major non-phenolic structures of lignin. MnP is an Mn-dependent enzyme that catalyzes the oxidation of various phenolic substrates but is not capable of oxidizing the more recalcitrant non-phenolic lignin. VP enzymes combine the catalytic activities of both MnP and LiP and are able to oxidize Mn(2+) like MnP, and non-phenolic compounds like LiP. DyPs occur in both fungi and bacteria and are members of a new superfamily of heme peroxidases called DyPs. DyP enzymes oxidize high-redox potential anthraquinone dyes and were recently reported to oxidize lignin model compounds. The second major group of lignin-degrading enzymes, laccases, are found in plants, fungi, and bacteria and belong to the multicopper oxidase superfamily. They catalyze a one-electron oxidation with the concomitant four-electron reduction of molecular oxygen to water. Fungal laccases can oxidize phenolic lignin model compounds and have higher redox potential than bacterial laccases. In the presence of redox mediators, fungal laccases can oxidize non-phenolic lignin model compounds. In addition to the peroxidases and laccases, fungi produce other accessory oxidases such as aryl-alcohol oxidase and the glyoxal oxidase that generate the hydrogen peroxide required by the peroxidases. Lignin-degrading enzymes have attracted the attention for their valuable biotechnological applications especially in the pretreatment of recalcitrant lignocellulosic biomass for biofuel production. The use of lignin-degrading enzymes has been studied in various applications such as paper industry, textile industry, wastewater treatment and the degradation of herbicides. Copyright © 2013 Elsevier Inc. All rights reserved.

Nitric oxide coupling to generate N2O promoted by a single-heme system as examined by density functional theory.

PubMed

Yi, Jun; Campbell, Adam L O; Richter-Addo, George B

2016-11-30

Bacteria utilize a heme/non-heme enzyme system to detoxify nitric oxide (NO) to N 2 O. In order to probe the capacity of a single-heme system to mediate this NO-to-N 2 O transformation, various scenarios for addition of electrons, protons, and a second NO molecule to a heme nitrosyl to generate N 2 O were explored by density functional theory calculations. We describe, utilizing this single-heme system, several stepwise intermediates along pathways that enable the critical N-N bond formation step yielding the desired Fe-N 2 O product. We also report a hitherto unreported directional second protonation that results in either productive N 2 O formation with loss of water, or formation of a non-productive hyponitrous acid adduct Fe{HONNOH}. Copyright © 2016 Elsevier Inc. All rights reserved.

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells.

PubMed

Immenschuh, S; Tan, M; Ramadori, G

1999-01-01

Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the body's tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide. Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols. Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner. This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor N(G)-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells. The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.

Heme Recognition By a Staphylococcus Aureus IsdE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.

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 portionmore » 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.« less

Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice

PubMed Central

Constante, Marco; Fragoso, Gabriela; Calvé, Annie; Samba-Mondonga, Macha; Santos, Manuela M.

2017-01-01

Dietary heme can be used by colonic bacteria equipped with heme-uptake systems as a growth factor and thereby impact on the microbial community structure. The impact of heme on the gut microbiota composition may be particularly pertinent in chronic inflammation such as in inflammatory bowel disease (IBD), where a strong association with gut dysbiosis has been consistently reported. In this study we investigated the influence of dietary heme on the gut microbiota and inferred metagenomic composition, and on chemically induced colitis and colitis-associated adenoma development in mice. Using 16S rRNA gene sequencing, we found that mice fed a diet supplemented with heme significantly altered their microbiota composition, characterized by a decrease in α-diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes were similar to shifts seen in dextran sodium sulfate (DSS)-treated mice to induce colitis. In addition, dietary heme, but not systemically delivered heme, contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the azoxymethane/DSS colorectal cancer (CRC) mouse model. Using inferred metagenomics, we found that the microbiota alterations elicited by dietary heme resulted in non-beneficial functional shifts, which were also characteristic of DSS-induced colitis. Furthermore, a reduction in fecal butyrate levels was found in mice fed the heme supplemented diet compared to mice fed the control diet. Iron metabolism genes known to contribute to heme release from red blood cells, heme uptake, and heme exporter proteins, were significantly enriched, indicating a shift toward favoring the growth of bacteria able to uptake heme and protect against its toxicity. In conclusion, our data suggest that luminal heme, originating from dietary components or gastrointestinal bleeding in IBD and, to lesser extent in CRC, directly contributes to microbiota dysbiosis. Thus, luminal heme levels may further exacerbate colitis through the modulation of the gut microbiota and its metagenomic functional composition. Our data may have implications in the development of novel targets for therapeutic approaches aimed at lowering gastrointestinal heme levels through heme chelation or degradation using probiotics and nutritional interventions. PMID:28983289

Near-infrared autofluorescence induced by intraplaque hemorrhage and heme degradation as marker for high-risk atherosclerotic plaques.

PubMed

Htun, Nay Min; Chen, Yung Chih; Lim, Bock; Schiller, Tara; Maghzal, Ghassan J; Huang, Alex L; Elgass, Kirstin D; Rivera, Jennifer; Schneider, Hans G; Wood, Bayden R; Stocker, Roland; Peter, Karlheinz

2017-07-13

Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology that allows identification of atherosclerotic plaques with intraplaque hemorrhage and ultimately holds promise for detection of high-risk plaques in patients.Atherosclerosis diagnosis relies primarily on imaging and early detection of high-risk atherosclerotic plaques is important for risk stratification of patients and stabilization therapies. Here Htun et al. demonstrate that vulnerable atherosclerotic plaques generate near-infrared autofluorescence that can be detected via emission computed tomography.

Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon.

PubMed

Ijssennagger, Noortje; Belzer, Clara; Hooiveld, Guido J; Dekker, Jan; van Mil, Saskia W C; Müller, Michael; Kleerebezem, Michiel; van der Meer, Roelof

2015-08-11

Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.

Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury

PubMed Central

Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A.; Traylor, Amie; Agarwal, Anupam

2016-01-01

Abstract Aims: Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. Results: C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1−/−) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. Innovation: This is the first study delineating the role of heme in ALI caused by Br2. Conclusion: The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI. Antioxid. Redox Signal. 24, 99–112. PMID:26376667

Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury.

PubMed

Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A; Traylor, Amie; Agarwal, Anupam; Matalon, Sadis

2016-01-10

Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1-/-) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. This is the first study delineating the role of heme in ALI caused by Br2. The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI.

Fungal biodegradation and enzymatic modification of lignin

PubMed Central

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

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

Heme-Containing Metal-Organic Frameworks for the Oxidative Degradation of Chemical Warfare Agents

DTIC Science & Technology

2016-04-14

stability of the oxo without sacrificing its inherent reactivity, we have synthesized a new framework featuring fluorinated groups in the ortho...especially suitable for the degradation of electrophilic phosphorous center, leading to the cleavage of P-S or P-O bond present in VX nerve agents

Regulation of tolerance of Chlamydomonas reinhardtii to heavy metal toxicity by heme oxygenase-1 and carbon monoxide.

PubMed

Wei, Yuan Yuan; Zheng, Qi; Liu, Zhao Pu; Yang, Zhi Min

2011-09-01

Investigation of heavy metal tolerance genes in green algae is of great importance because heavy metals have become one of the major contaminants in the aquatic ecosystem. In plants, accumulation of heavy metals modifies many aspects of cellular functions. However, the mechanism by which heavy metals exert detrimental effects is poorly understood. In this study, we identified a role for HO-1 (encoding heme oxygenase-1) in regulating the response of Chlamydomonas reinhardtii, a unicellular green alga, to mercury (Hg). Transgenic algae overexpressing HO-1 showed high tolerance to Hg exposure, with a 48.2% increase in cell number over the wild type, but accumulated less Hg. Physiological analysis revealed that expression of HO-1 suppressed the Hg-induced generation of reactive oxygen species. We further identified the effect of carbon monoxide (CO), a product of HO-1-mediated heme degradation, on growth and physiological parameters. Interestingly, administration of exogenous CO at non-toxic levels also conferred the tolerance of algae to Hg exposure. The CO-mediated alleviation of Hg toxicity was closely related to the lower accumulation of Hg and free radical species. These results indicate that functional identification of HO-1 is useful for molecular breeding designed to improve plant tolerance to heavy metals and reduce heavy metal accumulation in plant cells.

Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.

PubMed

Hannibal, Luciana; Somasundaram, Ramasamy; Tejero, Jesús; Wilson, Adjele; Stuehr, Dennis J

2011-11-11

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.

Beneficial effect of prolonged heme oxygenase 1 activation in a rat model of chronic heart failure.

PubMed

Collino, Massimo; Pini, Alessandro; Mugelli, Niccolò; Mastroianni, Rosanna; Bani, Daniele; Fantozzi, Roberto; Papucci, Laura; Fazi, Marilena; Masini, Emanuela

2013-07-01

We and others have previously demonstrated that heme oxygenase 1 (HO-1) induction by acute hemin administration exerts cardioprotective effects. Here, we developed a rat model of heart failure to investigate whether a long-term induction of HO-1 by chronic hemin administration exerted protective effects. Sprague Dawley rats that underwent permanent ligation of the left coronary artery were closely monitored for survival rate analysis and sacrificed on day 28 post-operation. Administration of hemin (4 mg/kg body weight) every other day for 4 weeks induced a massive increase in HO-1 expression and activity, as shown by the increased levels of the two main metabolic products of heme degradation, bilirubin and carbon monoxide (CO). These effects were associated with significant improvement in survival and reduced the extension of myocardial damage. The ischemic hearts of the hemin-treated animals displayed reduced oxidative stress and apoptosis in comparison with the non-treated rats, as shown by the decreased levels of lipid peroxidation, free-radical-induced DNA damage, caspase-3 activity and Bax expression. Besides, chronic HO-1 activation suppressed the elevated levels of myeloperoxidase (MPO) activity, interleukin 1β (IL-1β) production and tumor necrosis factor-α (TNFα) production that were evoked by the ischemic injury, and increased the plasma level of the anti-inflammatory cytokine IL-10. Interestingly, HO-1 inhibitor zinc protoporphyrin IX (ZnPP-IX; 1 mg/kg) lowered bilirubin and CO concentrations to control values, thus abolishing all the cardioprotective effects of hemin. In conclusion, the results demonstrate that chronic HO-1 activation by prolonged administration of hemin improves survival and exerts protective effects in a rat model of myocardial ischemia by exerting a potent antioxidant activity and disrupting multiple levels of the apoptotic and inflammatory cascade.

In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coelho, Pedro S.; Brustad, Eric M.; Arnold, Frances H.

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 cellsmore » expressing the heme enzymes are also provided by the present invention.« less

Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease.

PubMed

Vinchi, Francesca; Costa da Silva, Milene; Ingoglia, Giada; Petrillo, Sara; Brinkman, Nathan; Zuercher, Adrian; Cerwenka, Adelheid; Tolosano, Emanuela; Muckenthaler, Martina U

2016-01-28

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease. © 2016 by The American Society of Hematology.

Dissociation of heme from gaseous myoglobin ions studied by infrared multiphoton dissociation spectroscopy and Fourier-transform ion cyclotron resonance mass spectrometry

NASA Astrophysics Data System (ADS)

Wang, Yi-Sheng; Sabu, Sahadevan; Wei, Shih-Chia; Josh Kao, C.-M.; Kong, Xianglei; Liau, Shing-Chih; Han, Chau-Chung; Chang, Huan-Cheng; Tu, Shih-Yu; Kung, A. H.; Zhang, John Z. H.

2006-10-01

Detachment of heme prosthetic groups from gaseous myoglobin ions has been studied by collision-induced dissociation and infrared multiphoton dissociation in combination with Fourier-transform ion cyclotron resonance mass spectrometry. Multiply charged holomyoglobin ions (hMbn +) were generated by electrospray ionization and transferred to an ion cyclotron resonance cell, where the ions of interest were isolated and fragmented by either collision with Ar atoms or irradiation with 3μm photons, producing apomyoglobin ions (aMbn +). Both charged heme loss (with [Fe(III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe(II)-heme] and aMbn + as the products) were detected concurrently for hMbn + produced from a myoglobin solution pretreated with reducing reagents. By reference to Ea=0.9eV determined by blackbody infrared radiative dissociation for charged heme loss of ferric hMbn +, an activation energy of 1.1eV was deduced for neutral heme loss of ferrous hMbn + with n =9 and 10.

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

PubMed

Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

2009-05-27

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Heme oxygenase-1: A new druggable target in the management of chronic and acute myeloid leukemia.

PubMed

Salerno, Loredana; Romeo, Giuseppe; Modica, Maria N; Amata, Emanuele; Sorrenti, Valeria; Barbagallo, Ignazio; Pittalà, Valeria

2017-12-15

Heme oxygenase-1 (HO-1) is the enzyme catalyzing the rate-limiting oxidative degradation of cellular heme into free iron, carbon monoxide (CO), and biliverdin, which is then rapidly converted into bilirubin. By means of these catabolic end-products and by removal of pro-oxidant heme, HO-1 exerts antioxidant, antiapoptotic, and immune-modulating effects, leading to overall cytoprotective and beneficial functions in mammalian cells. Therefore, HO-1 is considered a survival molecule in various stress-related conditions. By contrast, growing evidence suggests that HO-1 is a survival-enhancing molecule also in various solid and blood cancers, such as various types of leukemia, promoting carcinogenesis, tumor progression, and chemo-resistance. Among leukemias, chronic myeloid leukemia (CML) is currently therapeutically well treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM) and its congeners; nevertheless, resistance to all kinds of current drugs persist in a number of patients. Moreover, treatment outcomes for acute myeloid leukemia (AML) remain unsatisfactory, despite progress in chemotherapy and hematopoietic stem cell transplantation. Therefore, identification of new eligible targets that may improve leukemias therapy is of general interest. Several recent papers prove that inhibition of HO-1 through HO-1 inhibitors as well as modulation of other pathways involving HO-1 by a number of different new or known molecules, are critical for leukemia treatment. This review summarizes the current understanding of the pro-tumorigenic role of HO-1 and its potential as a molecular target for the treatment of leukemias. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hemin controls T cell polarization in sickle cell alloimmunization.

PubMed

Zhong, Hui; Bao, Weili; Friedman, David; Yazdanbakhsh, Karina

2014-07-01

Patients with sickle cell disease (SCD) often require transfusions to treat and prevent worsening anemia and other SCD complications. However, transfusions can trigger alloimmunization against transfused RBCs with serious clinical sequelae. Risk factors for alloimmunization in SCD remain poorly understood. We recently reported altered regulatory T cell (Treg) and Th responses with higher circulating Th1 (IFN-γ(+)) cytokines in chronically transfused SCD patients with alloantibodies as compared with those without alloantibodies. Because monocytes play a critical role in polarization of T cell subsets and participate in clearance of transfused RBCs, we tested the hypothesis that in response to the RBC breakdown product hemin, monocyte control of T cell polarization will differ between alloimmunized and non-alloimmunized SCD patients. Exogenous hemin induced Treg polarization in purified T cell/monocyte cocultures from healthy volunteers through the monocyte anti-inflammatory heme-degrading enzyme heme oxygenase-1. Importantly, hemin primarily through its effect on CD16+ monocytes induced an anti-inflammatory (higher Treg/lower Th1) polarization state in the non-alloimmunized SCD group, whereas it had little effect in the alloimmunized group. Non-alloimmunized SCD CD16+ monocytes expressed higher basal levels of heme oxygenase-1. Furthermore, IL-12, which contributed to a proinflammatory polarization state (low Treg/high Th1) in SCD, was dampened in hemin-treated stimulated monocytes from non-alloimmunized SCD patients, but not in the alloimmunized group. These data suggest that unlike alloimmunized patients, non-alloimmunized SCD CD16+ monocytes in response to transfused RBC breakdown products promote an anti-inflammatory state that is less conducive to alloimmunization. Copyright © 2014 by The American Association of Immunologists, Inc.

Rubber Oxygenase and Latex Clearing Protein Cleave Rubber to Different Products and Use Different Cleavage Mechanisms

PubMed Central

Birke, Jakob

2014-01-01

Two types of enzyme for oxidative cleavage of poly(cis-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by Xanthomonas sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome c peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833–13838, 2013, http://dx.doi.org/10.1073/pnas.1305560110). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of Streptomyces sp. strain K30 in a ΔroxA background of Xanthomonas sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C20 and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack. PMID:24907333

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. Copyright © 2015. Published by Elsevier Ltd.

Sampangine inhibits heme biosynthesis in both yeast and human

USDA-ARS?s Scientific Manuscript database

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...

Methane production from wheat straw with anaerobic sludge by heme supplementation.

PubMed

Xi, Yonglan; Chang, Zhizhou; Ye, Xiaomei; Xu, Rong; Du, Jing; Chen, Guangyin

2014-11-01

Wheat straw particles were directly used as substrate for batch anaerobic digestion with anaerobic sludge under 35°C to evaluate the effects of adding heme on methane production. When 1mg/l heme was added to the fermentation process with no agitated speed, a maximum cumulative methane production of 12227.8ml was obtained with cumulative methane yield of wheat straw was 257.4ml/g-TS (total solid), which was increased by 20.6% compared with 213.5ml/g-TS of no heme was added in the reactor. Meanwhile, oxido-reduction potential (ORP) level was decreased, the activity of coenzyme F420 was significantly improved and NADH/NAD(+) ratio were the highest than other experimental groups. These results suggest that heme-supplemented anaerobic sludge with no agitated speed may be providing a more reductive environment, which is a cost-effective method of anaerobic digestion from biomass waste to produce methane with less energy consuming. Copyright © 2014 Elsevier Ltd. All rights reserved.

Intracellular degradation of functionalized carbon nanotube/iron oxide hybrids is modulated by iron via Nrf2 pathway

PubMed Central

Elgrabli, Dan; Dachraoui, Walid; Marmier, Hélène de; Ménard-Moyon, Cécilia; Bégin, Dominique; Bégin-Colin, Sylvie; Bianco, Alberto; Alloyeau, Damien; Gazeau, Florence

2017-01-01

The in vivo fate and biodegradability of carbon nanotubes is still a matter of debate despite tremendous applications. In this paper we describe a molecular pathway by which macrophages degrade functionalized multi-walled carbon nanotubes (CNTs) designed for biomedical applications and containing, or not, iron oxide nanoparticles in their inner cavity. Electron microscopy and Raman spectroscopy show that intracellularly-induced structural damages appear more rapidly for iron-free CNTs in comparison to iron-loaded ones, suggesting a role of iron in the degradation mechanism. By comparing the molecular responses of macrophages derived from THP1 monocytes to both types of CNTs, we highlight a molecular mechanism regulated by Nrf2/Bach1 signaling pathways to induce CNT degradation via NOX2 complex activation and O2•−, H2O2 and OH• production. CNT exposure activates an oxidative stress-dependent production of iron via Nrf2 nuclear translocation, Ferritin H and Heme oxygenase 1 translation. Conversely, Bach1 was translocated to the nucleus of cells exposed to iron-loaded CNTs to recycle embedded iron. Our results provide new information on the role of oxidative stress, iron metabolism and Nrf2-mediated host defence for regulating CNT fate in macrophages. PMID:28120861

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H

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 cellsmore » expressing the heme enzymes are also provided by the present invention.« less

Oxidative stability of a heme iron-fortified bakery product: Effectiveness of ascorbyl palmitate and co-spray-drying of heme iron with calcium caseinate.

PubMed

Alemán, Mercedes; Bou, Ricard; Tres, Alba; Polo, Javier; Codony, Rafael; Guardiola, Francesc

2016-04-01

Fortification of food products with iron is a common strategy to prevent or overcome iron deficiency. However, any form of iron is a pro-oxidant and its addition will cause off-flavours and reduce a product's shelf life. A highly bioavailable heme iron ingredient was selected to fortify a chocolate cream used to fill sandwich-type cookies. Two different strategies were assessed for avoiding the heme iron catalytic effect on lipid oxidation: ascorbyl palmitate addition and co-spray-drying of heme iron with calcium caseinate. Oxidation development and sensory acceptability were monitored in the cookies over one-year of storage at room temperature in the dark. The addition of ascorbyl palmitate provided protection against oxidation and loss of tocopherols and tocotrienols during the preparation of cookies. In general, ascorbyl palmitate, either alone or in combination with the co-spray-dried heme iron, prevented primary oxidation and hexanal formation during storage. The combination of both strategies resulted in cookies that were acceptable from a sensory point of view after 1year of storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

PubMed

Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target

PubMed Central

Zhang, Shiming; Gerhard, Glenn S.

2009-01-01

Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics. PMID:19862332

Animal pigment bilirubin discovered in plants.

PubMed

Pirone, Cary; Quirke, J Martin E; Priestap, Horacio A; Lee, David W

2009-03-04

The bile pigment bilirubin-IXalpha is the degradative product of heme, distributed among mammals and some other vertebrates. It can be recognized as the pigment responsible for the yellow color of jaundice and healing bruises. In this paper we present the first example of the isolation of bilirubin in plants. The compound was isolated from the brilliant orange-colored arils of Strelitzia nicolai, the white bird of paradise tree, and characterized by HPLC-ESMS, UV-visible, (1)H NMR, and (13)C NMR spectroscopy, as well as comparison with an authentic standard. This discovery indicates that plant cyclic tetrapyrroles may undergo degradation by a previously unknown pathway. Preliminary analyses of related plants, including S. reginae, the bird of paradise, also revealed bilirubin in the arils and flowers, indicating that the occurrence of bilirubin is not limited to a single species or tissue type.

Haemophilus Responses to Nutritional Immunity: Epigenetic and Morphological Contribution to Biofilm Architecture, Invasion, Persistence and Disease Severity

PubMed Central

Szelestey, Blake R.; Heimlich, Derek R.; Raffel, Forrest K.; Justice, Sheryl S.; Mason, Kevin M.

2013-01-01

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites. PMID:24130500

Ruffling of Metalloporphyrins Bound to IsdG And IsdI, Two Heme Degrading Enzymes in Staphylococcus Aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, W.C.; Reniere, M.L.; Skaar, E.P.

2009-05-19

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe{sup 3+}-protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 {angstrom} or better resolution. These structures show that the metalloporphyrins are buried into similarmore » deep clefts such that the propionic acids form salt bridges to two Arg residues. His{sup 77} (IsdG) or His{sup 76} (IsdI), a critical residue required for activity, is coordinated to the Fe{sup 3+} or Co{sup 3+} atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the {beta}- and {delta}-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe{sup 3+} is pentacoordinate, and the distal side is occluded by the side chain of Ile{sup 55}. However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile{sup 55}. The chloride ion participates in a hydrogen bond to the side chain amide of Asn{sup 6}. Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the {beta}- or {delta}-meso carbon of the hemin.« less

TMEM14C is required for erythroid mitochondrial heme metabolism

PubMed Central

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

2014-01-01

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

Histidine at Position 195 is Essential for Association of Heme-b in Lcp1VH2

NASA Astrophysics Data System (ADS)

Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

2018-03-01

The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12, 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly(cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme-b in the central region of Lcp1VH2.

Histidine at Position 195 is Essential for Association of Heme- b in Lcp1VH2

NASA Astrophysics Data System (ADS)

Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

2018-05-01

The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12 , 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly( cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme- b in the central region of Lcp1VH2.

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.

RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli

PubMed Central

Kouse, Andrew B.; Righetti, Francesco; Kortmann, Jens; Narberhaus, Franz; Murphy, Erin R.

2013-01-01

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5′ untranslated region. In addition, we have identified similar nucleotide sequences within the 5′ untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5′ untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source. PMID:23704938

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

Heme-mediated cell activation: the inflammatory puzzle of sickle cell anemia.

PubMed

Guarda, Caroline Conceição da; Santiago, Rayra Pereira; Fiuza, Luciana Magalhães; Aleluia, Milena Magalhães; Ferreira, Júnia Raquel Dutra; Figueiredo, Camylla Vilas Boas; Yahouedehou, Setondji Cocou Modeste Alexandre; Oliveira, Rodrigo Mota de; Lyra, Isa Menezes; Gonçalves, Marilda de Souza

2017-06-01

Hemolysis triggers the onset of several clinical manifestations of sickle cell anemia (SCA). During hemolysis, heme, which is derived from hemoglobin (Hb), accumulates due to the inability of detoxification systems to scavenge sufficiently. Heme exerts multiple harmful effects, including leukocyte activation and migration, enhanced adhesion molecule expression by endothelial cells and the production of pro-oxidant molecules. Area covered: In this review, we describe the effects of heme on leukocytes and endothelial cells, as well as the features of vascular endothelial cells related to vaso-occlusion in SCA. Expert commentary: Free Hb, heme and iron, potent cytotoxic intravascular molecules released during hemolysis, can exacerbate, modulate and maintain the inflammatory response, a main feature of SCA. Endothelial cells in the vascular environment, as well as leukocytes, can become activated via the molecular signaling effects of heme. Due to the hemolytic nature of SCA, hemolysis represents an interesting therapeutic target for heme-scavenging purposes.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

PubMed Central

Pishchany, Gleb; Haley, Kathryn P.; Skaar, Eric P.

2013-01-01

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens. PMID:23426144

Expression and characteristics of manganese peroxidase from Ganoderma lucidum in Pichia pastoris and its application in the degradation of four dyes and phenol.

PubMed

Xu, Hui; Guo, Meng-Yuan; Gao, Yan-Hua; Bai, Xiao-Hui; Zhou, Xuan-Wei

2017-02-23

Manganese peroxidase (MnP) of white rot basidiomycetes, an extracellular heme enzyme, is part of a peroxidase superfamily that is capable of degrading the different phenolic compounds. Ganoderma, a white rot basidiomycete widely distributed worldwide, could secrete lignin-modifying enzymes (LME), including laccase (Lac), lignin peroxidases (LiP) and MnP. After the selection of a G. lucidum strain from five Ganoderma strains, the 1092 bp full-length cDNA of the MnP gene, designated as G. lucidum MnP (GluMnP1), was cloned from the selected strain. We subsequently constructed an eukaryotic expression vector, pAO815:: GlMnP, and transferred it into Pichia pastoris SMD116. Recombinant GluMnP1 (rGluMnP1) was with a yield of 126 mg/L and a molecular weight of approximately 37.72 kDa and a specific enzyme activity of 524.61 U/L. The rGluMnP1 could be capable of the decolorization of four types of dyes and the degradation of phenol. Phenol and its principal degradation products including hydroquinone, pyrocatechol, resorcinol, benzoquinone, were detected successfully in the experiments. The rGluMnP1 could be effectively expressed in Pichia pastoris and with a higher oxidation activity. We infer that, in the initial stages of the reaction, the catechol-mediated cycle should be the principal route of enzymatic degradation of phenol and its oxidation products. This study highlights the potential industrial applications associated with the production of MnP by genetic engineering methods, and the application of industrial wastewater treatment.

Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae.

PubMed

Liu, Lifang; Martínez, José L; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

2014-01-01

Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (α2β2). In this work we evaluated the expression of different combinations of α and β peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group. © 2013 Published by International Metabolic Engineering Society on behalf of International Metabolic Engineering Society.

Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant

PubMed Central

Mezoian, Taylor; Hunt, Taylor M.; Keane, Meaghan L.; Leonard, Jessica N.; Scola, Shelby E.; Beer, Emma N.; Perdue, Sarah; Pellock, Brett J.

2014-01-01

The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step. PMID:25356668

Resonance Raman detection of the heme Fe(II)-NO/2-nitrovinyl species in myoglobin

NASA Astrophysics Data System (ADS)

Ioannou, Androulla; Pinakoulaki, Eftychia

2018-01-01

The six-coordinate heme Fe(II)-NO/2-nitrovinyl species in myoglobin has been detected and characterized by resonance Raman spectroscopy. The Fe(II)-14NO and 15N-O stretching frequencies of the ferrous heme nitrosyl/2-nitrovinyl species are detected at 560 and 1587 cm-1, frequencies that are similar to those observed in the Mb heme Fe(II)-NO species. For the 2-nitrovinyl (Ca=CbNO2) moiety, which is formed upon H-abstraction from the -CbH2 group, the νs(NO2) is observed at 1322 cm-1, the νas(NO2) at 1516 cm-1 and the ν(Ca=Cb14NO2)/ ν(Ca=Cb15NO2) at 1623/1615 cm-1. The frequencies of the 2-nitrovinyl are largely unaffected by NO2-/NO binding to the heme Fe(II)/(III). The properties of the six-coordinate heme Fe(II)-NO/2-nitrovinyl species are compared to those of six-coordinate heme Fe(II)-NO and the five-coordinate heme Fe(II)-NO species isolated from meat products.

Heme oxygenase-1 induction by (S)-enantiomer of YS-51 (YS-51S), a synthetic isoquinoline alkaloid, inhibits nitric oxide production and nuclear factor-kappaB translocation in ROS 17/2.8 cells activated with inflammatory stimulants.

PubMed

Chaea, Han-Jung; Kim, Hyung-Ryong; Kang, Young Jin; Hyun, Kwang Chul; Kim, Hye Jung; Seo, Han Geuk; Lee, Jae Heun; Yun-Choi, Hye Sook; Chang, Ki Churl

2007-12-05

Activation of the inducible nitric oxide synthase (iNOS) pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis. We investigated the mechanism of action by which YS-51S, a synthetic isoquinoline alkaloid, inhibits iNOS expression and nitric oxide (NO) production in ROS 17/28 osteoblast cells activated with the mixture of TNF-alpha, IFN-gamma and LPS (MIX). YS-51S, concentration- and time-dependently, increased heme oxygenase (HO-1) expression. Treatment with YS-51S 1 h prior to MIX significantly reduced MIX-induced NO production and iNOS expression with the IC50 to NO production of 47+/-3.3 microM. Electrophoretic mobility shift assay (EMSA) and western blot analysis showed that YS-51S inhibited MIX-mediated activation and translocation of NF-kappaB to nucleus by suppressing the degradation of its inhibitory protein IkappaBalpha in cytoplasm. YS-51S also reduced NF-kappaB-luciferase activity. In addition, an HO-1 inhibitor ZnPPIX, antagonized the inhibitory effect of YS-51S on iNOS expression and DNA strand break induced by MIX, indicating prevention of NO production by YS-51S is associated with HO-1 activity. Moreover, YS-51S inhibited the oxidation of cytochrome c(2+) by peroxynitrite (PN). Our results indicated that YS-51S may be beneficial in NO-mediated inflammatory conditions such as rheumatoid arthritis by alleviating iNOS expression and NO-mediated cell death of osteoblast with 1) inducing HO-1 expression, 2) interfering the activation of NF-kappaB and 3) quenching of PN.

Coordination Chemistry of Linear Oligopyrrolic Fragments Inspired by Heme Metabolites

NASA Astrophysics Data System (ADS)

Gautam, Ritika

Linear oligopyrroles are degradation products of heme, which is converted in the presence of heme oxygenase to bile pigments, such as biliverdin and bilirubin. These tetrapyrrolic oligopyrroles are ubiquitously present in biological systems and find applications in the fields of catalysis and sensing. These linear tetrapyrrolic scaffolds are further degraded into linear tripyrrolic and dipyrrolic fragments. Although these lower oligopyrroles are abundantly present, their coordination chemistry requires further characterization. This dissertation focuses mainly on two classes of bioinspired linear oligopyrroles, propentdyopent and tripyrrindione, and their transition metal complexes, which present a rich ligand-based redox chemistry. Chapter 1 offers an overview of heme degradation to different classes of linear oligopyrroles and properties of their transition metal complexes. Chapter 2 is focused on the tripyrrin-1,14-dione scaffold of the urinary pigment uroerythrin, which coordinates divalent transition metals palladium and copper with square planar geometry. Specifically, the tripyrrin-1, 14-dione ligand binds Cu(II) and Pd(II) as a dianionic organic radical under ambient conditions. The electrochemical study confirms the presence of ligand based redox chemistry, and one electron oxidation or reduction reactions do not alter the planar geometry around the metal center. The X-Ray analysis and the electron paramagnetic resonance (EPR) studies of the complexes in the solid and solution phase reveals intermolecular interactions between the ligand based unpaired electrons and therefore formation of neutral pi-pi dimers. In Chapter 3, the antioxidant activity and the fluorescence sensor properties of the tripyrrin-1,14-dione ligand in the presence of superoxide are described. We found that the tripyrrindione ligand undergoes one-electron reduction in the presence of the superoxide radical anion (O2•- ) to form highly fluorescent H3TD1•- radical anion, which emits at 635 nm. This reaction also explains the antioxidant properties of the linear tripyrrin-1,14-dione ligand, which acts as a scavenger of O2•-. In Chapter 4, the zinc binding properties of the tripyrrin-1,14-dione ligand are described. The tripyrrolic ligand coordinates as a dianionic ligand with the divalent Zn(II) ion in both organic and aqueous buffered conditions. The complex formed is highly fluorescent with a long wavelength emission band at 648 nm. The X-Ray crystallography analysis indicates the existence of dinuclear complex [Zn(TD1•)(H2O)]2, featuring a distorted square planar geometry around the Zn(II) center. In Chapter 5, the coordination chemistry of the dipyrrin-1,9-dione fragment of propentdyopent ligand is shown with a series of transition metals like (e.g., Co(II), Ni(II), Cu(II) and Zn(II)), which form homoleptic tetrahedral complexes. The spectroscopic and electrochemical characterization confirms that the complexes shows ligand-based redox chemistry and acts as reservoirs for unpaired electrons. Chapter 6 describes the formation of the fluorescent BODIPY complex of propentdyopent ligand. The dipyrrin-1,9-dione scaffold of heme metabolite propendyopent undergoes a one-pot reaction with borontrifluoride etherate in toluene to form a green fluorescent [(pdp)BF2] complex. Spectroscopic studies reveal that the meso-unsubstituted [(pdp)BF2] complex is stable in tetrahydrofuran and has a quantum yield of 0.13. Electrochemical studies confirm that the complex undergoes ligand-based reduction and acts as a host for an unpaired electron.

Carbon monoxide: present and future indications for a medical gas

PubMed Central

Choi, Augustine M. K.

2013-01-01

Gaseous molecules continue to hold new promise in molecular medicine as experimental and clinical therapeutics. The low molecular weight gas carbon monoxide (CO), and similar gaseous molecules (e.g., H2S, nitric oxide) have been implicated as potential inhalation therapies in inflammatory diseases. At high concentration, CO represents a toxic inhalation hazard, and is a common component of air pollution. CO is also produced endogenously as a product of heme degradation catalyzed by heme oxygenase enzymes. CO binds avidly to hemoglobin, causing hypoxemia and decreased oxygen delivery to tissues at high concentrations. At physiological concentrations, CO may have endogenous roles as a signal transduction molecule in the regulation of neural and vascular function and cellular homeostasis. CO has been demonstrated to act as an effective anti-inflammatory agent in preclinical animal models of inflammation, acute lung injury, sepsis, ischemia/reperfusion injury, and organ transplantation. Additional experimental indications for this gas include pulmonary fibrosis, pulmonary hypertension, metabolic diseases, and preeclampsia. The development of chemical CO releasing compounds constitutes a novel pharmaceutical approach to CO delivery with demonstrated effectiveness in sepsis models. Current and pending clinical evaluation will determine the usefulness of this gas as a therapeutic in human disease. PMID:23525151

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production.

PubMed

Nogueira, Natália P; Saraiva, Francis M S; Oliveira, Matheus P; Mendonça, Ana Paula M; Inacio, Job D F; Almeida-Amaral, Elmo E; Menna-Barreto, Rubem F; Laranja, Gustavo A T; Torres, Eduardo J Lopes; Oliveira, Marcus F; Paes, Marcia C

2017-07-01

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation. Copyright © 2017. Published by Elsevier Inc.

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew

Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations inmore » 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.« less

Discovery and characterization of heme enzymes from unsequenced bacteria: application to microbial lignin degradation.

PubMed

Brown, Margaret E; Walker, Mark C; Nakashige, Toshiki G; Iavarone, Anthony T; Chang, Michelle C Y

2011-11-16

Bacteria and other living organisms offer a potentially unlimited resource for the discovery of new chemical catalysts, but many interesting reaction phenotypes observed at the whole organism level remain difficult to elucidate down to the molecular level. A key challenge in the discovery process is the identification of discrete molecular players involved in complex biological transformations because multiple cryptic genetic components often work in concert to elicit an overall chemical phenotype. We now report a rapid pipeline for the discovery of new enzymes of interest from unsequenced bacterial hosts based on laboratory-scale methods for the de novo assembly of bacterial genome sequences using short reads. We have applied this approach to the biomass-degrading soil bacterium Amycolatopsis sp. 75iv2 ATCC 39116 (formerly Streptomyces setonii and S. griseus 75vi2) to discover and biochemically characterize two new heme proteins comprising the most abundant members of the extracellular oxidative system under lignin-reactive growth conditions.

Hemin-utilizing G-quadruplex DNAzymes are strongly active in organic co-solvents.

PubMed

Canale, Thomas D; Sen, Dipankar

2017-05-01

The widespread use of organic solvents in industrial processes has focused in recent years on the utility of "green" solvents - those with less harmful environmental, health, and safety properties - such as methanol and formamide. However, protein enzymes, regarded as green catalysts, are often incompatible with organic solvents. Herein, we have explored the oxidative properties of a Fe(III)-heme, or hemin, utilizing catalytic DNA (heme·DNAzyme) in different green solvent-water mixtures. We find that the peroxidase and peroxygenase activities of the heme·DNAzyme are strongly enhanced in 20-30% v/v methanol or formamide, relative to water alone. Protic solvent content of >30% v/v gradually diminishes heme·DNAzyme catalytic activity; however, the heme·DNAzyme is still active in as high as 80% v/v methanol. In contrast to protic solvents, aqueous dimethylformamide solutions largely inhibit heme·DNAzyme activity. In view of the strong catalytic activity of heme·DNAzyme in aqueous methanol, we were able to determine that a 60% v/v methanol-water mixture gives the most optimal yield of the dibenzothiophene sulfoxide (DBTO) oxidation product of petroleum-derived dibenzothiophene (DBT). The high product yield reflects both DNAzyme catalysis and a high substrate availability. Overall, these results emphasize the excellent promise of G-quadruplex forming DNA catalysts in application to "greener" industrial chemistry. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vlasova, Irina I., E-mail: irina.vlasova@yahoo.com; Vakhrusheva, Tatyana V.; Sokolov, Alexey V.

Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acidmore » (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of neutrophils in blood causes an increase in plasma MPO concentration. ► Intraperitoneal injection of PEG-SWCNTs in mice induces an inflammatory response.« less

Cerebroprotective functions of HO-2.

PubMed

Parfenova, Helena; Leffler, Charles W

2008-01-01

The constitutive isoform of heme oxygenase, HO-2, is highly expressed in the brain and in cerebral vessels. HO-2 functions in the brain have been evaluated using pharmacological inhibitors of the enzyme and HO-2 gene deletion in in vivo animal models and in cultured cells (neurons, astrocytes, cerebral vascular endothelial cells). Rapid activation of HO-2 via post-translational modifications without upregulation of HO-2 expression or HO-1 induction coincides with the increase in cerebral blood flow aimed at maintaining brain homeostasis and neuronal survival during seizures, hypoxia, and hypotension. Pharmacological inhibition or gene deletion of brain HO-2 exacerbates oxidative stress induced by seizures, glutamate, and inflammatory cytokines, and causes cerebral vascular injury. Carbon monoxide (CO) and bilirubin, the end products of HO-catalyzed heme degradation, have distinct cytoprotective functions. CO, by binding to a heme prosthetic group, regulates the key components of cell signaling, including BK(Ca) channels, guanylyl cyclase, NADPH oxidase, and the mitochondria respiratory chain. Cerebral vasodilator effects of CO are mediated via activation of BK(Ca) channels and guanylyl cyclase. CO, by inhibiting the major components of endogenous oxidant-generating machinery, NADPH oxidase and the cytochrome C oxidase of the mitochondrial respiratory chain, blocks formation of reactive oxygen species. Bilirubin, via redox cycling with biliverdin, is a potent oxidant scavenger that removes preformed oxidants. Overall, HO-2 has dual housekeeping cerebroprotective functions by maintaining autoregulation of cerebral blood flow aimed at improving neuronal survival in a changing environment, and by providing an effective defense mechanism that blocks oxidant formation and prevents cell death caused by oxidative stress.

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation*

PubMed Central

Fernandez, Patricia L.; Dutra, Fabianno F.; Alves, Letícia; Figueiredo, Rodrigo T.; Mourão-Sa, Diego; Fortes, Guilherme B.; Bergstrand, Sophie; Lönn, David; Cevallos, Ricardo R.; Pereira, Renata M. S.; Lopes, Ulisses G.; Travassos, Leonardo H.; Paiva, Claudia N.; Bozza, Marcelo T.

2010-01-01

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases. PMID:20729208

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages

PubMed Central

Sawle, Philip; Foresti, Roberta; Mann, Brian E; Johnson, Tony R; Green, Colin J; Motterlini, Roberto

2005-01-01

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10–100 μM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-α, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation. PMID:15880142

S-Nitrosylation and the Development of Pulmonary Hypertension

DTIC Science & Technology

2010-02-01

cleavage, catalyzed by transition metal ions and reactive oxygen species, or through enzymatic degradation. One specific enzyme that regulates S...Hb causes a change in its conformation, resulting in the transfer of nitrosonium (NO+) equivalents from the heme (Fe+2) to the thiol (cysteine β93

Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

PubMed Central

Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

2014-01-01

Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted. PMID:24416467

Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage

PubMed Central

Wang, Jian; Doré, Sylvain

2008-01-01

Because heme oxygenase (HO) is the rate limiting enzyme in the degradation of the pro-oxidant hemin/heme from blood, here we investigated the contribution of the inducible HO-1 to early brain injury produced by intracerebral haemorrhage (ICH). We found that after induction of ICH, HO-1 proteins were highly detectable in the peri-ICH region predominantly in microglia/macrophages and endothelial cells. Remarkably, the injury volume was significantly smaller in HO-1 knockout (HO-1−/−) mice than in wild-type controls 24 and 72 h after ICH. Although the brain water content did not appear to be significantly different, the protection in HO-1−/− mice was associated with a marked reduction in ICH-induced leucocyte infiltration, microglia/macrophage activation and free radical levels. These data reveal a previously unrecognized role of HO-1 in early brain injury after ICH. Thus, modulation of HO-1 signalling should be assessed further in clinical settings, especially for haemorrhagic states. PMID:17525142

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

PubMed

Smirnov, Valeriy V; Roth, Justine P

2014-10-01

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

Heme-Induced ROS in Trypanosoma Cruzi Activates CaMKII-Like That Triggers Epimastigote Proliferation. One Helpful Effect of ROS

PubMed Central

Nogueira, Natália Pereira de Almeida; de Souza, Cintia Fernandes; Saraiva, Francis Monique de Souza; Sultano, Pedro Elias; Dalmau, Sergio Ranto; Bruno, Roberta Eitler; de Lima Sales Gonçalves, Renata; Laranja, Gustavo Augusto Travassos; Leal, Luís Henrique Monteiro; Coelho, Marsen Garcia Pinto; Masuda, Claudio A.; Oliveira, Marcus F.; Paes, Marcia Cristina

2011-01-01

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time-and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism. PMID:22022475

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis*

PubMed Central

Adamczack, Julia; Hoffmann, Martin; Papke, Ulrich; Haufschildt, Kristin; Nicke, Tristan; Bröring, Martin; Sezer, Murat; Weimar, Rebecca; Kuhlmann, Uwe; Hildebrandt, Peter; Layer, Gunhild

2014-01-01

Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1. PMID:25204657

The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

PubMed

Terentis, Andrew C; Freewan, Mohammed; Sempértegui Plaza, Tito S; Raftery, Mark J; Stocker, Roland; Thomas, Shane R

2010-01-26

The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory role by catalyzing the oxidative degradation of l-tryptophan. Here we show that the selenezal drug ebselen is a potent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a manner independent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDO (rIDO) with an apparent inhibition constant of 94 +/- 17 nM. Optical and resonance Raman spectroscopy showed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron from the predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch of the CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed that ebselen enhanced nonproductive l-tryptophan binding, while circular dichroism indicated that the drug reduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed that ebselen reacted with multiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitol reversed the effects of the drug on the heme environment and significantly restored enzyme activity. These findings indicate that ebselen inhibits IDO activity by reacting with the enzyme's cysteine residues that result in changes to protein conformation and active site heme, leading to an increase in the level of nonproductive substrate binding. This study highlights that modification of cysteine residues is a novel and effective means of inhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents a previously unrecognized in vivo target of ebselen.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Te-Chang; Institute of Pharmacology, Yang Min University, Taipei, Taiwan; Cheng, I-Cheng

Arsenic trioxide (ATO) treatment is a useful therapy against human acute promyelocytic leukemia (APL), however, it concomitantly brings potential adverse consequences including serious side effect, human carcinogenicity and possible development of resistance. This investigation revealed that those problems might be relaxed by simultaneous application with (-)-epigallocatechin-3-gallate (EGCG), one of the major components from green tea. EGCG significantly lowered down the ATO concentration required for an effective control of APL cells, HL-60. The simultaneous treatment of ATO with EGCG induced a mitochondria-dependent apoptosis in HL-60 cells significantly, which accounted for more than 70% of the cell death in the treatment. Themore » mechanism of apoptosis induction was elucidated. EGCG in HL-60 cells acted as a pro-oxidant enhancing intracellular hydrogen peroxide significantly. ATO, on the other hand, induced heme oxygenase-1 (HO-1) to catalyze heme degradation, thereby provided ferrous iron for EGCG-induced hydrogen peroxide to precede Fenton reaction, which in turn generated deleterious reactive oxygen species to damage cell. In addition, EGCG inhibited expression of ferritin, which supposedly to sequester harmful ferrous iron, thereby augmented the occurrence of Fenton reaction. This investigation also provided evidence that ATO, since mainly acted to induce HO-1 in simultaneous treatment with EGCG, could be replaced by other HO-1 inducer with much less human toxicity. Furthermore, several of our preliminary investigations revealed that the enhanced cytotoxicity induced by combining heme degradation and Fenton reaction is selectively toxic to malignant but not non-malignant cells.« less

A peroxidase mimic with atom transfer radical polymerization activity constructed through the grafting of heme onto metal-organic frameworks.

PubMed

Jiang, Wei; Pan, Yue; Yang, Jiebing; Liu, Yong; Yang, Yan; Tang, Jun; Li, Quanshun

2018-07-01

Atom transfer radical polymerization (ATRP) has been considered to be an efficient strategy for constructing functional macromolecules owing to its simple operation and versatile monomers, and thus it is of great significance to develop ideal catalysts with higher activity and perfect reusability. We constructed a peroxidase mimic through the grafting of heme onto metal-organic frameworks UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. After the systematic characterization of structure, the composite Heme-ZrMOF was demonstrated to possess high peroxidase activity using 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and 3,3',5,5'-tetramethylbenzidine as substrates. The enzyme mimic was then used as catalysts in the ATRP reactions of different monomers, in which favorable monomer conversion (44.6-98.0%) and product molecular weight (8600-25,600 g/mol) could be obtained. Compared to free heme, Heme-ZrMOF could efficiently achieve the easy separation of heme from the catalytic system and facilitate the ATRP reaction in an aqueous environment to avoid the utilization of organic solvents. In conclusion, the enzyme mimic Heme-ZrMOF could be potentially used as an effective catalyst for preparing well-defined polymers with biomedical applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Detergent-Mediated Formation of β-Hematin: Heme Crystallization Promoted by Detergents Implicates Nanostructure Formation for Use as a Biological Mimic

PubMed Central

2016-01-01

Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of β-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate β-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent’s molecular structure and the corresponding kinetics of β-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent’s ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote β-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway. PMID:27175104

A Model for the Flexibility of the Distal Histidine in Dehaloperoxidase-Hemoglobin A Based on X-ray Crystal Structures of the Carbon Monoxide Adduct

PubMed Central

2015-01-01

Dehaloperoxidase hemoglobin A (DHP A) is a multifunctional hemoglobin that appears to have evolved oxidative pathways for the degradation of xenobiotics as a protective function that complements the oxygen transport function. DHP A possesses at least two internal binding sites, one for substrates and one for inhibitors, which include various halogenated phenols and indoles. Herein, we report the X-ray crystallographic structure of the carbonmonoxy complex (DHPCO). Unlike other DHP structures with 6-coordinated heme, the conformation of the distal histidine (H55) in DHPCO is primarily external or solvent exposed, despite the fact that the heme Fe is 6-coordinated. As observed generally in globins, DHP exhibits two distal histidine conformations (one internal and one external). In previous structural studies, we have shown that the distribution of H55 conformations is weighted strongly toward the external position when the DHP heme Fe is 5-coordinated. The large population of the external conformation of the distal histidine observed in DHPCO crystals at pH 6.0 indicates that some structural factor in DHP must account for the difference from other globins, which exhibit a significant external conformation only when pH < 4.5. While the original hypothesis suggested that interaction with a heme-Fe-bound ligand was the determinant of H55 conformation, the current study forces a refinement of that hypothesis. The external or open conformation of H55 is observed to have interactions with two propionate groups in heme, at distances of 3.82 and 2.73 Å, respectively. A relatively weak hydrogen bonding interaction between H55 and CO, combined with strong interactions with heme propionate (position 6), is hypothesized to strengthen the external conformation of H55. Density function theory (DFT) calculations were conducted to test whether there is a weaker hydrogen bond interaction between H55 and heme bonded CO or O2. Molecular dynamics simulations were conducted to examine how the tautomeric forms of H55 affect the dynamic motions of the distal histidine that govern the switching between open and closed conformations. The calculations support the modified hypothesis suggesting a competition between the strength of interactions with heme ligand and the heme propionates as the factors that determine the conformation of the distal histidine. PMID:24670063

The role of myoglobin degradation in nephrotoxicity after rhabdomyolysis.

PubMed

Zorova, Ljubava D; Pevzner, Irina B; Chupyrkina, Anastasia A; Zorov, Savva D; Silachev, Denis N; Plotnikov, Egor Y; Zorov, Dmitry B

2016-08-25

The fate of myoglobin in renal cells was explored in an animal model of rhabdomyolysis known as the pathology highly related to oxidative stress resulting in impairment of renal functioning. The working hypothesis was that the proper degradation of myoglobin in rhabdomyolytic kidney can activate the reparative processes in the tissue. We found that incubation of myoglobin with kidney cells causes its accumulation in the cytoplasm. In rhabdomyolytic rats, the level of heme and free iron in cytoplasm and mitochondria of kidney cells is remarkably increased while inhibition of proteolysis results in further elevation of myoglobin content in the renal tissue. Heme oxygenase and ferritin levels were found to be increased in the kidney tissue at rhabdomyolysis and simulating conditions performed by i/v injection of myoglobin. In addition, the level of peroxidized lipids was high in rhabdomyolytic kidney and became even higher after inhibition of proteolysis by aprotinin. Elevated levels of carbonylated proteins were also observed after rhabdomyolysis, however, if prior to induction of rhabdomyolysis the injection of myoglobin was done, the level of carbonylated proteins dropped versus unprimed kidney tissue thus affording protection to the kidney against oxidative stress. Injection of myoglobin to the rat results in impairment of renal functioning and inhibition of myoglobin degradation in the rhabdomyolytic animal aggravates acute renal failure, demonstrating that degradation of myoglobin is somehow beneficial although it may result in undesired release of free iron which can participate in toxic redox cycling. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

CARBON MONOXIDE REVERSIBLY DISRUPTS IRON HOMEOSTATIS AND RESPIRATORY EPITHELIAL CELLS FUNCTION

EPA Science Inventory

Iron dissociation from heme is a major factor in iron metabolism and cellular concentrations of the metal correlate inversely with the expression of heme oxygenase (HO). We tested the hypothesis that 1) exposure to a product of HO, carbon monoxide (CO), disturbs iron homeostas...

Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change*s

PubMed Central

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

2008-01-01

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H2. 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 H2, 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

Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs

PubMed Central

Quan, Shuo; Yang, Liming; Abraham, Nader G.; Kappas, Attallah

2001-01-01

Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin. PMID:11593038

Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

PubMed

Quan, S; Yang, L; Abraham, N G; Kappas, A

2001-10-09

Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

PubMed

Manoj, Kelath Murali; Parashar, Abhinav; Venkatachalam, Avanthika; Goyal, Sahil; Satyalipsu; Singh, Preeti Gunjan; Gade, Sudeep K; Periyasami, Kalaiselvi; Jacob, Reeba Susan; Sardar, Debosmita; Singh, Shanikant; Kumar, Rajan; Gideon, Daniel A

2016-06-01

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Heme oxygenase is not involved in the anti-proliferative effects of statins on pancreatic cancer cells.

PubMed

Vanova, K; Boukalova, S; Gbelcova, H; Muchova, L; Neuzil, J; Gurlich, R; Ruml, T; Vitek, L

2016-05-12

Pancreatic cancer is recognized as one of the most fatal tumors due to its aggressiveness and resistance to therapy. Statins were previously shown to inhibit the proliferation of cancer cells via various signaling pathways. In healthy tissues, statins activate the heme oxygenase pathway, nevertheless the role of heme oxygenase in pancreatic cancer is still controversial. The aim of this study was to evaluate, whether anti-proliferative effects of statins in pancreatic cancer cells are mediated via the heme oxygenase pathway. In vitro effects of various statins and hemin, a heme oxygenase inducer, on cell proliferation were evaluated in PA-TU-8902, MiaPaCa-2 and BxPC-3 human pancreatic cancer cell lines. The effect of statins on heme oxygenase activity was assessed and heme oxygenase-silenced cells were used for pancreatic cancer cell proliferation studies. Cell death rate and reactive oxygen species production were measured in PA-TU-8902 cells, followed by evaluation of the effect of cerivastatin on GFP-K-Ras trafficking and expression of markers of invasiveness, osteopontin (SPP1) and SOX2. While simvastatin and cerivastatin displayed major anti-proliferative properties in all cell lines tested, pravastatin did not affect the cell growth at all. Strong anti-proliferative effect was observed also for hemin. Co-treatment of cerivastatin and hemin increased anti-proliferative potential of these agents, via increased production of reactive oxygen species and cell death compared to individual treatment. Heme oxygenase silencing did not prevent pancreatic cancer cells from the tumor-suppressive effect of cerivastatin or hemin. Cerivastatin, but not pravastatin, protected Ras protein from trafficking to the cell membrane and significantly reduced expressions of SPP1 (p < 0.05) and SOX2 (p < 0.01). Anti-proliferative effects of statins and hemin on human pancreatic cancer cell lines do not seem to be related to the heme oxygenase pathway. While hemin triggers reactive oxygen species-induced cell death, cerivastatin targets Ras protein trafficking and affects markers of invasiveness.

Significance of heme-based respiration in meat spoilage caused by Leuconostoc gasicomitatum.

PubMed

Jääskeläinen, Elina; Johansson, Per; Kostiainen, Olli; Nieminen, Timo; Schmidt, Georg; Somervuo, Panu; Mohsina, Marzia; Vanninen, Paula; Auvinen, Petri; Björkroth, Johanna

2013-02-01

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) which causes spoilage in cold-stored modified-atmosphere-packaged (MAP) meat products. In addition to the fermentative metabolism, L. gasicomitatum is able to respire when exogenous heme and oxygen are available. In this study, we investigated the respiration effects on growth rate, biomass, gene expression, and volatile organic compound (VOC) production in laboratory media and pork loin. The meat samples were evaluated by a sensory panel every second or third day for 29 days. We observed that functional respiration increased the growth (rate and yield) of L. gasicomitatum in laboratory media with added heme and in situ meat with endogenous heme. Respiration increased enormously (up to 2,600-fold) the accumulation of acetoin and diacetyl, which are buttery off-odor compounds in meat. Our transcriptome analyses showed that the gene expression patterns were quite similar, irrespective of whether respiration was turned off by excluding heme from the medium or mutating the cydB gene, which is essential in the respiratory chain. The respiration-based growth of L. gasicomitatum in meat was obtained in terms of population development and subsequent development of sensory characteristics. Respiration is thus a key factor explaining why L. gasicomitatum is so well adapted in high-oxygen packed meat.

Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation

PubMed Central

Majtan, Tomas; Frerman, Frank E.

2011-01-01

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS. PMID:21184140

Prolonged Neutrophil Dysfunction Following Plasmodium falciparum Malaria is Related to Hemolysis and Heme Oxygenase-1 Induction1

PubMed Central

Cunnington, Aubrey J.; Njie, Madi; Correa, Simon; Takem, Ebako N.; Riley, Eleanor M.; Walther, Michael

2012-01-01

It is not known why people are more susceptible to bacterial infections such as non-Typhoid Salmonella (NTS) during and after a malaria infection but, in mice, malarial hemolysis impairs resistance to NTS by impairing the neutrophil oxidative burst. This acquired neutrophil dysfunction is a consequence of induction of the cytoprotective, heme degrading enzyme heme oxygenase-1 (HO-1) in neutrophil progenitors in bone marrow. In this study, we assessed whether neutrophil dysfunction occurs in humans with malaria and how this relates to hemolysis. We evaluated neutrophil function in 58 Gambian children with Plasmodium falciparum malaria (55 (95%) with uncomplicated disease), and examined associations with erythrocyte count, haptoglobin, hemopexin, plasma heme, expression of receptors for heme uptake, and HO-1 induction. Malaria caused the appearance of a dominant population of neutrophils with reduced oxidative burst activity, which gradually normalized over 8 weeks of follow-up. The degree of neutrophil impairment correlated significantly with markers of hemolysis and HO-1 induction. HO-1 expression was increased in blood during acute malaria, but at a cellular level HO-1 expression was modulated by changes in surface expression of the haptoglobin receptor (CD163). These findings demonstrate that neutrophil dysfunction occurs in P. falciparum malaria and support the relevance of the mechanistic studies in mice. Furthermore, they suggest the presence of a regulatory pathway to limit HO-1 induction by hemolysis in the context of infection, and indicate new targets for therapeutic intervention to abrogate the susceptibility to bacterial infection in the context of hemolysis in humans. PMID:23100518

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.

Biosynthesis of Lincosamide Antibiotics: Reactions Associated with Degradation and Detoxification Pathways Play a Constructive Role.

PubMed

Zhang, Daozhong; Tang, Zhijun; Liu, Wen

2018-06-19

Natural products typically are small molecules produced by living organisms. These products possess a wide variety of biological activities and thus have historically played a critical role in medicinal chemistry and chemical biology either as chemotherapeutic agents or as useful tools. Natural products are not synthesized for use by human beings; rather, living organisms produce them in response to various biochemical processes and environmental concerns, both internal and external. These processes/concerns are often dynamic and thus motivate the diversification, optimization, and selection of small molecules in line with changes in biological function. Consequently, the interactions between living organisms and their environments serve as an engine that drives coevolution of natural products and their biological functions and ultimately programs the constant theme of small-molecule development in nature based on biosynthesis generality and specificity. Following this theme, we herein review the biosynthesis of lincosamide antibiotics and dissect the process through which nature creates an unusual eight-carbon aminosugar (lincosamide) and then functionalizes this common high-carbon chain-containing sugar core with diverse l-proline derivatives and sulfur appendages to form individual members, including the clinically useful anti-infective agent lincomycin A and its naturally occurring analogues celesticetin and Bu-2545. The biosynthesis of lincosamide antibiotics is unique in that it results from an intersection of anabolic and catabolic chemistry. Many reactions that are usually involved in degradation and detoxification play a constructive role in biosynthetic processes. Formation of the trans-4-propyl-l-proline residue in lincomycin A biosynthesis requires an oxidation-associated degradation-like pathway composed of heme peroxidase-catalyzed ortho-hydroxylation and non-heme 2,3-dioxygenase-catalyzed extradiol cleavage for l-tyrosine processing prior to the building-up process. Mycothiol (MSH) and ergothioneine (EGT), two small-molecule thiols that are known for their redox-relevant roles in protection against various endogenous and exogenous stresses, function through two unusual S-glycosylations to mediate an eight-carbon aminosugar transfer, activation, and modification during the molecular assembly and tailoring processes in lincosamide antibiotic biosynthesis. Related intermediates include an MSH S-conjugate, mercapturic acid, and a thiomethyl product, which are reminiscent of intermediates found in thiol-mediated detoxification metabolism. In these biosynthetic pathways, "old" protein folds can result in "new" enzymatic activity, such as the DinB-2 fold protein for thiol exchange between EGT and MSH, the γ-glutamyltranspeptidase homologue for C-C bond cleavage, and the pyridoxal-5'-phosphate-dependent enzyme for diverse S-functionalization, generating interest in how nature develops remarkably diverse biochemical functions using a limited range of protein scaffolds. These findings highlight what we can learn from natural product biosynthesis, the recognition of its generality and specificity, and the natural theme of the development of bioactive small molecules, which enables the diversification process to advance and expand small-molecule functions.

Salivary proline-rich protein may reduce tannin-iron chelation: a systematic narrative review.

PubMed

Delimont, Nicole M; Rosenkranz, Sara K; Haub, Mark D; Lindshield, Brian L

2017-01-01

Tannins are often cited for antinutritional effects, including chelation of non-heme iron. Despite this, studies exploring non-heme iron bioavailability inhibition with long-term consumption have reported mixed results. Salivary proline-rich proteins (PRPs) may mediate tannin-antinutritional effects on non-heme iron bioavailability. To review evidence regarding biochemical binding mechanisms and affinity states between PRPs and tannins, as well as effects of PRPs on non-heme iron bioavailability with tannin consumption in vivo. Narrative systematic review and meta-analysis. Common themes in biochemical modeling and affinity studies were collated for summary and synthesis; data were extracted from in vivo experiments for meta-analysis. Thirty-two studies were included in analysis. Common themes that positively influenced tannin-PRP binding included specificity of tannin-PRP binding, PRP and tannin stereochemistry. Hydrolyzable tannins have different affinities than condensed tannins when binding to PRPs. In vivo, hepatic iron stores and non-heme iron absorption are not significantly affected by tannin consumption ( d  = -0.64-1.84; -2.7-0.13 respectively), and PRP expression may increase non-heme iron bioavailability with tannin consumption. In vitro modeling suggests that tannins favor PRP binding over iron chelation throughout digestion. Hydrolyzable tannins are not representative of tannin impact on non-heme iron bioavailability in food tannins because of their unique structural properties and PRP affinities. With tannin consumption, PRP production is increased, and may be an initial line of defense against tannin-non-heme iron chelation in vivo . More research is needed to compare competitive binding of tannin-PRP to tannin-non-heme iron complexes, and elucidate PRPs' role in adaption to non-heme iron bioavailability in vivo.

Heme oxygenase-1 in tumor biology and therapy.

PubMed

Was, Halina; Dulak, Jozef; Jozkowicz, Alicja

2010-12-01

Heme oxygenase-1 (HO-1) degrades heme to carbon monoxide (CO), biliverdin, and ferrous iron. As HO-1 expression is highly increased by stressful conditions, the major role of the enzyme is the protection against oxidative injury. Additionally, it regulates cell proliferation, modulates inflammatory response and facilitates angiogenesis. Beneficial activities of HO-1 have been recognized in many pathological states e.g. atherosclerosis, diabetes, ischemia/reperfusion injury or organ transplantation. Interestingly HO-1 expression is very often boosted in tumor tissues and could be further elevated in response to radio-, chemo-, or photodynamic therapy. A growing body of evidence suggests that HO-1 may play a role in tumor induction and can potently improve the growth and spread of tumors. This review discusses the implications of HO-1 properties for tumor proliferation and cell death, differentiation, angiogenesis and metastasis, and tumor-related inflammation. Finally, it suggests that pharmacological agents that regulate HO activity or HO-1 gene silencing may become powerful tools for preventing the onset or progression of various cancers and sensitize them to anticancer therapies.

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

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

PubMed

Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

2016-02-01

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis. © 2015 The Protein Society.

Bioavailability of a heme-iron concentrate product added to chocolate biscuit filling in adolescent girls living in a rural area of Mexico.

PubMed

González-Rosendo, Guillermina; Polo, Javier; Rodríguez-Jerez, José Juan; Puga-Díaz, Rubén; Reyes-Navarrete, Eduardo G; Quintero-Gutiérrez, Adrián G

2010-04-01

A heme-iron concentrate product derived from swine hemoglobin was used to enrich the chocolate-flavored filling of biscuits and the bioavailability of this source of heme-iron was assessed in adolescent girls. The placebo control (PC) group consisted of 35 teenagers with the highest baseline hemoglobin concentrations. The supplemented groups were randomized to receive biscuits fortified with iron sulfate (IS, n = 37) or heme-iron concentrate (HIC, n = 40). Both groups were supplemented with 10.3 mg Fe/d for 7 wk. Blood chemistry and hematology analyses were performed at baseline and at the end of the study. The baseline prevalence of anemia (hemoglobin <12 g/dl) in the entire group was 3.9% and by the end of the study it had fallen to 2.3%. The hemoglobin levels in both supplemented groups increased (P < 0.05) during the study period from 13.6 and 13.5 g/dl for HIC and IS, respectively, at baseline to 14 g/dl at the end of the study. Serum ferritin concentrations decreased by the end of the study in both the PC and IS groups (P < 0.05), but not in the heme group. In conclusion, iron bioavailability from HIC-fortified biscuits was calculated to be 23.7% higher than that observed for IS, as shown by the differences observed in serum ferritin levels during the study. The iron contained in the heme-iron concentrate was well absorbed and tolerated by the adolescents included in the study.

Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis

PubMed Central

Moore, Lee R.; Fujioka, Hisashi; Williams, P. Stephen; Chalmers, Jeffrey J.; Grimberg, Brian; Zimmerman, Peter; Zborowski, Maciej

2013-01-01

During intra-erythrocytic development, malaria trophozoites digest hemoglobin, which leads to parasite growth and asexual replication while accumulating toxic heme. To avoid death, the parasite synthesizes insoluble hemozoin crystals in the digestive vacuole through polymerization of β-hematin dimers. In the process, the heme is converted to a high-spin ferriheme whose magnetic properties were studied as early as 1936 by Pauling et al. Here, by magnetophoretic cell motion analysis, we provide evidence for a graduated increase of live cell magnetic susceptibility with developing blood-stage parasites, compatible with the increase in hemozoin content and the mechanism used by P. falciparum to avoid heme toxicity. The measured magnetophoretic mobility of the erythrocyte infected with a late-stage schizont form was m = 2.94 × 10−6 mm3 s/kg, corresponding to the net volume magnetic susceptibility (relative to water) of Δχ = 1.80 × 10−6, significantly higher than that of the oxygenated erythrocyte (−0.18×10−6) but lower than that of the fully deoxygenated erythrocyte (3.33×10−6). The corresponding fraction of hemoglobin converted to hemozoin, calculated based on the known magnetic susceptibilities of hemoglobin heme and hemozoin ferriheme, was 0.50, in agreement with the published biochemical and crystallography data. Magnetophoretic analysis of live erythrocytes could become significant for antimalarial drug susceptibility and resistance determination. PMID:16461330

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, Amit V., E-mail: amit@pandeylab.org; 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 formmore » 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.« less

The Bordetella bhu Locus Is Required for Heme Iron Utilization

PubMed Central

Vanderpool, Carin K.; Armstrong, Sandra K.

2001-01-01

Bordetella pertussis and Bordetella bronchiseptica are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (bhu, for Bordetella heme utilization) were identified in a B. pertussis genomic sequence database, and the corresponding DNA was isolated from a virulent strain of B. pertussis. A B. pertussis bhuR mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain, bhuR mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned bhuRSTUV genes restored heme utilization to wild-type levels. In parallel studies, B. bronchiseptica bhu sequences were also identified and a B. bronchiseptica bhuR mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type B. bronchiseptica parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the bhuR mutant. Furthermore, production of BhuR by iron-starved B. bronchiseptica was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the bhu cluster identified open reading frames predicted to encode homologs of the Escherichia coli ferric citrate uptake regulators FecI and FecR. These putative Bordetella regulators may mediate heme-responsive positive transcriptional control of the bhu genes. PMID:11418569

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Carbon monoxide alleviates lipopolysaccharide-induced oxidative stress injury through suppressing the expression of Fis1 in NR8383 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Jia; Yu, Jian-bo, E-mail: yujianbo11@126.com; Liu, Wei

Acute respiratory distress syndrome (ARDS) is one of the most devastating complications of sepsis lacking of effective therapy. Mitochondrial dynamics undergoing continuous fusion and fission play a crucial role in mitochondrial structure and function. Fis1, as a small protein located on the outer membrane of mitochondria, has been thought to be an important protein mediated mitochondrial fission. During ARDS, alveolar macrophages suffer from increased oxidative stress and apoptosis, and also accompanied by disrupted mitochondrial dynamics. In addition, as one of the products of heme degradation catalyzed by heme oxygenase, carbon monoxide (CO) possesses powerful protective properties in vivo or inmore » vitro models, such as anti-inflammatory, antioxidant and anti-apoptosis function. However, there is little evidence that CO alleviates oxidative stress damage through altering mitochondrial fission in alveolar macrophages. In the present study, our results showed that CO increased cell vitality, improved mitochondrial SOD activity, reduced reactive oxygen species (ROS) production and inhibited cell apoptosis in NR8383 exposed to LPS. Meanwhile, CO decreased the expression of Fis1, increased mitochondrial membrane potential and sustained elongation of mitochondria in LPS-incubated NR8383. Overall, our study underscored a critical role of CO in suppressing the expression of Fis1 and alleviating LPS- induced oxidative stress damage in alveolar macrophages. - Highlights: • LPS exposure triggered cell injury in NR8383. • CO alleviated LPS-induced oxidative stress damage in alveolar macrophages. • CO inhibited Fis1 levels and improved mitochondrial function in LPS-induced NR8383.« less

Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages.

PubMed

De Maere, Hannelore; Chollet, Sylvie; De Brabanter, Jos; Michiels, Chris; Paelinck, Hubert; Fraeye, Ilse

2018-01-01

Nitrite is commonly used in meat products due to its plural technological advantages. However, it is controversial because of its detrimental side effects on health. Within the context of nitrite reduction, zinc protoporphyrin IX (Zn(II)PPIX) formation in meat products as natural red colouring agent has been suggested. This investigation presents the evaluation of naturally occurring pigments, namely Zn(II)PPIX, protoporphyrin IX (PPIX) and heme in nitrite-free dry fermented sausages in function of time, meat source (pork, horsemeat and a combination of both meat sources) and pH condition. In function of time, Zn(II)PPIX and PPIX were formed and heme content decreased. Higher pH conditions promoted Zn(II)PPIX and PPIX formation, whereas the influence of pH on heme was less clear. The use of horsemeat also promoted Zn(II)PPIX formation. Moreover, even similar amounts were formed when it was combined with pork. Product redness, however, could not be related to Zn(II)PPIX formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of ROS Production and Vascular Function by Carbon Monoxide

PubMed Central

Choi, Yoon Kyung; Por, Elaine D.; Kwon, Young-Guen; Kim, Young-Myeong

2012-01-01

Carbon monoxide (CO) is a gaseous molecule produced from heme by heme oxygenase (HO). CO interacts with reduced iron of heme-containing proteins, leading to its involvement in various cellular events via its production of mitochondrial reactive oxygen species (ROS). CO-mediated ROS production initiates intracellular signal events, which regulate the expression of adaptive genes implicated in oxidative stress and functions as signaling molecule for promoting vascular functions, including angiogenesis and mitochondrial biogenesis. Therefore, CO generated either by exogenous delivery or by HO activity can be fundamentally involved in regulating mitochondria-mediated redox cascades for adaptive gene expression and improving blood circulation (i.e., O2 delivery) via neovascularization, leading to the regulation of mitochondrial energy metabolism. This paper will highlight the biological effects of CO on ROS generation and cellular redox changes involved in mitochondrial metabolism and angiogenesis. Moreover, cellular mechanisms by which CO is exploited for disease prevention and therapeutic applications will also be discussed. PMID:22928087

Abscisic Acid Participates in the Control of Cell Cycle Initiation Through Heme Homeostasis in the Unicellular Red Alga Cyanidioschyzon merolae.

PubMed

Kobayashi, Yuki; Ando, Hiroyuki; Hanaoka, Mitsumasa; Tanaka, Kan

2016-05-01

ABA is a phytohormone that is synthesized in response to abiotic stresses and other environmental changes, inducing various physiological responses. While ABA has been found in unicellular photosynthetic organisms, such as cyanobacteria and eukaryotic algae, its function in these organisms is poorly understood. Here, we found that ABA accumulated in the unicellular red alga Cyanidioschyzon merolae under conditions of salt stress and that the cell cycle G1/S transition was inhibited when ABA was added to the culture medium. A gene encoding heme-scavenging tryptophan-rich sensory protein-related protein (CmTSPO; CMS231C) was positively regulated by ABA, as in Arabidopsis, and CmTSPO bound heme in vitro. The intracellular content of total heme was increased by addition of ABA, but unfettered heme decreased, presumably due to scavenging by CmTSPO. The inhibition of DNA replication by ABA was negated by addition of heme to the culture medium. Thus, we propose a regulatory role for ABA and heme in algal cell cycle initiation. Finally, we found that a C. merolae mutant that is defective in ABA production was more susceptible to salt stress, indicating the importance of ABA to stress resistance in red algae. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

PubMed Central

2014-01-01

Chlorite dismutases (Clds) are heme b-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of “Candidatus Nitrospira defluvii” (NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in Escherichia coli. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O–O bond formation. PMID:24754261

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

PubMed Central

Kathiresan, Meena; Martins, Dorival; English, Ann M.

2014-01-01

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

PubMed

Kathiresan, Meena; Martins, Dorival; English, Ann M

2014-12-09

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

Pivotal Advance: Heme oxygenase 1 expression by human CD4+ T cells is not sufficient for their development of immunoregulatory capacity.

PubMed

Biburger, Markus; Theiner, Gabi; Schädle, Mirjam; Schuler, Gerold; Tiegs, Gisa

2010-02-01

HO-1 is the only inducible one of three isoenzymes that catalyzes the oxidative degradation of heme. HO-1 is inducible by various cellular stress factors and exerts cytoprotective and immunomodulatory effects. Recent publications demonstrated that HO-1 is constitutively expressed by CD4(+)CD25(+) T(regs) and induced in CD4(+)CD25(-) T cells upon FoxP3 transfection. Here, we investigated whether HO-1 was essential and sufficient for human T(regs) to exert immunosuppression in vitro. PGJ(2) induced pronounced expression of HO-1 in CD4(+)CD25(-) T cells without accompanying FoxP3 induction. Treatment of CD4(+)CD25(-) T cells with PGJ(2) decreased their proliferation, whereas the HO-1 inhibitor SnPP enhanced the proliferation of HO-1-expressing T(regs), suggesting that HO-1 may modulate the proliferative capacity of T lymphocytes. HO-1 modulation by SnPP treatment of T(regs) or PGJ(2) treatment of CD4(+)CD25(-) T cells neither suppressed nor induced immune-modulatory function in these cells, respectively, as measured by responder-cell proliferation and/or IL-2 production. In summary, these data suggest that HO-1 expression by T(regs) might contribute to their typical reluctance to proliferate but does not account independently for their suppressive functions.

Role of nuclear factor-κB and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells

PubMed Central

Alcaraz, María José; Vicente, Ana María; Araico, Amparo; Dominguez, José N; Terencio, María Carmen; Ferrándiz, María Luisa

2004-01-01

The synthetic chalcone 3′,4′,5′,3,4,5-hexamethoxy-chalcone (CH) is an anti-inflammatory compound able to reduce nitric oxide (NO) production by inhibition of inducible NO synthase protein synthesis. In this work, we have studied the mechanisms of action of this compound. CH (10–30 μM) prevents the overproduction of NO in RAW 264.7 macrophages stimulated with lipopolysaccharide (1 μg ml−1) due to the inhibition of nuclear factor κB (NF-κB) activation. We have shown that treatment of cells with CH results in diminished degradation of the NF-κB–IκB complex leading to inhibition of NF-κB translocation into the nucleus, DNA binding and transcriptional activity. We also demonstrate the ability of this compound to activate NfE2-related factor (Nrf2) and induce heme oxygenase-1 (HO-1). Our results indicate that CH determines a rapid but nontoxic increase of intracellular oxidative species, which could be responsible for Nrf2 activation and HO-1 induction by this chalcone derivative.  This novel anti-inflammatory agent simultaneously induces a cytoprotective response (HO-1) and downregulates an inflammatory pathway (NF-κB) with a mechanism of action different from antioxidant chalcones. PMID:15249426

Determination of the chirality of the saturated pyrrole in sulfmyoglobin using the nuclear Overhauser effect.

PubMed

Parker, W O; Chatfield, M J; La Mar, G N

1989-02-21

The interproton nuclear Overhauser effect (NOE) and paramagnetic dipolar relaxation rates for hyperfine-shifted resonances in the proton NMR spectra of sperm whale met-cyano sulfmyoglobin have led to the location and assignment of the proton signals of the heme pocket residue isoleucine 99 (FG5) in two sulfmyoglobin isomers. Dipolar relaxation rates of these protein signals indicate a highly conserved geometry of the heme pocket upon sulfmyoglobin formation, while the similar upfield direction of dipolar shifts for this residue to that observed in native sperm whale myoglobin reflects largely retained magnetic properties. Dipolar connectivity of this protein residue to the substituents of the reacted heme pyrrole ring B defines the stereochemistry of the puckered thiolene ring found in one isomer, with the 3-CH3 tilted out of the heme plane proximally. The chirality of the saturated carbons of pyrrole ring B in both the initial sulfmyoglobin product and the terminal alkaline product is consistent with a mechanism of formation in which an atom of sulfur is incorporated distally to form an episulfide across ring B, followed by reaction of the vinyl group to yield the thiolene ring that retains the C3 chirality.

Low concentrations of bilirubin inhibit activation of hepatic stellate cells in vitro.

PubMed

Tang, Yinhe; Zhang, Qiyu; Zhu, Yefan; Chen, Gang; Yu, Fuxiang

2017-04-01

Hepatic stellate cell (HSC) activation serves a key role in liver fibrosis, and is associated with chronic liver diseases. Bilirubin, a product of heme degradation, has been demonstrated to have antioxidant properties. The present study investigated the effects of physiological concentrations of bilirubin on rat HSC activation. Rat HSCs were isolated and cultured for several generations to induce activation. The activated HSCs were subsequently treated with 0, 1, 10 or 20 mg/l bilirubin and assayed for parameters of cell activation. As the bilirubin concentration increased, HSCs demonstrated reduced production of reactive oxygen species, reduced protein expression levels of α‑smooth muscle actin, a decreased mRNA expression ratio of tissue inhibitor of matrix metalloproteinase‑1/matrix metalloproteinase‑2, decreased proliferation and increased apoptosis. In conclusion, elevated bilirubin levels, within its physiological concentration range, appeared to inhibit HSC activation. These findings suggested a potential role for bilirubin in the treatment of fibrosis that requires further investigation.

Targetting the hemozoin synthesis pathway for antimalarial drug and detected by TEM (Transmission electron microscope)

NASA Astrophysics Data System (ADS)

Abbas, Jamilah; Artanti, Nina; Sundowo, Andini; Dewijanti, Indah Dwiatmi; Hanafi, Muhammad; Lisa, Syafrudin, Din

2017-11-01

Malaria is a major public health problem mainly due to the development of resistance by the most lethal causative parasite species, the alarming spread of drug resistance and limited number of effective drug available now. Therefore it is important to discover new antimalarial drug. Malaria is caused by a singlecelled parasite from the genus Plasmodium. Plasmodium falciparum parasite infect red blood cells, ingesting and degradation hemoglobin in the acidic food vacuola trough a sequential metabolic process involving multiple proteases. During these process, hemoglobin is utilized as the predominant source of nutrition. Proteolysis of hemoglobin yields amino acid for protein synthesis as well as toxic heme. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite has evolved a distinct mechanism for detoxification of heme through conversion into insoluble crystalline pigment, known as hemozoin (β hematoin). Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarial drug. TEM (Transmission Electron Microscope) technology for hemozoin formation in vitro assay was done in this research. Calophyllum aerophyllum Lauterb as medicinal plants was used as a source of antimalarial drug. Acetone extracts of C. lowii showed growth inhibition against parasite P. falciparum with IC50 = 5.2 µg/mL. Whereas from hexane, acetone and methanol fraction of C. aerophyllum showed growth inhibition with IC50 = 0.054, 0.055 and 0.0054 µg/mL respectively. New drug from Calophyllum might have potential compounds that have unique structures and mechanism of action which required to develop new drug for treatment of sensitive and drug resistant strain of malaria.

Effect of ionizing radiation on the protein and lipid quality characteristics of mutton kheema treated with rice bran oil and sunflower oil

NASA Astrophysics Data System (ADS)

Jalarama Reddy, K.; Jayathilakan, K.; Pandey, M. C.

2015-12-01

Effect of rice bran oil (RBO) and irradiation (0, 1, 2 and 3 kGy) on lipid and protein quality of ready-to-eat mutton kheema were established during refrigerated storage (4±1 °C). Total carbonyls, thiobarbituric acid reactive substance (TBARS), non-heme iron and total volatiles in irradiated RBO samples were significantly lower (p<0.05) from the corresponding sunflower oil (SFO) treated samples initially and during storage. Product with RBO and Flaxseed oil (FSO) at the optimized level yielded a designer meat product having an SFA:MUFA:PUFA and n-6/n-3 ratio of 1:1.3:1.3 and 3.6:1 respectively. Degradation in PUFA levels in SFO samples were significantly higher (p<0.05) and an increase of 31% in metmyoglobin after 50 days was noticed in comparison with RBO samples. Non-linear correlation analysis of chemical markers established polynomial fit equations. 2 kGy radiation processing with RBO yielded a product having 50 days of shelf stability in terms of its chemical characteristics.

Chemical and biosynthetic studies of chlorophylls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huster, M.S.

1988-01-01

Chlorophyll occurrence, structure, biosynthesis, and degradation are discussed. Degradation and ring cleavage of heme is also discussed. The author examines the formation of dihydrobiliverdins by alkaline hydrolysis of zinc(II) meso-trifluoroacetoxypheophoribides, as a possible model for chlorophyll catabolism. {sup 18}O{sub 2}-labelling experiments show that the dihydrobiliverdin terminal lactam oxygens are derived from two different dioxygen molecules, also analogous to the Two Oxygen Molecular mechanism observed in heme degradation. The initially obtained dihydrobiliverdin readily undergoes an isomeric structural transformation, which is proposed as a model for the P{sub R}-P{sub FR} interconversion of the light sensor pigment phytochrome. The generality of the ring-openingmore » reaction is demonstrated with various chlorophyll-derived zinc(II) trifluoroacetoxychlorins, and side reactions of the isocyclic ring are discussed. The synthesis and properties of a chlorophyll-derived meso-oxochlorin are described. Facile one-electron oxidation, and its inhibition by protonation, is demonstrated by NMR, ESR, and cyclic voltammetry studies. Cyclic voltammetry is also used to measure redox potentials of a range of pheophorbide and meso-trifluoroacetoxypheophorbide metal complexes, including an oxochlorin nickel(II) complex. The results are presented of biosynthetic feeding studies of green sulfur bacteria with {sup 13}C- and {sup 14}C-labelled glutamate, glycine, and methionine. This study examines an unusual oxidation of a bacteriomethylpheophorbide 5-ethyl substituent, and describes attempts to elucidate the mechanism by {sup 18}O-labelling studies. Attempts to similarily derivatize a pyropheophorbide 5-methyl substituent are discussed.« less

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

PubMed Central

Ibrahim, Mohammed; Kuchinskas, Michael; Youn, Hwan; Kerby, Robert L.; Roberts, Gary P.; Poulos, Thomas L.; Spiro, Thomas G.

2007-01-01

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions. PMID:17720248

Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

PubMed Central

Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B.; Pedersen, Per Dedenroth; Dal Bello, Fabio

2013-01-01

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

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

The induction of heme oxygenase-1 suppresses heat shock protein 90 and the proliferation of human breast cancer cells through its byproduct carbon monoxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Wen-Ying; Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; Chen, Yen-Chou

2014-01-01

Heme oxygenase (HO)-1 is an oxidative stress-response enzyme which catalyzes the degradation of heme into bilirubin, ferric ion, and carbon monoxide (CO). Induction of HO-1 was reported to have antitumor activity; the inhibitory mechanism, however, is still unclear. In the present study, we found that treatment with [Ru(CO){sub 3}Cl{sub 2}]{sub 2} (RuCO), a CO-releasing compound, reduced the growth of human MCF7 and MDA-MB-231 breast cancer cells. Analysis of growth-related proteins showed that treatment with RuCO down-regulated cyclinD1, CDK4, and hTERT protein expressions. Interestingly, RuCO treatment resulted in opposite effects on wild-type and mutant p53 proteins. These results were similar tomore » those of cells treated with geldanamycin (a heat shock protein (HSP)90 inhibitor), suggesting that RuCO might affect HSP90 activity. Moreover, RuCO induced mutant p53 protein destabilization accompanied by promotion of ubiquitination and proteasome degradation. The induction of HO-1 by cobalt protoporphyrin IX (CoPP) showed consistent results, while the addition of tin protoporphyrin IX (SnPP), an HO-1 enzymatic inhibitor, diminished the RuCO-mediated effect. RuCO induction of HO-1 expression was reduced by a p38 mitogen-activated protein kinase inhibitor (SB203580). Additionally, treatment with a chemopreventive compound, curcumin, induced HO-1 expression accompanied with reduction of HSP90 client protein expression. The induction of HO-1 by curcumin inhibited 12-O-tetradecanoyl-13-acetate (TPA)-elicited matrix metalloproteinase-9 expression and tumor invasion. In conclusion, we provide novel evidence underlying HO-1's antitumor mechanism. CO, a byproduct of HO-1, suppresses HSP90 protein activity, and the induction of HO-1 may possess potential as a cancer therapeutic. - Highlights: • CO and HO-1 inhibited the growth of human breast cancer cells. • CO and HO-1 attenuated HSP90 and its client proteins expression. • CO induced mutant p53 protein ubiquitination and degradation. • Curcumin induced HO-1 expression and attenuated HSP90's client proteins expression.« less

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

PubMed

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-03-16

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

PubMed Central

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-01-01

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

Convergence of hepcidin deficiency, systemic iron overloading, heme accumulation, and REV-ERBα/β activation in aryl hydrocarbon receptor-elicited hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fader, Kelly A.; Nault, Rance

Persistent aryl hydrocarbon receptor (AhR) agonists elicit dose-dependent hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis in mice. Iron (Fe) promotes AhR-mediated oxidative stress by catalyzing reactive oxygen species (ROS) production. To further characterize the role of Fe in AhR-mediated hepatotoxicity, male C57BL/6 mice were orally gavaged with sesame oil vehicle or 0.01–30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. Duodenal epithelial and hepatic RNA-Seq data were integrated with hepatic AhR ChIP-Seq, capillary electrophoresis protein measurements, and clinical chemistry analyses. TCDD dose-dependently repressed hepatic expression of hepcidin (Hamp and Hamp2), the master regulator of systemic Fe homeostasis, resultingmore » in a 2.6-fold increase in serum Fe with accumulating Fe spilling into urine. Total hepatic Fe levels were negligibly increased while transferrin saturation remained unchanged. Furthermore, TCDD elicited dose-dependent gene expression changes in heme biosynthesis including the induction of aminolevulinic acid synthase 1 (Alas1) and repression of uroporphyrinogen decarboxylase (Urod), leading to a 50% increase in hepatic hemin and a 13.2-fold increase in total urinary porphyrins. Consistent with this heme accumulation, differential gene expression suggests that heme activated BACH1 and REV-ERBα/β, causing induction of heme oxygenase 1 (Hmox1) and repression of fatty acid biosynthesis, respectively. Collectively, these results suggest that Hamp repression, Fe accumulation, and increased heme levels converge to promote oxidative stress and the progression of TCDD-elicited hepatotoxicity. - Highlights: • TCDD represses hepatic hepcidin expression, leading to systemic iron overloading. • Dysregulation of heme biosynthesis is consistent with heme and porphyrin accumulation. • Heme-activated REV-ERBα/β repress circadian-regulated hepatic lipid metabolism. • Disruption of iron homeostasis promotes TCDD-elicited steatohepatitis with fibrosis.« less

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

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

PubMed

Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

2016-06-15

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath†

PubMed Central

Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.

1999-01-01

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

Heme and menaquinone induced electron transport in lactic acid bacteria.

PubMed

Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

2009-05-29

For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

Mutation of Phe413 to Tyr in catalase KatE from Escherichia coli leads to side chain damage and main chain cleavage.

PubMed

Jha, Vikash; Donald, Lynda J; Loewen, Peter C

2012-09-15

The monofunctional catalase KatE of Esherichia coli exhibits exceptional resistance to heat denaturation and proteolytic degradation. During an investigation of subtle conformation changes in Arg111 and Phe413 on the proximal side of the heme induced by H(2)O(2), variants at position R111, T115 and F413 were constructed. Because the residues are not situated in the distal side heme cavity where catalysis occurs, significant changes in reactivity were not expected and indeed, only small changes in the kinetic characteristics were observed in all of the variants. However, the F413Y variant was found to have undergone main chain cleavage whereas the R111A, T115A, F413E and F413K variants had not. Two sites of cleavage were identified in the crystal structure and by mass spectrometry at residues 111 and 115. In addition to main chain cleavage, modifications to the side chains of Tyr413, Thr115 and Arg111 were suggested by differences in the electron density maps compared to maps of the native and inactive variant H128N/F413Y. The inactive variant H128N/F413Y and the active variant T115A/F413Y both did not exhibit main chain cleavage and the R11A/F413Y variant exhibited less cleavage. In addition, the apparent modification of three side chains was largely absent in these variants. It is also significant that all three F413 single variants contained heme b suggesting that the fidelity of the phenyl group was important for mediating heme b oxidation to heme d. The reactions are attributed to the introduction of a new reactive center possibly involving a transient radical on Tyr413 formed during catalytic turn over. Copyright © 2011 Elsevier Inc. All rights reserved.

Mn(ii) mediated degradation of artemisinin based on Fe3O4@MnSiO3-FA nanospheres for cancer therapy in vivo

NASA Astrophysics Data System (ADS)

Chen, Jian; Zhang, Weijie; Zhang, Min; Guo, Zhen; Wang, Haibao; He, Mengni; Xu, Pengping; Zhou, Jiajia; Liu, Zhenbang; Chen, Qianwang

2015-07-01

Artemisinin (ART) is a natural drug with potent anticancer activities related with Fe2+ mediated cleavage of the endoperoxide bridge in ART. Herein, we reported that Mn2+ could substitute for Fe2+ to react with ART and generate toxic products, inducing a much higher anticancer efficiency. On this basis, we prepared pH-responsive Fe3O4@MnSiO3-FA nanospheres which can efficiently deliver hydrophobic ART into tumors in mice models. Mn2+ was released in acidic tumor environments and intracellular lysosomes, interacting with ART to kill cancer cells. The ART-loaded nanocarriers could suppress tumor growth more efficiently than free ART, which could be further illustrated by magnetic resonance imaging (MRI). Histological analysis revealed that the drug delivery system had no obvious effect on the major organs of mice. ART has been reported to have lower toxicity than chemotherapeutics. The ART-loaded nanocarriers are promising to be used in improving the survival of chemotherapy patients, providing a novel method for clinical tumor therapy.Artemisinin (ART) is a natural drug with potent anticancer activities related with Fe2+ mediated cleavage of the endoperoxide bridge in ART. Herein, we reported that Mn2+ could substitute for Fe2+ to react with ART and generate toxic products, inducing a much higher anticancer efficiency. On this basis, we prepared pH-responsive Fe3O4@MnSiO3-FA nanospheres which can efficiently deliver hydrophobic ART into tumors in mice models. Mn2+ was released in acidic tumor environments and intracellular lysosomes, interacting with ART to kill cancer cells. The ART-loaded nanocarriers could suppress tumor growth more efficiently than free ART, which could be further illustrated by magnetic resonance imaging (MRI). Histological analysis revealed that the drug delivery system had no obvious effect on the major organs of mice. ART has been reported to have lower toxicity than chemotherapeutics. The ART-loaded nanocarriers are promising to be used in improving the survival of chemotherapy patients, providing a novel method for clinical tumor therapy. Electronic supplementary information (ESI) available: Iron mediated degradation mechanism for artemisinin, mechanism of alkylation of iron(ii)-heme or iron(ii)/heme dimethylester by artemisinin, mechanism of alkylation of the heme model MnIITPP by artemisinin, schematic illustration of the synthesis of ART-loaded Fe3O4@MnSiO3-FA nanospheres, further characterization such as XRD and EDX patterns, N2 adsorption and desorption isotherm and BJH pore distribution, FT-IR spectra, UV-vis spectra, DLS and parallel test results of flow cytometric detection are given in Fig. S1-S13, Fe2+ or Mn2+ release from Fe3O4@MnSiO3 nanospheres in PBS at different pHs is given in Table S1. See DOI: 10.1039/c5nr02402a

GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury.

PubMed

Duann, Pu; Lianos, Elias A

2009-09-01

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury

PubMed Central

Duann, Pu; Lianos, Elias A.

2009-01-01

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-β1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury. PMID:19587144

Heme oxygenase-1 regulates mitochondrial quality control in the heart

PubMed Central

Hull, Travis D.; Boddu, Ravindra; Guo, Lingling; Tisher, Cornelia C.; Traylor, Amie M.; Patel, Bindiya; Joseph, Reny; Prabhu, Sumanth D.; Suliman, Hagir B.; Piantadosi, Claude A.; George, James F.

2016-01-01

The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control. PMID:27110594

Heme oxygenase and carbon monoxide protect from muscle dystrophy.

PubMed

Chan, Mun Chun; Ziegler, Olivia; Liu, Laura; Rowe, Glenn C; Das, Saumya; Otterbein, Leo E; Arany, Zoltan

2016-11-28

Duchenne muscle dystrophy (DMD) is one of the most common lethal genetic diseases of children worldwide and is 100% fatal. Steroids, the only therapy currently available, are marred by poor efficacy and a high side-effect profile. New therapeutic approaches are urgently needed. Here, we leverage PGC-1α, a powerful transcriptional coactivator known to protect against dystrophy in the mdx murine model of DMD, to search for novel mechanisms of protection against dystrophy. We identify heme oxygenase-1 (HO-1) as a potential novel target for the treatment of DMD. Expression of HO-1 is blunted in the muscles from the mdx murine model of DMD, and further reduction of HO-1 by genetic haploinsufficiency worsens muscle damage in mdx mice. Conversely, induction of HO-1 pharmacologically protects against muscle damage. Mechanistically, HO-1 degrades heme into biliverdin, releasing in the process ferrous iron and carbon monoxide (CO). We show that exposure to a safe low dose of CO protects against muscle damage in mdx mice, as does pharmacological treatment with CO-releasing molecules. These data identify HO-1 and CO as novel therapeutic agents for the treatment of DMD. Safety profiles and clinical testing of inhaled CO already exist, underscoring the translational potential of these observations.

Host heme oxygenase-1: Friend or foe in tackling pathogens?

PubMed

Singh, Nisha; Ahmad, Zeeshan; Baid, Navin; Kumar, Ashwani

2018-05-14

Infectious diseases are a major challenge in management of human health worldwide. Recent literature suggests that host immune system could be modulated to ameliorate the pathogenesis of infectious disease. Heme oxygenase (HMOX1) is a key regulator of cellular signaling and it could be modulated using pharmacological reagents. HMOX1 is a cytoprotective enzyme that degrades heme to generate carbon monoxide (CO), biliverdin, and molecular iron. CO and biliverdin (or bilirubin derived from it) can restrict the growth of a few pathogens. Both of these also induce antioxidant pathways and anti-inflammatory pathways. On the other hand, molecular iron can induce proinflammatory pathway besides making the cellular environment oxidative in nature. Since microbial infections often induce oxidative stress in host cells/tissues, role of HMOX1 has been analyzed in the pathogenesis of number of infections. In this review, we have described the role of HMOX1 in pathogenesis of bacterial infections caused by Mycobacterium species, Salmonella and in microbial sepsis. We have also provided a succinct overview of the role of HMOX1 in parasitic infections such as malaria and leishmaniasis. In the end, we have also elaborated the role of HMOX1 in viral infections such as AIDS, hepatitis, dengue, and influenza. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

A central role of heme oxygenase-1 in cardiovascular protection.

PubMed

Wu, Meng-Ling; Ho, Yen-Chun; Yet, Shaw-Fang

2011-10-01

The intrinsic defense mechanisms of the body are critical in protecting tissues from injury in response to pathological stress. Heme oxygenase-1 (HO-1), a stress response protein, is induced in response to various pathological stimuli to serve a cytoprotective function. By degrading the oxidant heme and generating the antioxidant bilirubin and anti-inflammatory molecule carbon monoxide, HO-1 may protect cell from injury due to oxidative and pathological stress. Oxidative stress in the heart caused by ischemia and reperfusion leads to cardiomyocyte death and subsequent myocardial infarction. Vascular diseases including atherosclerosis, graft failure, and restenosis are all associated with reactive oxygen species-induced injury and inflammation. Given that cardiovascular disease is the leading cause of death worldwide, there is considerable interest in developing new strategies for preventing and treating cardiovascular disease. Since HO-1 is induced in the heart and blood vessels in response to various stresses, a role of HO-1 has been implicated in cardiovascular homeostasis. Numerous studies using pharmacological method or genetic approach have since demonstrated the cardiovascular protective function of HO-1. Importantly, a number of studies have associated human HO-1 gene promoter polymorphisms with risk for vascular diseases. Taken together, HO-1 has a great therapeutic potential for cardiovascular disease.

The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

PubMed

Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

2013-12-01

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome

PubMed Central

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A.G.

2015-01-01

Abstract Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls. The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs. The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population. PMID:26313808

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome.

PubMed

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A G

2015-08-01

Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS.We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls.The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs.The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population.

Engineering proximal vs. distal heme-NO coordination via dinitrosyl dynamics: implications for NO sensor design.

PubMed

Kekilli, Demet; Petersen, Christine A; Pixton, David A; Ghafoor, Dlzar D; Abdullah, Gaylany H; Dworkowski, Florian S N; Wilson, Michael T; Heyes, Derren J; Hardman, Samantha J O; Murphy, Loretta M; Strange, Richard W; Scrutton, Nigel S; Andrew, Colin R; Hough, Michael A

2017-03-01

Proximal vs. distal heme-NO coordination is a novel strategy for selective gas response in heme-based NO-sensors. In the case of Alcaligenes xylosoxidans cytochrome c' (AXCP), formation of a transient distal 6cNO complex is followed by scission of the trans Fe-His bond and conversion to a proximal 5cNO product via a putative dinitrosyl species. Here we show that replacement of the AXCP distal Leu16 residue with smaller or similar sized residues (Ala, Val or Ile) traps the distal 6cNO complex, whereas Leu or Phe residues lead to a proximal 5cNO product with a transient or non-detectable distal 6cNO precursor. Crystallographic, spectroscopic, and kinetic measurements of 6cNO AXCP complexes show that increased distal steric hindrance leads to distortion of the Fe-N-O angle and flipping of the heme 7-propionate. However, it is the kinetic parameters of the distal NO ligand that determine whether 6cNO or proximal 5cNO end products are formed. Our data support a 'balance of affinities' mechanism in which proximal 5cNO coordination depends on relatively rapid release of the distal NO from the dinitrosyl precursor. This mechanism, which is applicable to other proteins that form transient dinitrosyls, represents a novel strategy for 5cNO formation that does not rely on an inherently weak Fe-His bond. Our data suggest a general means of engineering selective gas response into biologically-derived gas sensors in synthetic biology.

Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c.

PubMed

Roberts, V A; Freeman, H C; Olson, A J; Tainer, J A; Getzoff, E D

1991-07-15

To understand the specificity and efficiency of protein-protein interactions promoting electron transfer, we evaluated the role of electrostatic forces in precollision orientation by the development of two new methods, computer graphics alignment of protein electrostatic fields and a systematic orientational search of intermolecular electrostatic energies for two proteins at present separation distances. We applied these methods to the plastocyanin/cytochrome c interaction, which is faster than random collision, but too slow for study by molecular dynamics techniques. Significant electrostatic potentials were concentrated on one-fourth (969 A2) of the plastocyanin surface, with the greatest negative potential centered on the Tyr-83 hydroxyl within the acidic patch, and on one-eighth (632 A2) of the cytochrome c surface, with the greatest positive potential centered near the exposed heme edge. Coherent electrostatic fields occurred only over these regions, suggesting that local, rather than global, charge complementarity controls productive recognition. The three energetically favored families of pre-collision orientations all directed the positive region surrounding the heme edge of cytochrome c toward the acidic patch of plastocyanin but differed in heme plane orientation. Analysis of electrostatic fields, electrostatic energies of precollision orientations with 12 and 6 A separation distances, and surface topographies suggested that the favored orientations should converge to productive complexes promoting a single electron-transfer pathway from the cytochrome c heme edge to Tyr-83 of plastocyanin. Direct interactions of the exposed Cu ligand in plastocyanin with the cytochrome c heme edge are not unfavorable sterically or electrostatically but should occur no faster than randomly, indicating that this is not the primary pathway for electron transfer.

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

PubMed

Sheng, Yinghong; Zhong, Linghao; Guo, Dahai; Lau, Gavin; Feng, Changjian

2015-12-01

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production. Copyright © 2015 Elsevier Inc. All rights reserved.

Dietary iron controls circadian hepatic glucose metabolism through heme synthesis.

PubMed

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

2015-04-01

The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Differential Regulation of the Two Ferrochelatase Paralogues in Shewanella loihica PV-4 in Response to Environmental Stresses

DOE PAGES

Qiu, Dongru; Xie, Ming; Dai, Jingcheng; ...

2016-06-10

Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. MostShewanellaspecies encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, were investigated inShewanellaloihicaPV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels.hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription ofhemH1was regulated bymore » the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H 2O 2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the Δ hemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the ΔhemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds.Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c -type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. In conclusion, our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by theShewanella loihicaPV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.« less

Differential Regulation of the Two Ferrochelatase Paralogues in Shewanella loihica PV-4 in Response to Environmental Stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Dongru; Xie, Ming; Dai, Jingcheng

Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. MostShewanellaspecies encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, were investigated inShewanellaloihicaPV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels.hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription ofhemH1was regulated bymore » the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H 2O 2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the Δ hemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the ΔhemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds.Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c -type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. In conclusion, our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by theShewanella loihicaPV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.« less

Synthetic Heme/Copper Assemblies: Toward an Understanding of Cytochrome c Oxidase Interactions with Dioxygen and Nitrogen Oxides

PubMed Central

Hematian, Shabnam; Garcia-Bosch, Isaac; Karlin, Kenneth D.

2016-01-01

Conspectus Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper and/or iron ions, those reacting with dioxygen (O2) and/or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2−)). As inspiration for this work, we turn to mitochondrial cytochrome c oxidase which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis and characterization of new O2-adducts whose further study will add insights into O2-reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO’s function, which is intimately tied to cellular O2-balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-FeIII-O-CuII(L) products; their properties are discussed. The O-atom is derived from dioxygen and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo-complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a “naked” synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active-sites. The other sector of research is focused on heme/Cu assemblies mediating the redox interplay between nitrite and NO(g). In the nitrite reductase chemistry, the cupric center serves as a Lewis acid while the heme is the redox active center providing the electron. The orientation of nitrite in approaching the ferrous heme center and N-atom binding are important. Also, detailed spectroscopic and kinetic studies of the NO(g) oxidase chemistry, in excellent agreement with theoretical calculations, reveal the intermediates and key mechanistic steps. Thus, we suggest that both chemical and biochemical heme/Cu mediated nitrite reductase and NO(g) oxidase chemistry require N-atom binding to a ferrous heme along with cupric ion O-atom coordination, proceeding via a three-membered O-Fe-N chelate ring transition state. These important mechanistic features of heme/Cu systems interconverting NO(g) and nitrite are discussed for the first time. PMID:26244814

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

PubMed

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry, and Reactivity

PubMed Central

2013-01-01

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O=O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation. PMID:24364531

Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

PubMed

Haynes, Richard K; Cheu, Kwan-Wing; N'Da, David; Coghi, Paolo; Monti, Diego

2013-08-01

The isolation of artemisinin from the traditional medicinal herb qīng hāo (Artemisia annua), its characterization as a peroxide and preparation of the derivatives dihydroartemisinin, artemether and artesunate in the 1970s and 1980s by Chinese scientists under the umbrella of Project 523 collectively represents one of the great events in medicine in the latter third of the 20(th) Century. Artemisinins have become the most important component of chemotherapy of malaria: although used initially in monotherapy, they are now used in combination therapies or ACTs with longer half-life quinolines or arylmethanols. Nevertheless, the recent emergence of artemisinin-tolerant strains of the malaria parasite as reflected in increased clearance times of parasitaemia in patients treated with ACTs represents the greatest threat to control of malaria since resistance to chloroquine was first reported over 55 years ago. Importantly, the event brings into sharp focus the realization that relatively little is precisely understood, as opposed to widely assumed, for the mechanism of drug action of artemisinins and their synthetic peroxide analogues. Thus, we review here their antimalarial activities, the use of artemisinins in combination therapies, drug-drug interactions with the quinolines and arylmethanols, and metabolism of the artemisinins and synthetic peroxides. The mechanism of action of quinolines and arylmethanols, in particular their ability to induce redistribution of heme into the parasite cytosol, is also highlighted. This collective information is then used as a counterpoint to screen the validity of two of the prevailing hypotheses of drug action of artemisinins and synthetic peroxides, namely i. 'the C-radical hypothesis' wherein the peroxide undergoes 'bioactivation' by ferrous iron to generate C-radicals that are held to be the cytotoxic agents and ii. the 'heme hypothesis' wherein ferrous heme may generate either the same type of 'cytotoxic' C-radical, or the peroxide forms heme adducts that apparently inherit the exquisite cytotoxicities of the parent peroxide in one way or another. In a subsequent review, we screen the third and fourth hypotheses: the SERCA hypothesis wherein artemisinins modulate operation of the malaria parasite sarcoendo plasmic reticulum calcium pump SERCA Ca(2+)-ATPase ATP6 and the co-factor hypothesis wherein artemisinins act as oxidant drugs through rapidly oxidizing reduced conjugates of flavin cofactors, or those of flavin cofactor precursors such as riboflavin, and other susceptible endogenous substrates that play a role in maintaining intraparasitic redox homeostasis. For the C-radical hypothesis, details of in vitro chemical studies in the context of established chemistry of C-radicals and their ability to react with radical trapping agents such as nitroso compounds, cyclic nitrones, persistent nitroxyl radicals and atmospheric oxygen (dioxygen) are summarized. Overall, there is no correlation between antimalarial activities and abilities of the derived C-radicals to react with trapping agents in a chemical flask. This applies in particular to the reactions of C-radicals from artemisinins and steroidal tetraoxanes with the trapping agents vis-a-vis those from adamantyl capped systems. In an intraparasitic medium, it is not possible to intercept C-radicals either through use of a vast excess of a nitroxyl radical or dioxygen. The lack of correlation of antimalarial activities also applies to the Fe(2+)-mediated decomposition of artemisinins and synthetic peroxides, where literature data taken as indicating otherwise are critically assessed. The antagonism to antimalarial activities of artemisinins exerted by desferrioxamine (DFO) and related Fe(3+)-chelating agents is due to formation of stable chelates with bioavailable Fe(3+) that shuts down redox cycling through Fe(2+) and the subsequent generation of reactive oxygen species (ROS) via the Fenton reaction. The generation of ROS by Fe(2+) complements the action of artemisinins, to be discussed in Part 2; there is no need to posit a reaction of Fe(2+) with the artemisinins to account for their antimalarial activity. The ability of artemisinins and synthetic peroxides to elicit membrane damage is examined in the light of established processes of autoxidation. The oxidant character of the intraparasitic environment is incompatible with the reducing conditions required for generation of C-radicals, and in contrast to the expectation raised by the C-radical hypothesis, and indeed by the heme hypothesis outlined below, antimalarial activities of artemisinins are enhanced under higher partial pressures of dioxygen. Structure-activity data from a wide variety of artemisinins and synthetic peroxides cannot be accommodated within the bounds of the C-radical hypothesis. Finally, the antimalarial Cradical construct sharply contrasts with that of the potently antitumour-active ene-diyne antibiotics such as neocarzinostatin. In an iron-free process, these compounds generate highly reactive aryl C-radicals that abstract H atoms from deoxyribose units in DNA to generate alkyl C-radicals. The last do react with dioxygen in a normal intracellular environment to initiate DNA strand cleavage. Overall, it must be concluded that the C-radical hypothesis as the basis for antimalarial activities of artemisinins and synthetic peroxides is untenable. Heme has been intensively studied as an 'activator' of artemisinins and other antimalarial peroxides, and indeed the hypothesis seemingly has become firmly embedded in the underlying brickwork of the scientific edifice. The locus of activity of the peroxides interacting with the heme is considered to be the parasite digestive vacuole. The basis for the nanomolar activities of artemisinins and synthetic peroxides is variously ascribed to heme-Fe(2+)-mediated generation of C-radicals from the peroxides, formation of heme-artemisinin adducts that are held either to engage in redox cycling with concomitant generation of ROS or to inhibit formation of hemozoin. In the last case, just like the aminoquinolines and arylmethanols, the peroxides are not the active agents, but exert their parasiticidal effects through allowing the build-up of free heme-Fe(3+), the ultimate cytotoxic entity. We assess the literature relating to generation of heme by hemoglobin digestion, and the stage at which this process becomes significant in the intraerythrocytic parasite. The claims of production of heme and conversion into hemozoin occurring in a lipid environment may have to be put aside based on recent literature data that indicates crystallization of hemozoin must take place an aqueous interface; association of lipids with the heme/hemozoin is likely to be a reflection of attractive van der Waals interactions involving the hydrophobic surface of the heme or hemozoin aggregates. In addition, the observation leading to the claim that hemozoin manufacture commences at the mid-ring stage cannot be independently verified. That the quinoline and arylmethanol antimalarials have essentially no activities on the ring stage parasites and exert greatest efficacy at the trophozoite stage where heme production is maximal is consistent with this. Conversely, artemisinins, and indeed redox active drugs such as methylene blue and others, are highly active against early ring stage parasites. Thus, there is a prominent disconnect between stage specificities of artemisinins vis-a-vis those of 4-aminoquinolines and arylmethanols suggesting that heme is not the target of the former class of drug. Further, the ability of the Fe(3+) chelate DFO to antagonize antimalarial activities of artemisinins, but not the activities of 4-aminoquinolines, cannot be explained by involvement of heme as a target for artemisinins. We critically examine the basis for formation of products obtained from reaction of heme with artemisinins and synthetic peroxides under conditions ranging from biomimetic - reactions employing catalytic reagents under aqueous or semi-aqueous conditions - to those conducted under highly reducing and eminently artificial conditions, usually in the solvent dimethyl sulfoxide (DMSO) that both forms well characterized complexes with heme-Fe(2+) and actually assists in driving single electron transfer processes. It is noted that alkylated products tend to form in high yields under the last conditions, and this aspect is readily explained. Irrespective of product yields obtained under various conditions, an overarching correlation between facility of the reaction of the peroxide with heme and their antimalarial activities does not exist. The is underscored by the reproducible outcomes of reactions conducted under biomimetic conditions indicating adducts cannot form in physiologically meaningful concentrations and that heme is a recalcitrant reaction partner to artemisinins in general. Again, as in the case of the C-radical hypothesis, structure-activity data from a wide variety of artemisinins and synthetic peroxides is difficult to reconcile with the heme hypothesis. This applies in particular to dimeric and trimeric artemisinin derivatives where the ascribing of biological activity to reactions of the derived radicals or to the vastly encumbered artemisinin-heme adducts is physically unrealistic. Finally, the facile metabolism and induction of metabolism of the current clinically used artemisinins by members of the CYP superfamily - heme proteins that require an intimate interaction of the heme with the artemisinin for metabolism to occur - is incompatible with the oft-cited proclivity of the peroxide to associate via complex formation with heme as a prelude to its 'activation' as an antimalarial agent within the malaria parasite. (ABSTRACT TRUNCATED)

Heme and menaquinone induced electron transport in lactic acid bacteria

PubMed Central

Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

2009-01-01

Background For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. Conclusion We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species. PMID:19480672

Characterization of plasma labile heme in hemolytic conditions

PubMed Central

Gouveia, Zélia; Carlos, Ana R.; Yuan, Xiaojing; Aires-da-Silva, Frederico; Stocker, Roland; Maghzal, Ghassan J.; Leal, Sónia S.; Gomes, Cláudio M.; Todorovic, Smilja; Iranzo, Olga; Ramos, Susana; Santos, Ana C.; Hamza, Iqbal; Gonçalves, João; Soares, Miguel P.

2018-01-01

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (∼ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. PMID:28783254

Quercetin Reduces Tumor Necrosis Factor Alpha-Induced Muscle Atrophy by Upregulation of Heme Oxygenase-1.

PubMed

Kim, Yeji; Kim, Chu-Sook; Joe, Yeonsoo; Chung, Hun Taeg; Ha, Tae Youl; Yu, Rina

2018-06-01

The inflammatory cytokine tumor necrosis factor α (TNFα), upregulated in the obese condition, promotes protein degradation and is implicated in obesity-related skeletal muscle atrophy and age-related sarcopenia. Quercetin, a flavonoid, elicits antioxidative and anti-inflammatory activities. In this study, we investigated the effect of quercetin on TNFα-induced skeletal muscle atrophy as well as its potential mechanism of action. In this study, we observed that quercetin suppressed expression of TNFα-induced atrophic factors such as MAFbx/atrogin-1 and MuRF1 in myotubes, and it enhanced heme oxygenase-1 (HO-1) protein level accompanied by increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in myotubes. The HO-1 inhibitor ZnPP suppressed the inhibitory actions of quercetin on TNFα-induced atrophic responses and degradation of IκB-α in myotubes. Moreover, quercetin supplementation to high-fat diet-fed obese mice inhibited obesity-induced atrophic responses in skeletal muscle, accompanied by upregulation of HO-1 and inactivation of nuclear factor-kappa B (NF-κB), and the quercetin actions were attenuated in Nrf2-deficient mice. These findings suggest that quercetin protects against TNFα-induced muscle atrophy under obese conditions through Nrf2-mediated HO-1 induction accompanied by inactivation of NF-κB. Quercetin may be used as a dietary supplement to protect against obesity-induced skeletal muscle atrophy.

Heterologous Expression of Peroxidases

NASA Astrophysics Data System (ADS)

de Weert, Sandra; Lokman, B. Christien

The industrial importance of peroxidases has led to much research in the past two decades on the development of a cost effective and efficient production process for peroxidases. Unfortunately, even today, no clear answers can be given to questions such as (1) should the peroxidase be expressed in bacteria, yeast, or fungi? (2) which is the optimal production strain (e.g., protease deficient, heme overproducing)? (3) which expression vector should be chosen? and (4) what purification method should be used? Strategies that have proven successful for one peroxidase can fail for another one; for each individual peroxidase, a new strategy has to be developed. This chapter gives an overview of the heterologous production of heme containing peroxidases in various systems. It focuses on the heterologous production of fungal peroxidases as they have been subject of considerable research for their industrial and environmental applications. An earlier study has also been performed by Conesa et al. [1] and is extended with recent proceedings.

Genome mining and functional genomics for siderophore production in Aspergillus niger.

PubMed

Franken, Angelique C W; Lechner, Beatrix E; Werner, Ernst R; Haas, Hubertus; Lokman, B Christien; Ram, Arthur F J; van den Hondel, Cees A M J J; de Weert, Sandra; Punt, Peter J

2014-11-01

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins.

PubMed

Nath, Karl A; Belcher, John D; Nath, Meryl C; Grande, Joseph P; Croatt, Anthony J; Ackerman, Allan W; Katusic, Zvonimir S; Vercellotti, Gregory M

2018-05-01

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4 +/+ and TLR4 -/- mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

Heme as a danger molecule in pathogen recognition.

PubMed

Wegiel, Barbara; Hauser, Carl J; Otterbein, Leo E

2015-12-01

Appropriate control of redox mechanisms are critical for and effective innate immune response, which employs multiple cell types, receptors and molecules that recognize danger signals when they reach the host. Recognition of pathogen-associated pattern molecules (PAMPs) is a fundamental host survival mechanism for efficient elimination of invading pathogens and resolution of the infection and inflammation. In addition to PAMPs, eukaryotic cells contain a plethora of intracellular molecules that are normally secured within the confines of the plasma membrane, but if liberated and encountered in the extracellular milieu can provoke rapid cell activation. These are known as Alarmins or Danger-Associated Molecular Patterns (DAMPs) and can be released actively by cells or passively as a result of sterile cellular injury after trauma, ischemia, or toxin-induced cell rupture. Both PAMPs and DAMPs are recognized by a series of cognate receptors that increase the generation of free radicals and activate specific signaling pathways that result in regulation of a variety of stress response, redox sensitive genes. Multiple mediators released, as cells die include, but are not limited to ATP, hydrogen peroxide, heme, formyl peptides, DNA or mitochondria provide the second signal to amplify immune responses. In this review, we will focus on how sterile and infective stimuli activate the stress response gene heme oxygenase-1 (Hmox1, HO-1), a master gene critical to an appropriate host response that is now recognized as one with enormous therapeutic potential. HO-1 gene expression is regulated in large part by redox-sensitive proteins including but not limited to nrf2. Both PAMPs and DAMPs increase the activation of nrf2 and HO-1. Heme is a powerful pro-oxidant and as such should be qualified as a DAMP. With its degradation by HO-1a molecule of carbon monoxide (CO) is generated that in turn serves as a bioactive signaling molecule. PAMPs such as bacterial endotoxin activate HO-1, and the CO that is generated diffuses into the extracellular milieu where it interacts with bacteria, altering their behavior to increase production of ATP, which then functions as a second signal danger molecule. This two-hit cycle scenario results in efficient and effective activation of host leukocytes to attack and clear bacteria in part via enhanced reactive oxygen species generation. We discuss this intimate communication that occurs between host and bacteria and how these molecules serve as critical regulators of the acute inflammatory response, the overall redox status of the cell, and survival of the host. Copyright © 2015 Elsevier Inc. All rights reserved.

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

PubMed

Joubert, Laetitia; Dagieu, Jean-Baptiste; Fernandez, Annabelle; Derré-Bobillot, Aurélie; Borezée-Durant, Elise; Fleurot, Isabelle; Gruss, Alexandra; Lechardeur, Delphine

2017-01-16

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

PubMed Central

Kim, Junghyun; Zarjou, Abolfazl; Traylor, Amie M.; Bolisetty, Subhashini; Jaimes, Edgar A.; Hull, Travis D.; George, James F.; Mikhail, Fady M.; Agarwal, Anupam

2012-01-01

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation but whether such control occurs in vivo is not known. To enable such analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome Conformation Capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases. PMID:22495295

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation

PubMed Central

Wang, Wenqian; Tabu, Kouichi; Hagiya, Yuichiro; Sugiyama, Yuta; Kokubu, Yasuhiro; Murota, Yoshitaka; Ogura, Shun-ichiro; Taga, Tetsuya

2017-01-01

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism. PMID:28169355

Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis

PubMed Central

Choby, Jacob E.; Grunenwald, Caroline M.; Celis, Arianna I.; Gerdes, Svetlana Y.; DuBois, Jennifer L.

2018-01-01

ABSTRACT Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy in S. aureus heme biosynthesis. The first committed enzyme in the S. aureus heme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by which S. aureus responds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation of hemX leads to increased heme synthesis. Excess heme synthesis in a ΔhemX mutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates that S. aureus regulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX. PMID:29437922

Glucose metabolism during fasting is altered in experimental porphobilinogen deaminase deficiency.

PubMed

Collantes, María; Serrano-Mendioroz, Irantzu; Benito, Marina; Molinet-Dronda, Francisco; Delgado, Mercedes; Vinaixa, María; Sampedro, Ana; Enríquez de Salamanca, Rafael; Prieto, Elena; Pozo, Miguel A; Peñuelas, Iván; Corrales, Fernando J; Barajas, Miguel; Fontanellas, Antonio

2016-04-01

Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

ApoHRP-based assay to measure intracellular regulatory heme.

PubMed

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

2015-02-01

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

Induction of Heme Oxygenase-1 by Sodium 9-Hydroxyltanshinone IIA Sulfonate Derivative Contributes to Inhibit LPS-Mediated Inflammatory Response in Macrophages.

PubMed

Liu, Xin-Hua; Wang, Xi-Ling; Xin, Hong; Wu, Dan; Xin, Xiao-Ming; Miao, Lei; Zhang, Qiu-Yan; Zhou, Yang; Liu, Qian; Zhang, Qian; Zhu, Yi-Zhun

2015-01-01

Sodium 9-acetoxyltanshinone IIA sulfonate (ZY-1A4), a novel compound derived from sodium 9-hydroxyltanshinone IIA sulfonate, was synthesized with potential biological activities. This study aimed to explore the effects of ZY-1A4 on lipopolysaccharide (LPS)-triggered inflammatory response and the underlying mechanisms. Activation of RAW264.7 macrophages was induced by LPS. The effects of ZY-1A4 on inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) generation, nuclear factor-κB (NF-κB) activation, heme oxygenase-1 (HO-1) expression, and nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway were evaluated to elucidate its underlying mechanisms on inflammatory responses. ZY-1A4 concentration-dependently reduced iNOS expression and NO production, and inhibited c-Jun-N-terminal kinase 1/2 (JNK1/2) phosphorylation and NF-κB activation in LPS-stimulated macrophages. In addition, ZY-1A4 concentration- and time-dependently induced HO-1 expression associated with degradation of Kelch-like ECH-associated protein 1 (Keap1) and nuclear translocation of Nrf2, while the effect of ZY-1A4 was abolished by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Intriguingly, pharmacological inactivation of HO-1 with zinc protoporphyrin IX reversed anti-inflammatory effect of ZY- 1A4, but the anti-inflammatory effect of ZY-1A4 was largely mimicked by HO-1 by-products carbon monoxide and bilirubin. Furthermore, the inhibitory effect of ZY-1A4 on LPS-induced iNOS expression and NO release was abolished by HO-1 siRNA or LY294002. Our results demonstrated that ZY-1A4 suppressed LPS-induced iNOS expression and NO generation via modulation of NF-κB activation and HO-1 expression. This new finding might shed light to the prevention and therapy of cardiovascular diseases. © 2015 S. Karger AG, Basel.

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

PubMed Central

Pollock, W B; Loutfi, M; Bruschi, M; Rapp-Giles, B J; Wall, J D; Voordouw, G

1991-01-01

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes. Images PMID:1846136

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

PubMed

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-08-15

To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant

PubMed Central

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-01-01

AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to clone and express them and analyze their activities. METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5α . Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of △hHO-1 was reduced 91.21% after mutation compared with whHO-1. CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. △hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. △hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia. PMID:15285018

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening.

PubMed

Shin, Inchul; Ambler, Brett R; Wherritt, Daniel; Griffith, Wendell P; Maldonado, Amanda C; Altman, Ryan A; Liu, Aimin

2018-03-28

Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1 H NMR, 13 C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.

The radical SAM protein HemW is a heme chaperone.

PubMed

Haskamp, Vera; Karrie, Simone; Mingers, Toni; Barthels, Stefan; Alberge, François; Magalon, Axel; Müller, Katrin; Bill, Eckhard; Lubitz, Wolfgang; Kleeberg, Kirstin; Schweyen, Peter; Bröring, Martin; Jahn, Martina; Jahn, Dieter

2018-02-16

Radical S -adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c -type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Red Meat, Dietary Heme Iron, and Risk of Type 2 Diabetes: The Involvement of Advanced Lipoxidation Endproducts12

PubMed Central

White, Desley L.; Collinson, Avril

2013-01-01

There is growing evidence of disordered iron homeostasis in the diabetic condition, with links proposed between dietary iron intakes and both the risk of disease and the risk of complications of advanced disease. In the United States, Britain, and Canada, the largest dietary contributors of iron are cereals and cereal products and meat and meat products. This review discusses the findings of cohort studies and meta-analyses of heme iron and red meat intakes and the risk of type 2 diabetes. These suggest that processed red meat is associated with increased risk, with high intakes of red meat possibly also associated with a small increased risk. Historically, humans have relied on large quantities of heme iron and red meat in their diets, and therefore it is paradoxical that iron from meat sources should be associated with the risk of type 2 diabetes. A reason for this association may be drawn from studies of dietary advanced glycation and lipoxidation endproducts present in processed food and the mechanisms by which insulin output by pancreatic islet cells might be influenced by the protein modifications present in processed red meat. PMID:23858089

Challenging Density Functional Theory Calculations with Hemes and Porphyrins.

PubMed

de Visser, Sam P; Stillman, Martin J

2016-04-07

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.

Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

PubMed

Wang, Baowei; Cheng, Fansheng; Gao, Shun; Ge, Wenhua; Zhang, Mingai

2017-02-15

Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Succinic acid production by Actinobacillus succinogenes NJ113 using corn steep liquor powder as nitrogen source.

PubMed

Xi, Yong-lan; Chen, Ke-quan; Dai, Wen-yu; Ma, Jiang-feng; Zhang, Min; Jiang, Min; Wei, Ping; Ouyang, Ping-Kai

2013-05-01

In this study, corn steep liquor powder (CSL) was used as nitrogen source to replace the relatively costly yeast extract typically used for the production of succinic acid with Actinobacillus succinogenes NJ113. Moreover, when heme was added to the fermentation medium and the culture was agitated at a low speed, a maximum succinic acid concentration of 37.9 g/l was obtained from a glucose concentration of 50 g/l, and a productivity of 0.75 g/l/h was achieved. These yields are almost as high as for fermentation with glucose and yeast extract. These results suggest that heme-supplemented CSL may be a suitable alternative nitrogen source for a cost-effective method of producing succinic acid with A. succinogenes NJ113 while consuming less energy than previous methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prebiotics increase heme iron bioavailability and do not affect non-heme iron bioavailability in humans.

PubMed

Weinborn, Valerie; Valenzuela, Carolina; Olivares, Manuel; Arredondo, Miguel; Weill, Ricardo; Pizarro, Fernando

2017-05-24

The aim of this study was to establish the effect of a prebiotic mix on heme and non-heme iron (Fe) bioavailability in humans. To this purpose, twenty-four healthy women were randomized into one of two study groups. One group ate one yogurt per day for 12 days with a prebiotic mix (prebiotic group) and the other group received the same yogurt but without the prebiotic mix (control group). Before and after the intake period, the subjects participated in Fe absorption studies. These studies used 55 Fe and 59 Fe radioactive isotopes as markers of heme Fe and non-heme Fe, respectively, and Fe absorption was measured by the incorporation of radioactive Fe into erythrocytes. The results showed that there were no significant differences in heme and non-heme Fe bioavailability in the control group. Heme Fe bioavailability of the prebiotic group increased significantly by 56% post-prebiotic intake. There were no significant differences in non-heme Fe bioavailability in this group. We concluded that daily consumption of a prebiotic mix increases heme Fe bioavailability and does not affect non-heme iron bioavailability.

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide from Prevotella intermedia

PubMed Central

Cho, Yun-Jung

2013-01-01

Purpose Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. Methods LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory κB (IκB)-α degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Results Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IκB-α induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Conclusions Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-κB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease. PMID:24040572

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide from Prevotella intermedia.

PubMed

Cho, Yun-Jung; Kim, Sung-Jo

2013-08-01

Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory κB (IκB)-α degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IκB-α induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-κB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

Heme acquisition in the parasitic filarial nematode Brugia malayi.

PubMed

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

2016-10-01

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

Heme acquisition in the parasitic filarial nematode Brugia malayi

PubMed Central

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

2016-01-01

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

Cyanide binding to human plasma heme-hemopexin: A comparative study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it; Istituto Nazionale di Biostrutture e Biosistemi, Roma; Leboffe, Loris

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,more » 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.« less

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

Heme-Coordinating Inhibitors of Neuronal Nitric Oxide Synthase. Iron-Thioether Coordination is Stabilized by Hydrophobic Contacts Without Increased Inhibitor Potency

PubMed Central

Martell, Jeffrey D.; Li, Huiying; Doukov, Tzanko; Martásek, Pavel; Roman, Linda J.; Soltis, Michael; Poulos, Thomas L.; Silverman, Richard B.

2010-01-01

The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge, the first crystal structure of a thioether inhibitor complexed to any heme enzyme. A series of related potential inhibitors (4-8) also were evaluated. Compounds 4-8 were all found to be type I (non-heme-coordinating) inhibitors of ferric nNOS, but 4 and 6-8 were found to switch to type II upon heme reduction to the ferrous state, reflecting the higher affinity of thioethers for ferrous heme than for ferric heme. Contrary to what has been widely thought, thioether-heme ligation was found not to increase inhibitor potency, illustrating the intrinsic weakness of the thioether-ferric heme linkage. Subtle changes in the alkyl groups attached to the thioether sulfur caused drastic changes in binding conformation, indicating that hydrophobic contacts play a crucial role in stabilizing the thioether-heme coordination. PMID:20014790

Mini Heme-Proteins: Designability of Structure and Diversity of Functions.

PubMed

Rai, Jagdish

2017-08-30

Natural heme proteins may have heme bound to poly-peptide chain as a cofactor via noncovalent forces or heme as a prosthetic group may be covalently bound to the proteins. Nature has used porphyrins in diverse functions like electron transfer, oxidation, reduction, ligand binding, photosynthesis, signaling, etc. by modulating its properties through diverse protein matrices. Synthetic chemists have tried to utilize these molecules in equally diverse industrial and medical applications due to their versatile electro-chemical and optical properties. The heme iron has catalytic activity which can be modulated and enhanced for specific applications by protein matrix around it. Heme proteins can be designed into novel enzymes for sterio specific catalysis ranging from oxidation to reduction. These designed heme-proteins can have applications in industrial catalysis and biosensing. A peptide folds around heme easily due to hydrophobic effect of the large aromatic ring of heme. The directional property of co-ordinate bonding between peptide and metal ion in heme further specifies the structure. Therefore heme proteins can be easily designed for targeted structure and catalytic activity. The central aromatic chemical entity in heme viz. porphyrin is a very ancient molecule. Its presence in the prebiotic soup and in all forms of life suggests that it has played a vital role in the origin and progressive evolution of living organisms. Porphyrin macrocycles are highly conjugated systems composed of four modified pyrrole subunits interconnected at their α -carbon atoms via methine (=CH-) bridges. Initial minimalist models of hemoproteins focused on effect of heme-ligand co-ordinate bonding on chemical reactivity, spectroscopy, electrochemistry and magnetic properties of heme. The great sensitivity of these spectroscopic features of heme to its surrounding makes them extremely useful in structural elucidation of designed heme-peptide complexes. Therefore heme proteins are easier to work on for designing novel proteins for industrial and medical applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

PubMed

Sil, Debangsu; Rath, Sankar Prasad

2015-10-07

Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

Heme Oxygenase-2 Modulates Early Pathogenesis after Traumatic Injury to the Immature Brain

PubMed Central

Yoneyama-Sarnecky, Tomoko; Olivas, Andrea D.; Azari, Soraya; Ferriero, Donna M.; Manvelyan, Hovhannes M.; Noble-Haeusslein, Linda J.

2010-01-01

We determined if heme oxygenase-2 (HO-2), an enzyme that degrades the pro-oxidant heme, confers neuroprotection in the developing brain after traumatic brain injury (TBI). Male HO-2 wild-type (WT) and homozygous knockout (KO) mice at postnatal day 21 were subjected to TBI and euthanized 1, 7, and 14 days later. Relative cerebral blood flow, measured by laser Doppler, cortical and hippocampal pathogenesis, and motor recovery were evaluated at all time points. Cerebral blood flow was found to be similar between experimental groups. Blood flow significantly decreased immediately after injury, returned to baseline by 1 day, and was significantly elevated by 7 days, post-injury. Nonheme iron preferentially accumulated in the ipsilateral cortex, hippocampus, and external capsule in both WT and KO brain-injured genotypes. There were, however, a significantly greater number of TUNEL-positive cells in the hippocampal dentate gyrus and a significantly greater cortical lesion volume in KOs relative to WTs within the first week post-injury. By 14 days post-injury, however, cortical lesion volume and cell density in the hippocampal CA3 region and dorsal thalamus were similar between the two groups. Assays of fine motor function (grip strength) over the first 2 weeks post-injury revealed a general pattern of decreased strength in the contralateral forelimbs of KOs as compared to WTs. Together, these findings demonstrate that deficiency in HO-2 alters both the kinetics of secondary damage and fine motor recovery after TBI. PMID:20389079

Examining potential side effects of therapeutic hypothermia in experimental intracerebral hemorrhage.

PubMed

Wowk, Shannon; Fagan, Kelly J; Ma, Yonglie; Nichol, Helen; Colbourne, Frederick

2017-08-01

Studies treating intracerebral hemorrhage (ICH) with therapeutic hypothermia (TH) have shown inconsistent benefits. We hypothesized that TH's anti-inflammatory effects may be responsible as inflammatory cells are essential for removing degrading erythrocytes. Here, we subjected rats to a collagenase-induced striatal ICH followed by whole-body TH (∼33℃ for 11-72 h) or normothermia. We used X-ray fluorescence imaging to spatially quantify total and peri-hematoma iron three days post-injury. At three and seven days, we measured non-heme iron levels. Finally, hematoma volume was quantified on one, three, and seven days. In the injured hemisphere, total iron levels were elevated ( p < 0.001) with iron increasing in the peri-hematoma region ( p = 0.007). Non-heme iron increased from three to seven days (p < 0.001). TH had no effect on any measure of iron ( p ≥ 0.479). At one and three days, TH did not affect hematoma volume ( p ≥ 0.264); however, at seven days there was a four-fold increase in hematoma volume in 40% of treated animals ( p = 0.032). Thus, even when TH does not interfere with initial increases in total and non-heme iron or its containment, TH can cause re-bleeding post-treatment. This serious complication could partly account for the intermittent protection previously observed. This also raises serious concerns for clinical usage of TH for ICH.

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

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

2012-03-06

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.

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

Isoporphyrin intermediate in heme oxygenase catalysis. Oxidation of alpha-meso-phenylheme.

PubMed

Evans, John P; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

2008-07-11

Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the alpha-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin pi-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of alpha-meso-phenylheme-IX, alpha-meso-(p-methylphenyl)-mesoheme-III, and alpha-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593-42604), only the alpha-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced alpha-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation.

Isoporphyrin Intermediate in Heme Oxygenase Catalysis

PubMed Central

Evans, John P.; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

2008-01-01

Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. PMID:18487208

Parasite-Mediated Degradation of Synthetic Ozonide Antimalarials Impacts In Vitro Antimalarial Activity.

PubMed

Giannangelo, Carlo; Stingelin, Lukas; Yang, Tuo; Tilley, Leann; Charman, Susan A; Creek, Darren J

2018-03-01

The peroxide bond of the artemisinins inspired the development of a class of fully synthetic 1,2,4-trioxolane-based antimalarials, collectively known as the ozonides. Similar to the artemisinins, heme-mediated degradation of the ozonides generates highly reactive radical species that are thought to mediate parasite killing by damaging critical parasite biomolecules. We examined the relationship between parasite dependent degradation and antimalarial activity for two ozonides, OZ277 (arterolane) and OZ439 (artefenomel), using a combination of in vitro drug stability and pulsed-exposure activity assays. Our results showed that drug degradation is parasite stage dependent and positively correlates with parasite load. Increasing trophozoite-stage parasitemia leads to substantially higher rates of degradation for both OZ277 and OZ439, and this is associated with a reduction in in vitro antimalarial activity. Under conditions of very high parasitemia (∼90%), OZ277 and OZ439 were rapidly degraded and completely devoid of activity in trophozoite-stage parasite cultures exposed to a 3-h drug pulse. This study highlights the impact of increasing parasite load on ozonide stability and in vitro antimalarial activity and should be considered when investigating the antimalarial mode of action of the ozonide antimalarials under conditions of high parasitemia. Copyright © 2018 American Society for Microbiology.

Heme iron uptake by Caco-2 cells is a saturable, temperature sensitive and modulated by extracellular pH and potassium.

PubMed

Arredondo, Miguel; Kloosterman, Janneke; Núñez, Sergio; Segovia, Fabián; Candia, Valeria; Flores, Sebastián; Le Blanc, Solange; Olivares, Manuel; Pizarro, Fernando

2008-11-01

It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K+ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low Vmax and Km as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K+ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.

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

PubMed

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

2015-06-11

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. © 2015 by The American Society of Hematology.

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

PubMed Central

2017-01-01

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs. PMID:29286645

Single or functionalized fullerenes interacting with heme group

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costa, Wallison Chaves; Diniz, Eduardo Moraes, E-mail: eduardo.diniz@ufma.br

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 groupsmore » (−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.« less

Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction.

PubMed

Stranava, Martin; Martínková, Markéta; Stiborová, Marie; Man, Petr; Kitanishi, Kenichi; Muchová, Lucie; Vítek, Libor; Martínek, Václav; Shimizu, Toru

2014-11-01

The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

PubMed Central

Ivanov, Vladimir N.; Hei, Tom K.

2015-01-01

Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20–40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5–20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation. PMID:21910007

The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption.

PubMed

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

2015-10-30

The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe) absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study). Study I focused on the effects of cereal proteins (zein, gliadin and glutelin) and study II on the effects of legume proteins (soy, pea and lentil) on heme Fe absorption. When heme was given alone (as a control), study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05). In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p < 0.05). These results suggest that with the exception of soy proteins, which decreased absorption, proteins derived from cereals and legumes do not affect heme Fe absorption.

Insulin enhances the peroxidase activity of heme by forming heme-insulin complex: Relevance to type 2 diabetes mellitus.

PubMed

Huang, Yi; Yang, Zhen; Xu, Huan; Zhang, Pengfei; Gao, Zhonghong; Li, Hailing

2017-09-01

Evidences have implicated the involvement of heme in the type 2 diabetes mellitus (T2Dm) pathogenesis, but possible mediators linking between heme and diabetes are still poorly understood. Here, we explored a potential mechanism that linked heme, insulin and diabetes. Our results demonstrated the formation of heme-insulin complex by two classical methods, i.e. UV-vis and capillary electrophoresis-frontal analysis (CE-FA). UV-vis results implied heme binding insulin via bis-histidine sites, and CE-FA further revealed that, when insulin uses two sites binding with heme, this interaction occurs at high affinity (K d =3.13×10 -6 M). Molecule docking supported that histidine-B5 of insulin binds with heme-Fe. In addition to that, tyrosine-B26, phenylalanine-B1 and valine-B2 are also contributed to binding heme. The binding amplified the peroxidase activity of heme itself. Under oxidative and nitrative stress, it affects pathogenesis of diabetes from two aspects: promoting insulin cross-linking that leads to permanent loss of insulin functionality on one hand, and enhancing protein tyrosine nitration that may result in inactivation of proteins associated with diabetes on the other hand. This study suggested that the enhanced peroxidase activity of heme through binding with insulin might be a previously unrecognized contributor to the pathogenesis of T2Dm in some heme-associated disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds.

PubMed

Rahmanpour, Rahman; Rea, Dean; Jamshidi, Shirin; Fülöp, Vilmos; Bugg, Timothy D H

2016-03-15

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a β-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH

PubMed Central

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M.

2017-01-01

Abstract Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. PMID:28961970

Revisiting the putative role of heme as a trigger of inflammation.

PubMed

Vallelian, Florence; Schaer, Christian A; Deuel, Jeremy W; Ingoglia, Giada; Humar, Rok; Buehler, Paul W; Schaer, Dominik J

2018-04-01

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.

Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance.

PubMed

Ryter, Stefan W; Otterbein, Leo E; Morse, Danielle; Choi, Augustine M K

2002-01-01

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO exists as constitutive (HO-2, HO-3) and inducible isoforms (HO-1), the latter which responds to regulation by multiple stress-stimuli. HO-1 confers protection in vitro and in vivo against oxidative cellular stress. Although the redox active compounds that are generated from HO activity (i.e. iron, biliverdin-IXalpha, and bilirubin-IXa) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for CO. Though not reactive, CO regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. The latter effects of CO depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic 3':5'-guanosine monophosphate. CO potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. Recent progress indicates that CO exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p38 mitogen activated protein kinase (MAPK)-signaling pathway. By virtue of these effects, CO confers protection in oxidative lung injury models, and likely plays a role in HO-1 mediated tissue protection.

Iron acquisition by Haemophilus influenzae.

PubMed Central

Pidcock, K A; Wooten, J A; Daley, B A; Stull, T L

1988-01-01

The mechanisms for acquisition of iron by Haemophilus influenzae and their role in pathogenesis are not known. Heme and nonheme sources of iron were evaluated for their effect on growth of type b and nontypable strains of H. influenzae in an iron-restricted, defined medium. All 13 strains acquired iron from heme, hemoglobin, hemoglobin-haptoglobin, and heme-hemopexin. Among nonheme sources of protein-bound iron, growth of H. influenzae was enhanced by partially saturated human transferrin but not by lactoferrin or ferritin. Purified ferrienterochelin and ferridesferrioxamine failed to provide iron to H. influenzae, and the supernatants of H. influenzae E1a grown in iron-restricted medium failed to enhance iron-restricted growth of siderophore-dependent strains of Escherichia coli, Salmonella typhimurium, and Arthrobacter terregens. Marked alterations in the profile of outer membrane proteins of H. influenzae were observed when the level of free iron was varied between 1 microM and 1 mM. Catechols were not detected in the supernatants of strain E1a; however, iron-related hydroxamate production was detected by two biochemical assays. We conclude that the sources of iron for H. influenzae are diverse. The significance of hydroxamate production and iron-related outer membrane proteins to H. influenzae iron acquisition is not yet clear. Images PMID:2964410

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.

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

PubMed Central

Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

2016-01-01

It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M−1·s−1 for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

KIEFL,CHRISTOPH; SCREERAMA,NARASIMHA; LU,YI

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 decompositionmore » 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.« less

RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp).

PubMed

Birke, Jakob; Röther, Wolf; Jendrossek, Dieter

2017-07-15

Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c -type cytochromes of ≈70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C 15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (≈40 kDa), are b -type cytochromes, and cleave polyisoprene to a mixture of C 20 , C 25 , C 30 , and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c -type hemes). However, RoxB Xsp differs from all currently known RoxAs in having a distinctive product spectrum of C 20 , C 25 , C 30 , and higher oligo-isoprenoids that has been observed only for Lcps so far. Purified RoxB Xsp revealed the highest specific activity of 4.5 U/mg (at 23°C) of all currently known rubber oxygenases and exerts a synergistic effect on the efficiency of polyisoprene cleavage by RoxA Xsp RoxB homologs were identified in several other Gram-negative rubber-degrading species, pointing to a prominent function of RoxB for the biodegradation of rubber in Gram-negative bacteria. IMPORTANCE The enzymatic cleavage of rubber (polyisoprene) is of high environmental importance given that enormous amounts of rubber waste materials are permanently released (e.g., by abrasion of tires). Research from the last decade has discovered rubber oxygenase A, RoxA, and latex clearing protein (Lcp) as being responsible for the primary enzymatic attack on the hydrophobic and water-insoluble biopolymer poly( cis -1,4-isoprene) in Gram-negative and Gram-positive rubber-degrading bacteria, respectively. Here, we provide evidence that a third type of rubber oxygenase is present in Gram-negative rubber-degrading species. Due to its characteristics, we suggest the designation RoxB for the new type of rubber oxygenase. Bioinformatic analysis of genome sequences indicates the presence of roxB homologs in other Gram-negative rubber degraders. Copyright © 2017 American Society for Microbiology.

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

In Vitro Assembly of Catalase*

PubMed Central

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-01-01

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. PMID:25148685

Formation of naturally occurring pigments during the production of nitrite-free dry fermented sausages.

PubMed

De Maere, Hannelore; Fraeye, Ilse; De Mey, Eveline; Dewulf, Lore; Michiels, Chris; Paelinck, Hubert; Chollet, Sylvie

2016-04-01

This study investigates the potential of producing red coloured dry fermented sausages without the addition of nitrite and/or nitrate. Therefore, the formation of zinc protoporphyrin IX (Zn(II)PPIX) as naturally occurring pigment, and the interrelated protoporphyrin IX (PPIX) and heme content were evaluated during nitrite-free dry fermented sausage production at different pH conditions. Zn(II)PPIX was only able to form in dry fermented sausages at pH conditions higher than approximately 4.9. Additionally, the presence of Zn(II)PPIX increased drastically at the later phase of the production process (up to day 177), confirming that in addition to pH, time is also a crucial factor for its formation. Similarly, PPIX also accumulated in the meat products at increased pH conditions and production times. In contrast, a breakdown of heme was observed. This breakdown was more gradual and independent of pH and showed no clear relationship with the formed amounts of Zn(II)PPIX and PPIX. A statistically significant relationship between Zn(II)PPIX formation and product redness was established. Copyright © 2015 Elsevier Ltd. All rights reserved.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular mechanism and genetic determinants of buprofezin degradation.

PubMed

Chen, Xueting; Ji, Junbin; Zhao, Leizhen; Qiu, Jiguo; Dai, Chen; Wang, Weiwu; He, Jian; Jiang, Jiandong; Hong, Qing; Yan, Xin

2017-07-14

Buprofezin is a widely used insect growth regulator whose residue has been frequently detected in the environment, posing a threat to aquatic organisms and non-target insects. Microorganisms play an important role in the degradation of buprofezin in the natural environment. However, the relevant catabolic pathway has not been fully characterized, and the molecular mechanism of catabolism is still completely unknown. Rhodococcus qingshengii YL-1 can utilize buprofezin as a sole source of carbon and energy for growth. In this study, the upstream catabolic pathway in strain YL-1 was identified using tandem mass spectrometry. Buprofezin is composed of a benzene ring and a heterocyclic ring. The degradation is initiated by the dihydroxylation of the benzene ring and continues via dehydrogenation, aromatic ring cleavage, breaking of an amide bond and the release of the heterocyclic ring 2- tert -butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one (2-BI). A buprofezin degradation-deficient mutant strain YL-0 was isolated. Comparative genomic analysis combined with gene deletion and complementation experiments revealed that the gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin. bfzA3A4A1A2 encodes a novel Rieske non-heme iron oxygenase (RHO) system that is responsible for the dihydroxylation of buprofezin at the benzene ring; bfzB is involved in dehydrogenation, and bfzC is in charge of benzene ring cleavage. Furthermore, the products of bfzBA3A4A1A2C can also catalyze dihydroxylation, dehydrogenation and aromatic ring cleavage of biphenyl, flavanone, flavone and bifenthrin. In addition, a transcriptional study revealed that bfzBA3A4A1A2C is organized in one transcriptional unit that is constitutively expressed in strain YL-1. Importance There is an increasing concern about the residue and environmental fate of buprofezin. Microbial metabolism is an important mechanism responsible for the buprofezin degradation in natural environment. However, the molecular mechanism and genetic determinants of microbial degradation of buprofezin has not been well identified. This work revealed that gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin in R. qingshengii YL-1. The products of bfzBA3A4A1A2C could also degrade bifenthrin, a widely used pyrethroid insecticide. These findings enhance our understanding of the microbial degradation mechanism of buprofezin and benefit the application of strain YL-1 and bfzBA3A4A1A2C in the bioremediation of buprofezin contamination. Copyright © 2017 American Society for Microbiology.

HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

DOE PAGES

Dailey, Harry A.; Gerdes, Svetlana

2015-02-21

Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. We find that the heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed.more » Furthermore, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.« less

Metals and lipid oxidation. Contemporary issues.

PubMed

Schaich, K M

1992-03-01

Lipid oxidation is now recognized to be a critically important reaction in physiological and toxicological processes as well as in food products. This provides compelling reasons to understand what causes lipid oxidation in order to be able to prevent or control the reactions. Redox-active metals are major factors catalyzing lipid oxidation in biological systems. Classical mechanisms of direct electron transfer to double bonds by higher valence metals and of reduction of hydroperoxides by lower valence metals do not always account for patterns of metal catalysis of lipid oxidation in multiphasic or compartmentalized biological systems. To explain why oxidation kinetics, mechanisms, and products in molecular environments which are both chemically and physically complex often do not follow classical patterns predicted by model system studies, increased consideration must be given to five contemporary issues regarding metal catalysis of lipid oxidation: hypervalent non-heme iron or iron-oxygen complexes, heme catalysis mechanism(s), compartmentalization of reactions and lipid phase reactions of metals, effects of metals on product mixes, and factors affecting the mode of metal catalytic action.

Production of Dioxygen in the Dark: Dismutases of Oxyanions

PubMed Central

Ojha, Sunil

2016-01-01

O2 generating reactions are exceedingly rare in biology and difficult to mimic synthetically. Perchlorate-respiring bacteria enzymatically detoxify chlorite (ClO2−), the end product of the perchlorate (ClO4−) respiratory pathway, by rapidly converting it to dioxygen (O2) and chloride (Cl−). This reaction is catalyzed by a heme-containing protein, called chlorite dismutase (Cld), which bears no structural or sequence relationships with known peroxidases or other heme proteins and is part of a large family of proteins with more than one biochemical function. The original assumptions from the 1990s that perchlorate is not a natural product and that perchlorate respiration might be confined to a taxonomically narrow group of species have been called into question, as have the roles of perchlorate respiration and Cld-mediated reactions in the global biogeochemical cycle of chlorine. In this chapter, the chemistry and biochemistry of Cld-mediated O2 generation, as well as the biological and geochemical context of this extraordinary reaction, are described. PMID:25707466

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

PubMed Central

Atamna, Hani; Killilea, David W.; Killilea, Alison Nisbet; Ames, Bruce N.

2002-01-01

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process. PMID:12417755

Lycopene inhibits NF-κB activation and adhesion molecule expression through Nrf2-mediated heme oxygenase-1 in endothelial cells.

PubMed

Yang, Po-Min; Chen, Huang-Zhi; Huang, Yu-Ting; Hsieh, Chia-Wen; Wung, Being-Sun

2017-06-01

The endothelial expression of cell adhesion molecules plays a leading role in atherosclerosis. Lycopene, a carotenoid with 11 conjugated double bonds, has been shown to have anti-inflammatory properties. In the present study, we demonstrate a putative mechanism for the anti-inflammatory effects of lycopene. We demonstrate that lycopene inhibits the adhesion of tumor necrosis factor α (TNFα)-stimulated monocytes to endothelial cells and suppresses the expression of intercellular cell adhesion molecule-1 (ICAM-1) at the transcriptional level. Moreover, lycopene was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein, IκBα, following 6 h of pre-treatment. In TNFα-stimulated endothelial cells, nuclear factor-κB (NF-κB) nuclear translocation and transcriptional activity were abolished by up to 12 h of lycopene pre-treatment. We also found that lycopene increased the intracellular glutathione (GSH) level and glutamate-cysteine ligase expression. Subsequently, lycopene induced nuclear factor-erythroid 2 related factor 2 (Nrf2) activation, leading to the increased expression of downstream of heme oxygenase-1 (HO-1). The use of siRNA targeting HO-1 blocked the inhibitory effects of lycopene on IκB degradation and ICAM-1 expression. The inhibitory effects of lycopene thus appear to be mediated through its induction of Nrf2-mediated HO-1 expression. Therefore, the findings of the present study indicate that lycopene suppresses the activation of TNFα-induced signaling pathways through the upregulation of Nrf2-mediated HO-1 expression.

Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms.

PubMed

Martínez-Sernández, Victoria; Mezo, Mercedes; González-Warleta, Marta; Perteguer, María J; Gárate, Teresa; Romarís, Fernanda; Ubeira, Florencio M

2017-05-26

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein ( e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani , also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

PubMed

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

2012-10-30

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

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

Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but not Disruption of the Distal Hydrogen Bonding Network†

PubMed Central

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

2008-01-01

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

Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

PubMed

Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

2008-11-01

Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

Up-Regulation of Heme Oxygenase-1 in Rat Spleen Following Aniline Exposure

PubMed Central

Wang, Jianling; Ma, Huaxian; Boor, Paul J.; Sadagopa Ramanujam, V. M.; Ansari, G.A.S.; Khan, M. Firoze

2010-01-01

Splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats. However, underlying mechanisms by which aniline elicits splenotoxic response are not well understood. Previously we have shown that aniline exposure causes oxidative damage to the spleen. To further explore the oxidative mechanism of aniline toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1), which catalyzes heme degradation and releases free iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4 or 7 days, while respective controls received water only. Aniline exposure led to significant increases in HO-1 mRNA expression in the spleen (2- and 2.4-fold at days 4 and 7, respectively) with corresponding increases in protein expression, as confirmed by ELISA and Western blot analyses. Furthermore, immunohistochemical assessment of spleen showed stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined mainly to the red pulp areas. No changes were observed in mRNA and protein levels of HO-1 following 1 day exposure. The increase in HO-1 expression was associated with increases in total iron (2.4- and 2.7- fold), free iron (1.9- and 3.5-fold), and ferritin levels (1.9- and 2.1-fold) at 4 and 7 days of aniline exposure. Our data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a contributing pro-oxidant mechanism, mediated through iron release, and leading to oxidative damage. PMID:19969074

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

Relationship between natural and heme-mediated antibody polyreactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh

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 polyreactivitymore » 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. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.« less

Hemolysis-induced lethality involves inflammasome activation by heme

PubMed Central

Dutra, Fabianno F.; Alves, Letícia S.; Rodrigues, Danielle; Fernandez, Patricia L.; de Oliveira, Rosane B.; Golenbock, Douglas T.; Zamboni, Dario S.; Bozza, Marcelo T.

2014-01-01

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K+ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release. PMID:25225402

Hemolysis-induced lethality involves inflammasome activation by heme.

PubMed

Dutra, Fabianno F; Alves, Letícia S; Rodrigues, Danielle; Fernandez, Patricia L; de Oliveira, Rosane B; Golenbock, Douglas T; Zamboni, Dario S; Bozza, Marcelo T

2014-09-30

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K(+) efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.

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

PubMed

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

2010-12-13

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

A Novel Pathway for the Biosynthesis of Heme in Archaea: Genome-Based Bioinformatic Predictions and Experimental Evidence

PubMed Central

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

2010-01-01

Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d 1 biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified. PMID:21197080

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

Metabolomics analyses identify platelet activating factors and heme breakdown products as Lassa fever biomarkers.

PubMed

Gale, Trevor V; Horton, Timothy M; Grant, Donald S; Garry, Robert F

2017-09-01

Lassa fever afflicts tens of thousands of people in West Africa annually. The rapid progression of patients from febrile illness to fulminant syndrome and death provides incentive for development of clinical prognostic markers that can guide case management. The small molecule profile of serum from febrile patients triaged to the Viral Hemorrhagic Fever Ward at Kenema Government Hospital in Sierra Leone was assessed using untargeted Ultra High Performance Liquid Chromatography Mass Spectrometry. Physiological dysregulation resulting from Lassa virus (LASV) infection occurs at the small molecule level. Effects of LASV infection on pathways mediating blood coagulation, and lipid, amino acid, nucleic acid metabolism are manifest in changes in the levels of numerous metabolites in the circulation. Several compounds, including platelet activating factor (PAF), PAF-like molecules and products of heme breakdown emerged as candidates that may prove useful in diagnostic assays to inform better care of Lassa fever patients.

Metabolomics analyses identify platelet activating factors and heme breakdown products as Lassa fever biomarkers

PubMed Central

Gale, Trevor V.; Horton, Timothy M.; Grant, Donald S.

2017-01-01

Lassa fever afflicts tens of thousands of people in West Africa annually. The rapid progression of patients from febrile illness to fulminant syndrome and death provides incentive for development of clinical prognostic markers that can guide case management. The small molecule profile of serum from febrile patients triaged to the Viral Hemorrhagic Fever Ward at Kenema Government Hospital in Sierra Leone was assessed using untargeted Ultra High Performance Liquid Chromatography Mass Spectrometry. Physiological dysregulation resulting from Lassa virus (LASV) infection occurs at the small molecule level. Effects of LASV infection on pathways mediating blood coagulation, and lipid, amino acid, nucleic acid metabolism are manifest in changes in the levels of numerous metabolites in the circulation. Several compounds, including platelet activating factor (PAF), PAF-like molecules and products of heme breakdown emerged as candidates that may prove useful in diagnostic assays to inform better care of Lassa fever patients. PMID:28922385

Catalase degradation in sunflower cotyledons during peroxisome transition from glyoxysomal to leaf peroxisomal function. [Helianthus annuus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eising, R.; Gerhardt, B.

1987-06-01

First order rate constant for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing /sup 14/C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day/sup -1/ in contrast to the constants ranging from 0.304 day/sup -1/ to 0.515 day/sup -1/ during the other developmentalmore » stages. Density labeling experiments comprising labeling of catalase with /sup 2/H/sub 2/O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of /sup 14/C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.« less

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP*

PubMed Central

Juillan-Binard, Céline; Picciocchi, Antoine; Andrieu, Jean-Pierre; Petit-Hartlein, Isabelle; Caux-Thang, Christelle; Vivès, Corinne; Nivière, Vincent

2017-01-01

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli. We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins. PMID:28028176

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer.

PubMed

Pintscher, Sebastian; Kuleta, Patryk; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

2016-03-25

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on hemebligand mutants of cytochromebc1in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemesbin this cytochrome and in other membranous cytochromesbact as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

2012-03-01

B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Ordine, Robert L.; Rydel, Timothy J.; Storek, Michael J.

2009-09-08

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O{sub 2} into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer ({alpha}{sub 3}) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While themore » Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co{sup 2+}, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 {angstrom}, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.« less

Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein.

PubMed Central

Ewing, J F; Maines, M D

1991-01-01

Catalytic activity of heme oxygenase (heme, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.3) isozymes, HO-1 and HO-2, permits production of physiologic isomers of bile pigments. In turn, bile pigments biliverdin and bilirubin are effective antioxidants in biological systems. In the rat brain we have identified only the HO-1 isozyme of heme oxygenase as a heat shock protein and defined hyperthermia as a stimulus that causes an increase in brain HO-1 protein. Exposure of male rats to 42 degrees C for 20 min caused a rapid and marked increase in brain 1.8-kilobase HO-1 mRNA. Specifically, a 33-fold increase in brain HO-1 mRNA was observed within 1 h and sustained for at least 6 h posttreatment. In contrast, the two HO-2 homologous transcripts (1.3 and 1.9 kilobases) did not respond to heat shock; neither the ratio nor the level of the two messages differed from that of the control when measured either at 1, 6, or 24 h after hyperthermia. The induction of a 1.8-kilobase HO-1 mRNA resulted in a pronounced increase in HO-1 protein 6 h after hyperthermia, as detected by both Western immunoblot and RIA. Immunocytochemistry of rat brain showed discrete localization of HO-1-like protein only in neurons of select brain regions. Six hours after heat shock, an intense increase in HO-1-like protein was observed in both Purkinje cells of the cerebellum and epithelial cells lining the cerebral aqueduct of the brain. We suggest that the increase in HO-1 protein, hence increased capacity to form bile pigments, represents a neuronal defense mechanism against heat shock stress. Images PMID:2052613

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

PubMed

Schroeder, Susanne; Lawrence, Andrew D; Biedendieck, Rebekka; Rose, Ruth-Sarah; Deery, Evelyne; Graham, Ross M; McLean, Kirsty J; Munro, Andrew W; Rigby, Stephen E J; Warren, Martin J

2009-02-20

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Suicide inactivation of hydroxylamine oxidoreductase of Nitrosomonas europaea by organohydrazines.

PubMed

Logan, M S; Hooper, A B

1995-07-18

In the presence of a suitable electron acceptor such as mammalian cytochrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophic bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxylamine or hydrazine to nitrite or dinitrogen, respectively. Each subunit of HAO contains 7 c-hemes and a chromophore of the active site called heme P460, a c-heme bridged from a methylene carbon to a ring carbon of a tyrosine of the peptide chain. Reaction with either substrate results in reduction of several c-hemes of HAO. The reaction of organohydrazines with HAO was investigated in this work. HAO was inactivated by (phenyl-, (methyl-, or (hydroxyethyl)hydrazine. The process followed first order kinetics and was inhibited by the substrates, hydroxylamine or hydrazine. Complete loss of enzyme activity and absorbancy characteristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylhydrazine and HAO. HAO was covalently derivatized by two molecules of [14C]-phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinity. c-Hemes were not derivatized except for the partial reaction of (hydroxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazine, incubation with organohydrazines resulted in reduction of c-heme of HAO. Derivatized minus native optical difference spectra of ferric or ferrous HAO revealed changes in the optical properties of heme P460 which were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

PubMed Central

Matuschewski, Kai; Haussig, Joana M.

2016-01-01

Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.

2012-12-07

Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-cmore » containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.« less

Characterization of the Heme Environment in Arabidopsis thaliana Fatty Acid α-Dioxygenase-1*

PubMed Central

Liu, Wen; Rogge, Corina E.; Bambai, Bijan; Palmer, Graham; Tsai, Ah-Lim; Kulmacz, Richard J.

2010-01-01

Plant α-dioxygenases (PADOX) are hemoproteins in the myeloperoxidase family. We have used a variety of spectroscopic, mutagenic, and kinetic approaches to characterize the heme environment in Arabidopsis thaliana PADOX-1. Recombinant PADOX-1 purified to homogeneity contained 1 mol of heme bound tightly but noncovalently per protein monomer. Electronic absorbance, electron paramagnetic resonance, and magnetic circular dichroism spectra showed a high spin ferric heme that could be reduced to the ferrous state by dithionite. Cyanide bound relatively weakly in the ferric PADOX-1 heme vicinity (Kd ~10 mm) but did not shift the heme to the low spin state. Cyanide was a very strong inhibitor of the fatty acid oxygenase activity (Ki ~5 µm) and increased the Km value for oxygen but not that for fatty acid. Spectroscopic analyses indicated that carbon monoxide, azide, imidazole, and a variety of substituted imidazoles did not bind appreciably in the ferric PADOX-1 heme vicinity. Substitution of His-163 and His-389 with cysteine, glutamine, tyrosine, or methionine resulted in variable degrees of perturbation of the heme absorbance spectrum and oxygenase activity, consistent with His-389 serving as the proximal heme ligand and indicating that the heme has a functional role in catalysis. Overall, A. thaliana PADOX-1 resembles a b-type cytochrome, although with much more restricted access to the distal face of the heme than seen in most other myeloperoxidase family members, explaining the previously puzzling lack of peroxidase activity in the plant protein. PADOX-1 is unusual in that it has a high affinity, inhibitory cyanide-binding site distinct from the distal heme face and the fatty acid site. PMID:15100225

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

PubMed Central

Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

2008-01-01

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

PubMed Central

Dailey, Tamara A.; Gerdes, Svetlana; Jahn, Dieter; O'Brian, Mark R.; Warren, Martin J.

2017-01-01

SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. PMID:28123057

Rapid and reliable determination of the halogenating peroxidase activity in blood samples.

PubMed

Flemmig, Jörg; Schwarz, Pauline; Bäcker, Ingo; Leichsenring, Anna; Lange, Franziska; Arnhold, Jürgen

2014-12-15

By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases. Copyright © 2014 Elsevier B.V. All rights reserved.

Electron Flow in Multiheme Bacterial Cytochromes is a Balancing Act Between Heme Electronic Interaction and Redox Potentials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen

The naturally widespread process of electron transfer from metal reducing bacteria to extracellular solid metal oxides entails unique biomolecular machinery optimized for long-range electron transport. To perform this function efficiently microorganisms have adapted multi-heme c-type cytochromes to arrange heme cofactors into wires that cooperatively span the cellular envelope, transmitting electrons along distances greater than 100 Angstroms. Implications and opportunities for bionanotechnological device design are self-evident. However, at the molecular level how these proteins shuttle electrons along their heme wires, navigating intraprotein intersections and interprotein interfaces effciently, remains a mystery so far inaccessible to experiment. To shed light on this criticalmore » topic, we carried out extensive computer simulations to calculate Marcus theory quantities for electron transfer along the ten heme cofactors in the recently crystallized outer membrane cytochrome MtrF. The combination of electronic coupling matrix elements with free energy calculations of heme redox potentials and reorganization energies for heme-to-heme electron transfer allows the step-wise and overall electron transfer rate to be estimated and understood in terms of structural and dynamical characteristics of the protein. By solving a master equation for electron hopping, we estimate an intrinsic, maximum possible electron flux through solvated MtrF of 104-105 s-1, consistent with recently measured rates for the related MtrCAB protein complex. Intriguingly, this flux must navigate thermodynamically uphill steps past low potential hemes. Our calculations show that the rapid electron transport through MtrF is the result of a clear correlation between heme redox potential and the strength of electronic coupling along the wire: Thermodynamically uphill steps occur only between electronically well connected stacked heme pairs. This suggests that the protein evolved to harbor low potential hemes, presumably necessary for reduction of certain soluble substrates, without slowing down electron ow. These findings are particularly profound in light of the apparently well conserved staggered cross heme wire structural motif in functionally related outer-membrane proteins.« less

Engineering out motion: introduction of a de novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b5.

PubMed

Storch, E M; Daggett, V; Atkins, W M

1999-04-20

A previous molecular dynamics (MD) simulation of cytochrome b5 (cyt b5) at 25 degrees C displayed localized dynamics on the surface of the protein giving rise to the periodic formation of a cleft that provides access to the heme through a protected hydrophobic channel [Storch and Daggett (1995) Biochemistry 34, 9682]. Here we describe the production and testing of mutants designed to prevent the cleft from opening using a combination of experimental and theoretical techniques. Two mutants have been designed to close the surface cleft: S18D to introduce a salt bridge and S18C:R47C to incorporate a disulfide bond. The putative cleft forms between two separate cores of the protein: one is structural in nature and can be monitored through the fluorescence of Trp 22, and the other binds the heme prosthetic group and can be tracked via heme absorbance. An increase in motion localized to the cleft region was observed for each protein, except for the disulfide-containing variant, in MD simulations at 50 degrees C compared to simulations at 25 degrees C. For the disulfide-containing variant, the cleft remained closed. Both urea and temperature denaturation curves were nearly identical for wild-type and mutant proteins when heme absorbance was monitored. In contrast, fluorescence studies revealed oxidized S18C:R47C to be considerably more stable based on the midpoints of the denaturation transitions, Tm and U1/2. Moreover, the fluorescence changes for each protein were complete at approximately 50 degrees C and a urea concentration of approximately 3.9 M, significantly below the temperature and urea concentration (62 degrees C, 5 M urea) required to observe heme release. In addition, solvent accessibility based on acrylamide quenching of Trp 22 was lower in the S18C:R47C mutant, particularly at 50 degrees C, before heme release [presented in the accompanying paper (58)]. The results suggest that a constraining disulfide bond can be designed to inhibit dynamic cleft formation on the surface of cyt b5. Located near the heme, the native dynamics of the cleft may be functionally important for protein-protein recognition and/or complex stabilization.

Heme oxygenase activity increases after exercise in healthy volunteers

EPA Science Inventory

AbstractHeme oxygenase (HO) is an essential, rate-limiting protein which participates in the catabolism of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge carbon of the heme is eliminated as CO which can be measured as blood carboxyhemoglobin (COHb)....

Structure and function of enzymes in heme biosynthesis.

PubMed

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

2010-06-01

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

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH.

PubMed

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M; Hansen, Lars Hestbjerg

2017-09-01

Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Enhancement of DEN-induced liver tumorigenesis in heme oxygenase-1 G143H mutant transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Jianfeng; Wang, Dayong; Xiao, Haifeng

Heme oxygenase (HO) is the rate-limiting enzyme in heme metabolism. HO-1 exhibits anti-oxidative and anti-inflammatory function via the actions of its metabolite, respectively. A growing body of evidence demonstrates that HO-1 is implicated in the pathogenesis and progression of several types of cancer. However, whether HO-1 takes part in healthy-premalignant-malignant transformation is still undefined. In this study, we took advantage of transgenic mice which over-expressed HO-1 dominant negative mutant (HO-1 G143H) and observed its susceptibility to DEN-induced hepatocarcinogenesis. Our results indicate that HO-1 G143H mutant accelerates the progression of tumorigenesis and tumor growth. The mechanism is closely related to enhancementmore » of ROS production which induce more hepatocytes death and secretion of inflammatory cytokines, proliferation of surviving hepatocytes. Our result provides the direct evidence that HO-1 plays an important protective role in liver carcinogenesis. Alternatively, we suggest the possible explanation on effect of HO-1 promoter polymorphism which involved in tumorigenesis. - Highlights: • Enhancement of DEN-induced hepatocarcinogenesis in HO-1 G143H Tg mice. • HO-1G143H mutant enhanced DEN-induced ROS production and liver injury. • HO-1G143H mutant aggravated DEN-induced changes of inflammatory factors and cell proliferation.« less

Design and engineering of a man-made diffusive electron-transport protein

PubMed Central

Fry, Bryan A.; Solomon, Lee A.; Dutton, P. Leslie

2016-01-01

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7 × 106 M−1s−1 to 1.2 × 109 M−1s−1 follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and −19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. PMID:26423266

[hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis].

PubMed

Xia, Zhen Wei; Cui, Wen Jun; Zhou, Wen Pu; Zhang, Xue Hong; Shen, Qing Xiang; Li, Yun Zhu; Yu, Shan Chang

2004-10-01

Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae.

PubMed

Buchensky, Celeste; Almirón, Paula; Mantilla, Brian Suarez; Silber, Ariel M; Cricco, Julia A

2010-11-01

Trypanosoma cruzi, the etiologic agent for Chagas’ disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Cloning and Expression of cDNA for Rat Heme Oxygenase

NASA Astrophysics Data System (ADS)

Shibahara, Shigeki; Muller, Rita; Taguchi, Hayao; Yoshida, Tadashi

1985-12-01

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in λ gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

PubMed Central

Pourkhalili, Azin; Rahimi, Ebrahim

2013-01-01

Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron. PMID:23737716

Heme iron content in lamb meat is differentially altered upon boiling, grilling, or frying as assessed by four distinct analytical methods.

PubMed

Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

2013-01-01

Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

Two-photon excited fluorescence emission from hemoglobin

NASA Astrophysics Data System (ADS)

Sun, Qiqi; Zeng, Yan; Zhang, Wei; Zheng, Wei; Luo, Yi; Qu, Jianan Y.

2015-03-01

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA.

PubMed

Zubieta, Chloe; Joseph, Rosanne; Krishna, S Sri; McMullan, Daniel; Kapoor, Mili; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Murphy, Kevin D; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; Trout, Christina V; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases. (c) 2007 Wiley-Liss, Inc.

Biotin deficiency inhibits heme synthesis and impairs mitochondria in human lung fibroblasts.

PubMed

Atamna, Hani; Newberry, Justin; Erlitzki, Ronit; Schultz, Carla S; Ames, Bruce N

2007-01-01

Four of the 5 biotin-dependent carboxylases (BDC) are in the mitochondria. BDC replace intermediates in the Krebs [tricarboxylic acid (TCA)] cycle that are regularly removed for the synthesis of key metabolites such as heme or amino acids. Heme, unlike amino acids, is not recycled to regenerate these intermediates, is not utilized from the diet, and must be synthesized in situ. We studied whether biotin deficiency (BD) lowers heme synthesis and whether mitochondria would be disrupted. Biotin-deficient medium was prepared by using bovine serum stripped of biotin with charcoal/dextran or avidin. Biotin-deficient primary human lung fibroblasts (IMR90) lost their BDC and senesced before biotin-sufficient cells. BD caused heme deficiency; there was a decrease in heme content and heme synthesis, and biotin-deficient cells selectively lost mitochondrial complex IV, which contains heme-a. Loss of complex IV, which is part of the electron transport chain, triggered oxidant release and oxidative damage, hallmarks of heme deficiency. Restoring biotin to the biotin-deficient medium prevented the above changes. Old cells were more susceptible to biotin shortage than young cells. These findings highlight the biochemical connection among biotin, heme, and iron metabolism, and the mitochondria, due to the role of biotin in maintaining the biochemical integrity of the TCA cycle. The findings are discussed in relation to aging and birth defects in humans.

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo; National Institute for Infectious Diseases I.R.C.C.S. 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Roma; Imperi, Francesco

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k{sub off}) is reported. In the absence of drugs, the value of k{sub off} is (1.3 {+-} 0.2) x 10{sup -4} s{sup -1}. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k{sub off} value increases to (8.6 {+-} 0.9) x 10{sup -4} s{sup -1}. From the dependence of k{sub off} on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NOmore » (i.e., K = (1.2 {+-} 0.2) x 10{sup -3} M and (6.2 {+-} 0.7) x 10{sup -5} M, respectively) were determined. The increase of k{sub off} values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.« less

[THe characteristics of the action of Khi-Mi on gastric and hepatic secretion].

PubMed

Rymshina, M V; Vasilevskaia, L S

1996-01-01

The peculiarities of action of He-Me (92% of sodium glutamate and 8% sodium inosinemonophosphate) on gastric and liver secretions was studied in experiments on dog with Pavlov's isolated ventricle and dogs with bile duct, taken out on the skin (operation by M. F. Nesterin). He-Me exerted promotional influence on gastric secretion provoked by pentagastrin increasing of secretion by 3-5 times with prolongation of secretion time. The gastric secretion in various stages (nervous, secretory, intestinal) was strengthened after adding of He-Me to dietary meat. In that way effect of He-Me on gastric secretion is the same as effect of sodium glutamate but effect He-Me more strong after oral intake. He-Me has also choleretic effect. The data allow to re recommend He-Me as diagnostic and therapeutic remedy.

Hemoglobin binding and catalytic heme extraction by IsdB near iron transporter domains.

PubMed

Bowden, Catherine F M; Verstraete, Meghan M; Eltis, Lindsay D; Murphy, Michael E P

2014-04-15

The Isd (iron-regulated surface determinant) system is a multiprotein transporter that allows bacterium Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains two near iron transporter (NEAT) domains, one of which binds heme. IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay into the bacterial cell. Using a series of recombinant IsdB constructs that included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB extracted heme only from oxidized metHb, although it also bound oxyHb and the Hb-CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed the transfer of heme from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 μN and a kcat of 0.22 ± 0.01 s(-1). The latter is consistent with the transfer of heme from metHb to IsdB being the rate-limiting step. With both NEAT domains and the linker region present in a single contiguous polypeptide, high-affinity Hb binding was achieved, rapid heme uptake was observed, and multiple turnovers of heme extraction from metHb and transfer to IsdA were conducted, representing all known Hb-heme uptake functions of the full-length IsdB protein.

Effects of illumination and packaging on non-heme iron and color attributes of sliced ham.

PubMed

Li, H; Li, C B; Xu, X L; Zhou, G H

2012-08-01

This study was designed to investigate effects of illumination and packaging on color of cooked cured sliced ham during refrigeration, and the possibility of decomposition of nitrosylheme under light and oxygen exposure. Three illumination levels and three packaging films with different oxygen transmission rates (OTRs) were used in two separate experiments during 35 days storage, and pH value, a* value, nitrosylheme, residual nitrite and non-heme iron were evaluated. Packaging OTRs had significant effects (P<0.01) on a* value, but illumination level and packaging OTR did not affect (P>0.05) nitrosylheme concentration during storage. For both groups, storage time had a significant effect (P<0.01) on a* value and nitrosylheme. Negative relationships between nitrosylheme and nitrite in the illumination group, and between nitrosylheme and non-heme iron in the packaging group were observed. Therefore, illumination level and packaging OTR had limited effects on overall pigment stability, but more discoloration and loss of redness occurred on the surface of products. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro assembly of catalase.

PubMed

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-10-10

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Organism-like formation of Schistosoma hemozoin and its function suggest a mechanism for anti-malarial action of artemisinin

PubMed Central

Sun, Jun; Li, Chen; Wang, Suwen

2016-01-01

The current theories of antimalarial mechanism of artemisinin are inadequate to fully explain the observed effects. In our study, “organism-like” formation of Schistosoma hemozoin granules by attaching to and utilizing erythrocytes to form new ones was observed. This indicates that heme iron is transferred from erythrocytes to hemozoin granules during their formation. However, as a disposal product of heme detoxification, these granules are not completely expelled from the Schistosoma gut, but decomposed again between microvilli in the posterior portion of the gut to transfer iron to eggs. Based on the function of iron transport supported by our observation of the unique process of Schistosoma hemozoin formation, here we propose a new viewpoint of antimalarial mechanism of artemisinin, which emphasizes the final outcome, i.e., interference of iron utilization in parasites by artemisinin, instead of focusing on the mode of interaction between artemisinin and heme or hemozoin. This suggests that artemisinin and its endoperoxides derivatives likely hit the Achilles’ heel of hemozoin-producing and iron-dependent organisms. PMID:27694940

Increased carboxyhemoglobin level during liver resection with inflow occlusion.

PubMed

Godai, Kohei; Hasegawa-Moriyama, Maiko; Kuniyoshi, Tamotsu; Matsunaga, Akira; Kanmura, Yuichi

2013-04-01

Controlling stress responses associated with ischemic changes due to bleeding and ischemia/reperfusion injury is essential for anesthetic management. Endogenous carboxyhemoglobin (COHb) is produced in the oxidative degradation of heme proteins by the stress-response enzyme heme oxygenase. Although the COHb level is elevated in critically ill patients, changes in endogenous COHb during anesthesia have not been well investigated. Therefore, we evaluated changes in endogenous COHb levels in patients undergoing liver resections with inflow occlusion. Levels of COHb were significantly increased after the Pringle maneuver. The inflow occlusion time in patients with increased COHb after the Pringle maneuver (∆COHb > 0.3 %) was significantly longer than in patients without increased COHb (∆COHb < 0.3 %) (P = 0.01). In addition, COHb changes were correlated with inflow occlusion time (P = 0.005, R(2) = 0.21). Neither total blood loss, transfusion volume of packed red blood cells, operation time, nor anesthetic time differed between patients with and without increased COHb. The results indicated that endogenous COHb levels were increased by inflow occlusion in patients undergoing liver resections, which suggests that changes in COHb may correlate with hepatic ischemia/reperfusion injury induced by inflow occlusion.

Effects of cooking and food matrix on estimated mineral bioavailability in Mloukhiya, a Mediterranean dish based on jute leaves and meat.

PubMed

Njoumi, Sondos; Bellagha, Sihem; Icard-Vernière, Christèle; Picq, Christian; Amiot, Marie Josèphe; Mouquet-Rivier, Claire

2018-03-01

Traditional Mediterranean plant-based dishes could allow tackling malnutrition while preserving the cultural heritage. To determine the effect of the cooking method on mineral bioavailability, the content in minerals and chelators of Mloukhiya, a Mediterranean dish based on jute leaves (Corchorus olitorius) that contains also meat, was monitored during the whole cooking process. Mineral bioaccessibility was assessed by measuring in vitro dialyzability. Model equation was also used to estimate mineral bioavailability. Comparison of Mloukhiya samples collected at different cooking time points showed that the dish total mineral content did not change despite the exchanges between sauce and meat during cooking. However, iron bioavailability decreased, because 58% of heme iron was degraded after 5h of cooking and non-heme iron showed poor bioaccessibility (1.2%), mainly due to its high content of phenolic compounds. The bioaccessibility of other minerals (zinc, calcium, magnesium and potassium) was high, indicating that the food matrix had no or little effect. The mineral bioavailability values predicted by using mathematical models were of the same order of magnitude as the bioaccessibility values. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Code of Federal Regulations, 2012 CFR

2012-04-01

... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated... 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...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated... 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...

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

Code of Federal Regulations, 2011 CFR

2011-04-01

... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated... 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...

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

Code of Federal Regulations, 2013 CFR

2013-04-01

... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated... 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...

O 2 Activation by Non-Heme Iron Enzymes

DOE PAGES

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

2016-10-28

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

O 2 Activation by Non-Heme Iron Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

Redox-dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase revealed by cyanide binding studies.

PubMed

Grönberg, Karin L C; Watmough, Nicholas J; Thomson, Andrew J; Richardson, David J; Field, Sarah J

2004-04-23

The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.

Tissue factor-dependent coagulation activation by heme: A thromboelastometry study.

PubMed

de Souza, Gleice Regina; Hounkpe, Bidossessi Wilfried; Fiusa, Maiara Marx Luz; Colella, Marina Pereira; Annichino-Bizzacchi, Joyce M; Traina, Fabiola; Costa, Fernando Ferreira; De Paula, Erich Vinicius

2017-01-01

Heme has been characterized as potent trigger of inflammation. In hemostasis, although heme has been shown to both induce and inhibit different compartments of hemostasis, its net effect on the hemostatic balance, and the biological relevance of these effects remain to be determined. Herein we evaluated the effect of heme on hemostasis using a global assay able to generate clinically relevant data in several other complex hemostatic diseases. Citrated whole blood samples from healthy participants were stimulated by heme or vehicle and incubated for 4h at 37°C. Rotational thromboelastometry was immediately performed. The participation of tissue factor in coagulation activation was evaluated using inhibitory antibody. Heme was able of inducing ex vivo coagulation activation in whole blood, affecting predominantly parameters associated with the initial phases of clot formation. This activation effect was at least partially dependent on hematopoietic tissue factor, since the effects of heme were partially abrogated by the inhibition of human tissue factor. In conclusion, using a global hemostasis assay, our study confirmed that heme is able to activate coagulation in whole blood, in a tissue factor-dependent way. These findings could explain the disturbance in hemostatic balance observed in conditions associated with the release of heme such as sickle cell disease.

Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy

PubMed Central

Huang, Qing; Al-Azzam, Wasfi; Griebenow, Kai; Schweitzer-Stenner, Reinhard

2003-01-01

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B1g distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B2g-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP. PMID:12719258

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

PubMed Central

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

2009-01-01

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 (Jiang, Y., Ortiz de Montellano, P.R., Inorg. Chem., 47, 3480-3482 (2008)), indicate that a selenyl radical is formed in the hHO1 His25SeCys mutant that adds to a heme vinyl group. PMID:19135260

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.

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

PubMed

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-10-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagao, Satoshi; Hirai, Yueki; Kawano, Shin

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 inmore » 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.« less

Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

PubMed

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

2014-12-01

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekworomadu, MarCia T.; Poor, Catherine B.; Owens, Cedric P.

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positivemore » hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3{sub 10}-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3{sub 10}-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.« less

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

DOE PAGES

Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.; ...

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 (HO2 O), 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 (HO2 R) 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.

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 (HO2 O), 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 (HO2 R) 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.« less

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1.

PubMed

Sam, Katharine A; Tolland, John D; Fairhurst, Shirley A; Higham, Christopher W; Lowe, David J; Thorneley, Roger N F; Allen, James W A; Ferguson, Stuart J

2008-07-11

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd(1) at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d(1)-heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d(1)Fe(II)-NO and 45% cFe(II)d(1)Fe(II)-NO(+). No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH.

Iron management and production of electricity by microorganisms.

PubMed

Folgosa, Filipe; Tavares, Pedro; Pereira, Alice S

2015-10-01

The increasing dependency on fossil fuels has driven researchers to seek for alternative energy sources. Renewable energy sources such as sunlight, wind, or water are the most common. However, since the 1990s, other sources for energy production have been studied. The use of microorganisms such as bacteria or archaea to produce energy is currently in great progress. These present several advantages even when compared with other renewable energy sources. Besides the energy production, they are also involved in bioremediation such as the removal of heavy metal contaminants from soils or wastewaters. Several research groups have demonstrated that these organisms are able to interact with electrodes via heme and non-heme iron proteins. Therefore, the role of iron as well as iron metabolism in these species must be of enormous relevance. Recently, the influence of cellular iron regulation by Fur in the Geobacter sulfurreducens growth and ability to produce energy was demonstrated. In this review, we aim to briefly describe the most relevant proteins involved in the iron metabolism of bacteria and archaea and relate them and their biological function with the ability of selected organisms to produce energy.

Analysis of the electrochemistry of hemes with Ems spanning 800 mV

PubMed Central

Zheng, Zhong; Gunner, M. R.

2009-01-01

The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of Ems with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b-, and c-type, with bis-His, His-Met, and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental Ems range over 800 mV from −350 mV in cytochrome c3 to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated Ems are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental Ems is 0.73 (R2 = 0.90), showing the method accounts for 73% of the observed Em range. Adding a +160 mV correction to the His-Met c-type hemes yields a slope of 0.97 (R2 = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and Ems shows both the lowest and highest potential hemes are c-type, whereas the b-type hemes are found in the middle Em range. In solution, bis-His ligation lowers the Em by ≈205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His, and His-Met ligated b-type hemes all cluster about Ems which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, whereas the high potential His-Met c-type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the Em, has been suggested to be a major factor in tuning in situ Ems. However, the calculated solvation energy vs. experimental Em shows a slope of 0.2 and R2 of 0.5 thus correlates weakly with Ems. All other individual interactions show even less correlation with Em. However the sum of these terms does reproduce the range of observed Ems. Therefore, different proteins use different aspects of their structures to modulate the in situ heme electrochemistry. This study also shows that the calculated Ems are relatively insensitive to different heme partial charges and to the protein dielectric constant used in the simulation. PMID:19003997

Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.

PubMed

Manoj, Kelath Murali; Parashar, Abhinav; Gade, Sudeep K; Venkatachalam, Avanthika

2016-01-01

Using oxygen and NADPH, the redox enzymes cytochrome P450 (CYP) and its reductase (CPR) work in tandem to carry out the phase I metabolism of a vast majority of drugs and xenobiotics. As per the erstwhile understanding of the catalytic cycle, binding of the substrate to CYP's heme distal pocket allows CPR to pump electrons through a CPR-CYP complex. In turn, this trigger (a thermodynamic push of electrons) leads to the activation of oxygen at CYP's heme-center, to give Compound I, a two-electron deficient enzyme reactive intermediate. The formation of diffusible radicals and reactive oxygen species (DROS, hitherto considered an undesired facet of the system) was attributed to the heme-center. Recently, we had challenged these perceptions and proposed the murburn ("mured burning" or "mild unrestricted burning") concept to explain heme enzymes' catalytic mechanism, electron-transfer phenomena and the regulation of redox equivalents' consumption. Murburn concept incorporates a one-electron paradigm, advocating obligatory roles for DROS. The new understanding does not call for high-affinity substrate-binding at the heme distal pocket of the CYP (the first and the most crucial step of the erstwhile paradigm) or CYP-CPR protein-protein complexations (the operational backbone of the erstwhile cycle). Herein, the dynamics of reduced nicotinamide nucleotides' consumption, peroxide formation and depletion, product(s) formation, etc. was investigated with various controls, by altering reaction variables, environments and through the incorporation of diverse molecular probes. In several CYP systems, control reactions lacking the specific substrate showed comparable or higher peroxide in milieu, thereby discrediting the foundations of the erstwhile hypothesis. The profiles obtained by altering CYP:CPR ratios and the profound inhibitions observed upon the incorporation of catalytic amounts of horseradish peroxidase confirm the obligatory roles of DROS in milieu, ratifying murburn as the operative concept. The mechanism of uncoupling (peroxide/water formation) was found to be dependent on multiple one and two electron equilibriums amongst the reaction components. The investigation explains the evolutionary implications of xenobiotic metabolism, confirms the obligatory role of diffusible reactive species in routine redox metabolism within liver microsomes and establishes that a redox enzyme like CYP enhances reaction rates (achieves catalysis) via a novel (hitherto unknown) modality.

Characterization of docosahexaenoic acid (DHA)-induced heme oxygenase-1 (HO-1) expression in human cancer cells: the importance of enhanced BTB and CNC homology 1 (Bach1) degradation.

PubMed

Wang, Shuai; Hannafon, Bethany N; Wolf, Roman F; Zhou, Jundong; Avery, Jori E; Wu, Jinchang; Lind, Stuart E; Ding, Wei-Qun

2014-05-01

The effect of docosahexaenoic acid (DHA) on heme oxygenase-1 (HO-1) expression in cancer cells has never been characterized. This study examines DHA-induced HO-1 expression in human cancer cell model systems. DHA enhanced HO-1 gene expression in a time- and concentration-dependent manner, with maximal induction at 21 h of treatment. This induction of HO-1 expression was confirmed in vivo using a xenograft nude mouse model fed a fish-oil-enriched diet. The increase in HO-1 gene transcription induced by DHA was significantly attenuated by the antioxidant N-acetyl cysteine, suggesting the involvement of oxidative stress. This was supported by direct measurement of lipid peroxide levels after DHA treatment. Using a human HO-1 gene promoter reporter construct, we identified two antioxidant response elements (AREs) that mediate the DHA-induced increase in HO-1 gene transcription. Knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression compromised the DHA-induced increase in HO-1 gene transcription, indicating the importance of the Nrf2 pathway in this event. However, the nuclear protein levels of Nrf2 remained unchanged upon DHA treatment. Further studies demonstrated that DHA reduces nuclear Bach1 protein expression by promoting its degradation and attenuates Bach1 binding to the AREs in the HO-1 gene promoter. In contrast, DHA enhanced Nrf2 binding to the AREs without affecting nuclear Nrf2 expression levels, indicating a new cellular mechanism that mediates DHA's induction of HO-1 gene transcription. To our knowledge, this is the first characterization of DHA-induced HO-1 expression in human malignant cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Purification and Characterization of Active-Site Components of the Putative p-Cresol Methylhydroxylase Membrane Complex from Geobacter metallireducens▿

PubMed Central

Johannes, Jörg; Bluschke, Alexander; Jehmlich, Nico; von Bergen, Martin; Boll, Matthias

2008-01-01

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β2 subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr394 of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low Km values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a Kic (competitive inhibition) value of 18 ± 9 μM and a Kiu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented. PMID:18658262

Downregulation of Heme Oxygenase 1 (HO-1) Activity in Hematopoietic Cells Enhances Their Engraftment After Transplantation.

PubMed

Adamiak, Mateusz; Moore, Joseph B; Zhao, John; Abdelbaset-Ismail, Ahmed; Grubczak, Kamil; Rzeszotek, Sylwia; Wysoczynski, Marcin; Ratajczak, Mariusz Z

2016-01-01

Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential, and HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. This finding is supported by our recent observation that hematopoietic stem progenitor cells (HSPCs) from HO-1 KO mice are easy mobilizers, since they respond better to peripheral blood chemotactic gradients than wild-type littermates. Based on these findings, we hypothesized that transient inhibition of HO-1 by nontoxic small-molecule inhibitors would enhance migration of HSPCs in response to bone marrow chemoattractants and thereby facilitate their homing. To directly address this issue, we generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule activators and inhibitors of HO-1. Our results indicate that HO-1 is an inhibitor of hematopoietic cell migration in response to crucial BM homing chemoattractants such as stromal-derived factor 1 (SDF-1) and sphingosine-1-phosphate (S1P). Most importantly, our in vitro and in vivo animal experiments demonstrate for the first time that transiently inhibiting HO-1 activity in HSPCs by small-molecule inhibitors improves HSPC engraftment. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve engraftment of HSPCs, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., when transplanting umbilical cord blood).

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

PubMed

Song, Linyang; Song, Wei; Schipper, Hyman M

2007-08-01

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

Iron metabolism modulation in Atlantic salmon infested with the sea lice Lepeophtheirus salmonis and Caligus rogercresseyi: A matter of nutritional immunity?

PubMed

Valenzuela-Muñoz, Valentina; Gallardo-Escárate, Cristian

2017-01-01

Sea lice are copepodid ectoparasites that produce high economic losses and environmental issues, thus impacting the salmon aquaculture worldwide. Atlantic salmon (Salmo salar) from Northern and Southern Hemispheres are primarily parasitized by Lepeophtheirus salmonis and Caligus rogercresseyi, respectively. To cope L. salmonis infestation, studies suggest that Atlantic salmon can restrict iron availability as a mechanism of nutritional immunity. However, no molecular studies of iron regulation from salmonids infected with C. rogercresseyi have been reported. The aim of this study was to determine if there are differences in the regulation of iron metabolism in Atlantic salmon infested with L. salmonis or C. rogercresseyi. For comparisons, skin and head kidney were profiled using qPCR of 15 genes related to iron regulation in Atlantic salmons infected with each sea louse species in Norway and Chile, respectively. Prior to infestation, no significant differences were observed between fish group. However, genes involved in iron transport and Heme biosynthesis were highly upregulated in Atlantic salmon infested with L. salmonis. Interestingly, hepcidin and Heme oxygenase, a component of the Heme degradation pathway, were upregulated during C. rogercresseyi infestation. Oxidative stress related genes were also evaluated, showing higher transcription activity in the head kidney than in the skin of Atlantic salmon infested with L. salmonis. These comparative results suggest pathogen-specific responses in infected Atlantic salmon, where iron metabolism is primarily regulated during the infestation with L. salmonis than C. rogercresseyi. Feeding behavior, for instance haematophagy, of the infesting sea lice species in relation to iron modulation is discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heme versus non-heme iron-nitroxyl {FeN(H)O}⁸ complexes: electronic structure and biologically relevant reactivity.

PubMed

Speelman, Amy L; Lehnert, Nicolai

2014-04-15

Researchers have completed extensive studies on heme and non-heme iron-nitrosyl complexes, which are labeled {FeNO}(7) in the Enemark-Feltham notation, but they have had very limited success in producing corresponding, one-electron reduced, {FeNO}(8) complexes where a nitroxyl anion (NO(-)) is formally bound to an iron(II) center. These complexes, and their protonated iron(II)-NHO analogues, are proposed key intermediates in nitrite (NO2(-)) and nitric oxide (NO) reducing enzymes in bacteria and fungi. In addition, HNO is known to have a variety of physiological effects, most notably in the cardiovascular system. HNO may also serve as a signaling molecule in mammals. For these functions, iron-containing proteins may mediate the production of HNO and serve as receptors for HNO in vivo. In this Account, we highlight recent key advances in the preparation, spectroscopic characterization, and reactivity of ferrous heme and non-heme nitroxyl (NO(-)/HNO) complexes that have greatly enhanced our understanding of the potential biological roles of these species. Low-spin (ls) heme {FeNO}(7) complexes (S = 1/2) can be reversibly reduced to the corresponding {FeNO}(8) species, which are stable, diamagnetic compounds. Because the reduction is ligand (NO) centered in these cases, it occurs at extremely negative redox potentials that are at the edge of the biologically feasible range. Interestingly, the electronic structures of ls-{FeNO}(7) and ls-{FeNO}(8) species are strongly correlated with very similar frontier molecular orbitals (FMOs) and thermodynamically strong Fe-NO bonds. In contrast, high-spin (hs) non-heme {FeNO}(7) complexes (S = 3/2) can be reduced at relatively mild redox potentials. Here, the reduction is metal-centered and leads to a paramagnetic (S = 1) {FeNO}(8) complex. The increased electron density at the iron center in these species significantly decreases the covalency of the Fe-NO bond, making the reduced complexes highly reactive. In the absence of steric bulk, monomeric high-spin {FeNO}(8) complexes decompose rapidly. Notably, in a recently prepared, dimeric [{FeNO}(7)]2 species, we observed that reduction leads to rapid N-N bond formation and N2O generation, which directly models the reactivity of flavodiiron NO reductases (FNORs). We have also made key progress in the preparation and stabilization of corresponding HNO complexes, {FeNHO}(8), using both heme and non-heme ligand sets. In both cases, we have taken advantage of sterically bulky coligands to stabilize these species. ls-{FeNO}(8) complexes are basic and easily form corresponding ls-{FeNHO}(8) species, which, however, decompose rapidly via disproportionation and H2 release. Importantly, we recently showed that we can suppress this reaction via steric protection of the bound HNO ligand. As a result, we have demonstrated that ls-{FeNHO}(8) model complexes are stable and amenable to spectroscopic characterization. Neither ls-{FeNO}(8) nor ls-{FeNHO}(8) model complexes are active for N-N coupling, and hence, seem unsuitable as reactive intermediates in nitric oxide reductases (NORs). Hs-{FeNO}(8) complexes are more basic than their hs-{FeNO}(7) precursors, but their electronic structure and reactivity is not as well characterized.

Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY

PubMed Central

2010-01-01

Background Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY. Results HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation. Conclusions Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation. PMID:20438645

A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

PubMed Central

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J.; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-01-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope. PMID:25275454

The effect of calcium on non-heme iron uptake, efflux, and transport in intestinal-like epithelial cells (Caco-2 cells).

PubMed

Gaitán, Diego Alejandro; Flores, Sebastian; Pizarro, Fernando; Olivares, Manuel; Suazo, Miriam; Arredondo, Miguel

2012-03-01

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of (55)Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.

Effect of disordered hemes and dimerization in isolated a-subunits of hemoglobin detected by time-resolved fluorescence spectroscopy in the picosecond range

NASA Astrophysics Data System (ADS)

Gryczynski, Zygmunt; Fronticelli, Clara; Gratton, Enrico; Lubkowski, Jacek; Bucci, Enrico

1994-08-01

Our recent linear dichroism study of transition moment directions for protoporphyrin derivatives [1,2] demonstrate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e. on the k2 orientation parameter of the Forster equation. Using the atomic coordinates of human hemoglobin and taking into account the direction of the transition moment of the near UV (300-380 nm) heme absorption band we have estimated the rate of energy transfer from tryptophan to heme in the isolated a chains, which are a single tryptophan protein. It appears that the rate of energy transfer is very sensitive to the orientation of the transition moment of the heme and similarly to myoglobin [3] natural heme disorder significantly reduces the transfer efficiency in isolated a subunits. On this basis we were able to predict very accurately the two lifetimes detectable in the systems, of 32 and 1050 ps respectively, where the amplitude of the longer lifetime is very consistent with the amount of disordered hemes found by La Mar [4,5] for the a subunits of hemoglobin.

Measurements of heme relaxation and ligand recombination in strong magnetic fields.

PubMed

Zhang, Zhenyu; Benabbas, Abdelkrim; Ye, Xiong; Yu, Anchi; Champion, Paul M

2009-08-06

Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 T). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O(2)), 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 O(2) 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 nonradiative 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 ( approximately 10(9) s(-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.

Factorization of the association rate coefficient in ligand rebinding to heme proteins

NASA Astrophysics Data System (ADS)

Young, Robert D.

1984-01-01

A stochastic theory of ligand migration in biomolecules is used to analyze the recombination of small ligands to heme proteins after flash photolysis. The stochastic theory is based on a generalized sequential barrier model in which a ligand binds by overcoming a series of barriers formed by the solvent protein interface, the protein matrix, and the heme distal histidine system. The stochastic theory shows that the association rate coefficient λon factorizes into three terms λon =γ12Nout, where γ12 is the rate coefficient from the heme pocket to the heme binding site, is the equilibrium pocket occupation factor, and Nout is the fraction of heme proteins which do not undergo geminate recombination of a flashed-off ligand. The factorization of λon holds for any number of barriers and with no assumptions regarding the various rate coefficients so long as the exponential solvent process occurs. Transitions of a single ligand are allowed between any two sites with two crucial exceptions: (i) the heme binding site acts as a trap so that thermal dissociation of a bound ligand does not occur within the time of the measurement; (ii) the final step in the rebinding process always has a ligand in the heme pocket from where the ligand binds to the heme iron.

Isorhamnetin inhibits Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages via anti-inflammatory heme oxygenase-1 induction and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1 activation.

PubMed

Jin, J Y; Choi, E Y; Park, H R; Choi, J I; Choi, I S; Kim, S J

2013-12-01

Interleukin-6 (IL-6) is a key proinflammatory cytokine that has been considered to be important in the pathogenesis of periodontal disease. Therefore, host-modulatory agents directed at inhibiting IL-6 appear to be beneficial in terms of attenuating periodontal disease progression and potentially improving disease susceptibility. In the current study, we investigated the effect of the flavonoid isorhamnetin on the production of IL-6 in murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Lipopolysaccharide from P. intermedia ATCC 25611 was isolated using the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time PCR to quantify IL-6 and heme oxygenase-1 (HO-1) mRNA expression. The expression of HO-1 protein and the levels of signaling proteins were monitored using immunoblot analyses. The DNA-binding activity of nuclear factor-κB (NF-κB) was analyzed using ELISA-based assay kits. Isorhamnetin significantly down-regulated P. intermedia LPS-induced production of IL-6 as well as its mRNA expression in RAW264.7 cells. Isorhamnetin up-regulated the expression of HO-1 at both gene transcription and translation levels in cells stimulated with P. intermedia LPS. In addition, inhibition of HO-1 activity by tin protoporphyrin IX blocked the inhibitory effect of isorhamnetin on IL-6 production. Isorhamnetin failed to prevent LPS from activating either c-Jun N-terminal kinase or p38 pathways. Isorhamnetin did not inhibit NF-κB transcriptional activity at the level of inhibitory κB-α degradation. Isorhamnetin suppressed NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and attenuated signal transducer and activator of transcription 1 signaling. Although further research is required to clarify the detailed mechanism of action, we propose that isorhamnetin may contribute to blockade of the host-destructive processes mediated by IL-6 and could be a highly efficient modulator of the host response in the treatment of inflammatory periodontal disease. Further research in animal models of periodontitis is required to better evaluate, the potential of isorhamnetin as a novel agent for treating periodontal disease. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ultrafast dynamics of the photo-excited hemes b and cn in the cytochrome b6f complex.

PubMed

Agarwal, Rachna; Chauvet, Adrien A P

2017-01-25

The dynamics of hemes b and c n within the cytochrome b 6 f complex are investigated by means of ultrafast broad-band transient absorption spectroscopy. On the one hand, the data reveal that, subsequent to visible light excitation, part of the b hemes undergoes pulse-limited photo-oxidation, with the liberated electron supposedly being transferred to one of the adjacent aromatic amino acids. Photo-oxidation is followed by charge recombination in about 8.2 ps. Subsequent to charge recombination, heme b is promoted to a vibrationally excited ground state that relaxes in about 4.6 ps. On the other hand, heme c n undergoes ultrafast ground state recovery in about 140 fs. Interestingly, the data also show that, in contrast to previous beliefs, Chl a is involved in the photochemistry of hemes. Indeed, subsequent to heme excitation, Chl a bleaches and recovers to its ground state in 90 fs and 650 fs, respectively. Chl a bleaching allegedly corresponds to the formation of a short lived Chl a anion. Beyond the previously suggested structural role, this study provides unique evidence that Chl a is directly involved in the photochemistry of the hemes.

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha.

PubMed

Handschin, Christoph; Lin, Jiandie; Rhee, James; Peyer, Anne-Kathrin; Chin, Sherry; Wu, Pei-Hsuan; Meyer, Urs A; Spiegelman, Bruce M

2005-08-26

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

PubMed

Zubieta, Chloe; Krishna, S Sri; Kapoor, Mili; Kozbial, Piotr; McMullan, Daniel; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Hampton, Eric; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). (c) 2007 Wiley-Liss, Inc.

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

Control of metazoan heme homeostasis by a conserved multidrug resistance protein

PubMed Central

Korolnek, Tamara; Zhang, Jianbing; Beardsley, Simon; Scheffer, George L; Hamza, Iqbal

2014-01-01

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally-derived heme and environmentally-acquired heme. Here, we show that the multidrug resistance protein, MRP-5/ABCC5, likely acts as a heme exporter and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development. PMID:24836561

Selenolate Complexes of CYP101 and the Heme-bound hHO-1/H25A Proximal Cavity Mutant

PubMed Central

Jiang, Yongying; Ortiz de Montellano, Paul R.

2009-01-01

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-visible spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions, but reduced the heme iron to the ferrous state as shown by formation of a ferrous-CO complex. Under aerobic conditions, the selenolate but not thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect. PMID:18376820

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

PubMed

Jiang, Yongying; Ortiz de Montellano, Paul R

2008-05-05

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

Isonitrile Formation by a Non-heme Iron(II)-Dependent Oxidase/Decarboxylase.

PubMed

Harris, Nicholas; Born, David; Cai, Wenlong; Huang, Yaobing; Martin, Joelle; Khalaf, Ryan; Drennan, Catherine; Zhang, Wenjun

2018-06-15

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, members of which are critical for the virulence of pathogenic mycobacteria. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heme compounds as iron sources for nonpathogenic Rhizobium bacteria.

PubMed Central

Noya, F; Arias, A; Fabiano, E

1997-01-01

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

Heme compounds as iron sources for nonpathogenic Rhizobium bacteria.

PubMed

Noya, F; Arias, A; Fabiano, E

1997-05-01

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

Porphyrins and Porphyria Diagnosis

MedlinePlus

... diagnosis The porphyrias are caused by deficiencies of enzymes of the heme biosynthetic pathway. This pathway, like ... heme. Heme is essential for life, and each enzyme is also essential, because it is responsible for ...

Electrostatic environment of hemes in proteins: pK(a)s of hydroxyl ligands.

PubMed

Song, Yifan; Mao, Junjun; Gunner, M R

2006-07-04

The pK(a)s of ferric aquo-heme and aquo-heme electrochemical midpoints (E(m)s) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK(a)s span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E(m)s range from -250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo-heme pK(a)s tend to have lower E(m)s. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo-heme E(m)s more than the pK(a)s. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water-heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu(B) in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK(a) calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK(a)s and E(m)s differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo-heme pK(a) is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner (2006) Biochemistry 45, 7959-7975].

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

PubMed

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

2012-07-01

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

Pulsed ENDOR Determination of Relative Orientation of g- and Molecular-Frames of Imidazole-Coordinated Heme Center of iNOS

PubMed Central

Astashkin, Andrei V.; Fan, Weihong; Elmore, Bradley O.; Guillemette, J. Guy; Feng, Changjian

2011-01-01

Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059–12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe – FMN radius-vector, RFe-FMN, with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g- and molecular frames, from which RFe-FMN can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, while the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works. PMID:21834532

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.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

PubMed Central

Maitra, Dhiman; Shaeib, Faten; Abdulhamid, Ibrahim; Abdulridha, Rasha M.; Saed, Ghassan M.; Diamond, Michael P.; Pennathur, Subramaniam; Abu-Soud, Husam M.

2013-01-01

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl−) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV–visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 μM), precluding the enzyme from functioning at maximum activity (80–90% inhibition). MPO heme destruction occurred only in the presence of Cl−. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO–Compound I–Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron. PMID:23624305

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

PubMed

Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

2008-02-26

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

The bhuQ Gene Encodes a Heme Oxygenase That Contributes to the Ability of Brucella abortus 2308 To Use Heme as an Iron Source and Is Regulated by Irr

PubMed Central

Ojeda, Jenifer F.; Martinson, David A.; Menscher, Evan A.

2012-01-01

The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl3, but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source. PMID:22636783

2.3 Å X-ray Structure of the Heme-Bound GAF Domain of Sensory Histidine Kinase DosT of Mycobacterium tuberculosis†

PubMed Central

Podust, Larissa M.; Ioanoviciu, Alexandra; Ortiz de Montellano, Paul R.

2009-01-01

Mycobacterium tuberculosis responds to the changes in environmental conditions through a two-component signaling system that detects reduced O2 tension and NO and CO exposures via the heme-binding GAF domains of two sensory histidine kinases, DosT and DevS, and the transcriptional regulator DosR. We report the first x-ray structure of the DosT heme-bound GAF domain (GAFDosT) in both oxy and deoxy forms determined to a resolution of 2.3 Å. In GAFDosT, heme binds in an orientation orthogonal to that in the PAS domains via a highly conserved motif including invariant H147 as a proximal heme axial ligand. On the distal side, invariant Y169 is in stacking interactions with the heme with its long axis parallel and the plane of the ring orthogonal to the heme plane. In one of the two protein monomers in an asymmetric unit, O2 binds as a second axial ligand to the heme iron, and is stabilized via an H-bond to the OH-group of Y169. The structure reveals two small tunnel-connected cavities and a pore on the protein surface that suggest a potential route for O2 access to the sensing pocket. The limited conformational differences observed between differently heme iron-ligated GAFDosT monomers in the asymmetric unit may result from crystal lattice limitations since atmospheric oxygen binding likely occurs in the crystal as a result of x-ray induced Fe3+ photoreduction during diffraction data collection. Determination of the GAFDosT structure sets up a framework in which to address ligand-recognition, discrimination, and signal propagation schemes in the heme-based GAF domains of biological sensors. PMID:18980385

Up-regulation of A1M/α1-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage.

PubMed

Olsson, Magnus G; Allhorn, Maria; Larsson, Jörgen; Cederlund, Martin; Lundqvist, Katarina; Schmidtchen, Artur; Sørensen, Ole E; Mörgelin, Matthias; Akerström, Bo

2011-01-01

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (K(d) = 0.96×10(-6) M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.

Triosephosphate isomerase tyrosine nitration induced by heme-NaNO2 -H2 O2 or peroxynitrite: Effects of different natural phenolic compounds.

PubMed

Gao, Wanxia; Zhao, Jie; Li, Hailing; Gao, Zhonghong

2017-06-01

Peroxynitrite and heme peroxidases (or heme)-H 2 O 2 -NaNO 2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H 2 O 2 -NaNO 2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H 2 O 2 -NaNO 2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H 2 O 2 -NaNO 2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H 2 O 2 -NaNO 2 -induced nitration. Our results indicated that peroxynitrite and heme-H 2 O 2 -NaNO 2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening. © 2017 Wiley Periodicals, Inc.

Bovine protoporphyria: documentation of autosomal recessive inheritance and comparison with the human disease through measurement of heme synthase activity.

PubMed Central

Bloomer, J R; Morton, K O; Reuter, R J; Ruth, G R

1982-01-01

Protoporphyria is an autosomal dominant disease in man in which protoporphyrin accumulated because of a defect in heme synthase (ferrochelatase) activity. A disease has been described in cattle that has the same manifestations as does the human disease. We measured heme synthase activity in sonicates of cultured skin fibroblasts and whole liver homogenates from animals with protoporphyria, their unaffected parents, and normal cattle in order to examine the mode of inheritance and compare it with human protoporphyria. The mean activity (+/- SEM) in fibroblasts from the three groups was 2.0 +/- 0.4, 47 +/- 12, and 149 +/- 10 pmol heme formed/mg protein per hr, respectively, consistent with autosomal recessive inheritance. Similarly, the levels of heme synthase activity in livers of the parents were intermediate to those of normal animals and of animals with protoporphyria. When compared with normal human fibroblasts and liver, the specific activity of heme synthase in normal bovine tissue was significantly higher. These studies indicate that manifestations of protoporphyria do not occur in cattle unless the animal is homozygous for the gene defect, whereas in humans, the heterozygous condition is sufficient. This is probably because the specific activity of heme synthase in cells of heterozygous animals is not reduced to a level that significantly alters heme metabolism. PMID:7072720

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

PubMed

Frébortova; Matsushita; Arata; Adachi

1998-01-27

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Ultraviolet-A1 irradiation therapy for systemic lupus erythematosus.

PubMed

McGrath, H

2017-10-01

Systemic lupus erythematosus (lupus, SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which bind to antigens and are deposited within tissues to fix complement, resulting in widespread systemic inflammation. The studies presented herein are consistent with hyperpolarized, adenosine triphosphate (ATP)-deficient mitochondria being central to the disease process. These hyperpolarized mitochondria resist the depolarization required for activation-induced apoptosis. The mitochondrial ATP deficits add to this resistance to apoptosis and also reduce the macrophage energy that is needed to clear apoptotic bodies. In both cases, necrosis, the alternative pathway of cell death, results. Intracellular constituents spill into the blood and tissues, eliciting inflammatory responses directed at their removal. What results is "autoimmunity." Ultraviolet (UV)-A1 photons have the capacity to remediate this aberrancy. Exogenous exposure to low-dose, full-body, UV-A1 radiation generates singlet oxygen. Singlet oxygen has two major palliative actions in patients with lupus and the UV-A1 photons themselves have several more. Singlet oxygen depolarizes the hyperpolarized mitochondrion, triggering non-ATP-dependent apoptosis that deters necrosis. Next, singlet oxygen activates the gene encoding heme oxygenase (HO-1), a major governor of systemic homeostasis. HO-1 catalyzes the degradation of the oxidant heme into biliverdin (converted to bilirubin), Fe, and carbon monoxide (CO), the first three of these exerting powerful antioxidant effects, and in conjunction with a fourth, CO, protecting against injury to the coronary arteries, the central nervous system, and the lungs. The UV-A1 photons themselves directly attenuate disease in lupus by reducing B cell activity, preventing the suppression of cell-mediated immunity, slowing an epigenetic progression toward SLE, and ameliorating discoid and subacute cutaneous lupus. Finally, a combination of these mechanisms reduces levels of anticardiolipin antibodies and protects during lupus pregnancy. Capping all of this is that UV-A1 irradiation is an essentially innocuous, highly manageable, and comfortable therapeutic agency.

Ultraviolet-A1 irradiation therapy for systemic lupus erythematosus

PubMed Central

2017-01-01

Systemic lupus erythematosus (lupus, SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which bind to antigens and are deposited within tissues to fix complement, resulting in widespread systemic inflammation. The studies presented herein are consistent with hyperpolarized, adenosine triphosphate (ATP)-deficient mitochondria being central to the disease process. These hyperpolarized mitochondria resist the depolarization required for activation-induced apoptosis. The mitochondrial ATP deficits add to this resistance to apoptosis and also reduce the macrophage energy that is needed to clear apoptotic bodies. In both cases, necrosis, the alternative pathway of cell death, results. Intracellular constituents spill into the blood and tissues, eliciting inflammatory responses directed at their removal. What results is “autoimmunity.” Ultraviolet (UV)-A1 photons have the capacity to remediate this aberrancy. Exogenous exposure to low-dose, full-body, UV-A1 radiation generates singlet oxygen. Singlet oxygen has two major palliative actions in patients with lupus and the UV-A1 photons themselves have several more. Singlet oxygen depolarizes the hyperpolarized mitochondrion, triggering non-ATP-dependent apoptosis that deters necrosis. Next, singlet oxygen activates the gene encoding heme oxygenase (HO-1), a major governor of systemic homeostasis. HO-1 catalyzes the degradation of the oxidant heme into biliverdin (converted to bilirubin), Fe, and carbon monoxide (CO), the first three of these exerting powerful antioxidant effects, and in conjunction with a fourth, CO, protecting against injury to the coronary arteries, the central nervous system, and the lungs. The UV-A1 photons themselves directly attenuate disease in lupus by reducing B cell activity, preventing the suppression of cell-mediated immunity, slowing an epigenetic progression toward SLE, and ameliorating discoid and subacute cutaneous lupus. Finally, a combination of these mechanisms reduces levels of anticardiolipin antibodies and protects during lupus pregnancy. Capping all of this is that UV-A1 irradiation is an essentially innocuous, highly manageable, and comfortable therapeutic agency. PMID:28480786

Substance P regulates macrophage inflammatory protein 3α/chemokine C-C ligand 20 (CCL20) with heme oxygenase-1 in human periodontal ligament cells

PubMed Central

Lee, S-K; Pi, S-H; Kim, S-H; Min, K-S; Lee, H-J; Chang, H-S; Kang, K-H; Kim, H-R; Shin, H-I; Lee, S-K; Kim, E-C

2007-01-01

Although substance P (SP), a potent proinflammatory peptide, is involved in inflammation and immune responses, the effect of SP on the expression of macrophage inflammatory protein 3α[MIP-3α, chemokine C-C ligand 20 (CCL20)] in periodontal ligament (PDL) cells is unknown. Equally enigmatic is the link between SP, the stress protein heme oxygenase-1 (HO-1), and CCL20 production. We investigated whether SP induces the release of chemokine CCL20 from immortalized PDL (IPDL) cells, and further clarify SP-mediated pathways. We also examined the relationship between HO-1 and CCL20 by treating PDL cells with SP. Incubating IPDL cells with SP increased expression of CCL20 mRNA and CCL20 protein in a dose–time-dependent manner. Highly selective p38 and extracellular-regulated kinase 1/2 (ERK1/2) inhibitors abrogated SP-induced expression of CCL20 in IPDL cells. SP is also responsible for initiating phosphorylation of IκB, degradation of IκB and activation of nuclear factor (NF)-κB. SP induced expression of HO-1 in both a concentration- and time-dependent manner, and CCL20 reflected similar patterns. The inductive effects of SP on HO-1 and CCL20 were enhanced by HO-1 inducer hemin and the membrane-permeable guanosine 3′,5′-monophosphate (cGMP) analogue 8-bromo-cGMP. Conversely, this pathway was inhibited by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). We report herein the pathway that connects SP along with other modulators of neuroimmunoregulation to the induction of HO-1 and the inflammatory mediator macrophage inflammatory protein (MIP)-3α/CCL20 in IPDL cells, which play an important role in the development of periodontitis or inflammation during orthodontic tooth movement. PMID:17924972

Structure of the dimerization domain of DiGeorge Critical Region 8

PubMed Central

Senturia, Rachel; Faller, Michael; Yin, Sheng; Loo, Joseph A; Cascio, Duilio; Sawaya, Michael R; Hwang, Daniel; Clubb, Robert T; Guo, Feng

2010-01-01

Maturation of microRNAs (miRNAs, ∼22nt) from long primary transcripts [primary miRNAs (pri-miRNAs)] is regulated during development and is altered in diseases such as cancer. The first processing step is a cleavage mediated by the Microprocessor complex containing the Drosha nuclease and the RNA-binding protein DiGeorge critical region 8 (DGCR8). We previously reported that dimeric DGCR8 binds heme and that the heme-bound DGCR8 is more active than the heme-free form. Here, we identified a conserved dimerization domain in DGCR8. Our crystal structure of this domain (residues 298–352) at 1.7 Å resolution demonstrates a previously unknown use of a WW motif as a platform for extensive dimerization interactions. The dimerization domain of DGCR8 is embedded in an independently folded heme-binding domain and directly contributes to association with heme. Heme-binding-deficient DGCR8 mutants have reduced pri-miRNA processing activity in vitro. Our study provides structural and biochemical bases for understanding how dimerization and heme binding of DGCR8 may contribute to regulation of miRNA biogenesis. PMID:20506313

Regulation of a glutamyl-tRNA synthetase by the heme status

PubMed Central

Levicán, Gloria; Katz, Assaf; de Armas, Merly; Núñez, Harold; Orellana, Omar

2007-01-01

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the C5 pathway. In this route Glu-tRNA is transformed to δ-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme. PMID:17360620

Anti-inflammatory and heme oxygenase-1 inducing activities of lanostane triterpenes isolated from mushroom Ganoderma lucidum in RAW264.7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Solip; Nguyen, Van Thu; Tae, Nara

Ganoderma lucidum is a popular medicinal mushroom used in traditional medicine for preventing or treating a variety of diseases. In the present study, we investigated the anti-inflammatory and heme oxygenase (HO)-1 inducing effects of 12 lanostane triterpenes from G. lucidum in RAW264.7 cells. Of these, seven triterpenes, butyl lucidenateE{sub 2}, butyl lucidenateD{sub 2} (GT-2), butyl lucidenate P, butyl lucidenateQ, Ganoderiol F, methyl ganodenate J and butyl lucidenate N induced HO-1 expression and suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Inhibiting HO-1 activity abrogated the inhibitory effects of these triterpenes on the production of NO in LPS-stimulated RAW264.7 cells, suggesting themore » involvement of HO-1 in the anti-inflammatory effects of these triterpenes. We further studied the anti-inflammatory and HO-1 inducing effects of GT-2. Mitogen-activated protein kinase inhibitors or N-acetylcysteine, an antioxidant, did not suppress GT-2-mediated HO-1 induction; however, LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked GT-2-induced HO-1 mRNA and protein expression. GT-2 increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and knockdown of Nrf2 by small interfering RNA blocked GT-2-mediated HO-1 induction, suggesting that GT-2 induced HO-1 expression via the PI3K/AKT-Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, GT-2 inhibited the production of tumor necrosis factor-α and interleukin-6, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression. These findings suggest that HO-1 inducing activities of these lanostane triterpenes may be important in the understanding of a novel mechanism for the anti-inflammatory activity of G. lucidum. - Highlights: • The anti-inflammatory effects of selected triterpenes from Ganoderma lucidum are demonstrated. • Heme oxygenase-1 induction is attributable to the anti-inflammatory properties of these triterpenes. • The triterpenes induce heme oxygenase-1 expression via the AKT-Nrf2 pathway. • The mechanism explains the anti-inflammatory effect of triterpenes from G. lucidum.« less

Staphylococcus aureus Redirects Central Metabolism to Increase Iron Availability

PubMed Central

Pishchany, Gleb; Whitwell, Corbin W; Torres, Victor J; Skaar, Eric P

2006-01-01

Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Δfur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus. PMID:16933993

Paeonol extracted from Paeonia suffruticosa Andr. ameliorated UVB-induced skin photoaging via DLD/Nrf2/ARE and MAPK/AP-1 pathway.

PubMed

Sun, ZhengWang; Du, Juan; Hwang, Eunson; Yi, Tae-Hoo

2018-05-10

Paeonia suffruticosa Andr. (PS) has been used in traditional Chinese medicine for a long time. However, there are no studies that investigate the preventive effects of PS on ultraviolet B (UVB)-induced photoaging. In this study, paeonol (PA) was detected the main compound in PS root. In vitro, PS and PA significantly inhibited UVB-induced phosphorylation of mitogen-activated protein kinase and activator protein 1 in keratinocytes, which consequently led to degradation of procollagen type I. On the other hand, PS and PA increased NAD(P)H:quinone oxidoreductase 1 and heme oxygenase-1 expression, confirmed by greater nuclear accumulation of nuclear factor E2-releated factor 2 (Nrf2). Furthermore, this study proved that the endogenous antioxidant system Nrf2/antioxidant response element was regulated by dihydrolipoamide dehydrogenase, a tricarboxylic acid (TCA) cycle-associated protein whose level was decreased after UVB exposure. PS and PA promoted the production of dihydrolipoamide dehydrogenase, as well as the activation of Nrf2 and antioxidant response element, resulting in preventing procollagen type I ruined caused by UVB. In vivo, topical application of PS and PA attenuated UVB-induced matrix metalloproteinase-1 production and promoted procollagen type I in hairless mice. These results suggested PA a promising botanical in protecting skin from UVB-induced photoaging. Copyright © 2018 John Wiley & Sons, Ltd.

Heme Regulates Allosteric Activation of the Slo1 BK Channel

PubMed Central

Horrigan, Frank T.; Heinemann, Stefan H.; Hoshi, Toshinori

2005-01-01

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state. PMID:15955873

Hemopexin and haptoglobin: allies against heme toxicity from hemoglobin not contenders

PubMed Central

Smith, Ann; McCulloh, Russell J.

2015-01-01

The goal here is to describe our current understanding of heme metabolism and the deleterious effects of “free” heme on immunological processes, endothelial function, systemic inflammation, and various end-organ tissues (e.g., kidney, lung, liver, etc.), with particular attention paid to the role of hemopexin (HPX). Because heme toxicity is the impetus for much of the pathology in sepsis, sickle cell disease (SCD), and other hemolytic conditions, the biological importance and clinical relevance of HPX, the predominant heme binding protein, is reinforced. A perspective on the function of HPX and haptoglobin (Hp) is presented, updating how these two proteins and their respective receptors act simultaneously to protect the body in clinical conditions that entail hemolysis and/or systemic intravascular (IVH) inflammation. Evidence from longitudinal studies in patients supports that HPX plays a Hp-independent role in genetic and non-genetic hemolytic diseases without the need for global Hp depletion. Evidence also supports that HPX has an important role in the prognosis of complex illnesses characterized predominantly by the presence of hemolysis, such as SCD, sepsis, hemolytic-uremic syndrome, and conditions involving IVH and extravascular hemolysis (EVH), such as that generated by extracorporeal circulation during cardiopulmonary bypass (CPB) and from blood transfusions. We propose that quantitating the amounts of plasma heme, HPX, Hb-Hp, heme-HPX, and heme-albumin levels in various disease states may aid in the diagnosis and treatment of the above-mentioned conditions, which is crucial to developing targeted plasma protein supplementation (i.e., “replenishment”) therapies for patients with heme toxicity due to HPX depletion. PMID:26175690

Structure and Reactivity of a Thermostable Prokaryotic Nitric-oxide Synthase That Forms a Long-lived Oxy-Heme Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sudhamsu,J.; Crane, B.

2006-01-01

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from L-arginine via the stable intermediate N-hydroxy L-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates L-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxymore » species. In single turnover experiments with NOHA, NO forms only in the presence of H4B. The crystal structure of gsNOS at 3.2 A Angstroms of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degC. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.« less

In vitro Activation of heme oxygenase-2 by menadione and its analogs

PubMed Central

2014-01-01

Background Previously, we reported that menadione activated rat, native heme oxygenase-2 (HO-2) and human recombinant heme oxygenase-2 selectively; it did not activate spleen, microsomal heme oxygenase-1. The purpose of this study was to explore some structure–activity relationships of this activation and the idea that redox properties may be an important aspect of menadione efficacy. Methods Heme oxygenase activity was determined in vitro using rat spleen and brain microsomes as the sources of heme oxygenase-1 and −2, respectively, as well as recombinant, human heme oxygenase-2. Results Menadione analogs with bulky aliphatic groups at position-3, namely vitamins K1 and K2, were not able to activate HO-2. In contrast, several compounds with similar bulky but less lipophilic moieties at position-2 (and −3) were able to activate HO-2 many fold; these compounds included polar, rigid, furan-containing naphthoquinones, furan-benzoxazine naphthoquinones, 2-(aminophenylphenyl)-3-piperidin-1-yl naphthoquinones. To explore the idea that redox properties might be involved in menadione efficacy, we tested analogs such as 1,4-dimethoxy-2-methylnaphthalene, pentafluoromenadione, monohalogenated naphthoquinones, α-tetralone and 1,4-naphthoquinone. All of these compounds were inactive except for 1,4-naphthoquinone. Menadione activated full-length recombinant human heme oxygenase-2 (FL-hHO-2) as effectively as rat brain enzyme, but it did not activate rat spleen heme oxygenase. Conclusions These observations are consistent with the idea that naphthoquinones such as menadione bind to a receptor in HO-2 and activate the enzyme through a mechanism that may involve redox properties. PMID:24533775

In vitro Activation of heme oxygenase-2 by menadione and its analogs.

PubMed

Vukomanovic, Dragic; Rahman, Mona N; Bilokin, Yaroslav; Golub, Andriy G; Brien, James F; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

2014-02-18

Previously, we reported that menadione activated rat, native heme oxygenase-2 (HO-2) and human recombinant heme oxygenase-2 selectively; it did not activate spleen, microsomal heme oxygenase-1. The purpose of this study was to explore some structure-activity relationships of this activation and the idea that redox properties may be an important aspect of menadione efficacy. Heme oxygenase activity was determined in vitro using rat spleen and brain microsomes as the sources of heme oxygenase-1 and -2, respectively, as well as recombinant, human heme oxygenase-2. Menadione analogs with bulky aliphatic groups at position-3, namely vitamins K1 and K2, were not able to activate HO-2. In contrast, several compounds with similar bulky but less lipophilic moieties at position-2 (and -3) were able to activate HO-2 many fold; these compounds included polar, rigid, furan-containing naphthoquinones, furan-benzoxazine naphthoquinones, 2-(aminophenylphenyl)-3-piperidin-1-yl naphthoquinones. To explore the idea that redox properties might be involved in menadione efficacy, we tested analogs such as 1,4-dimethoxy-2-methylnaphthalene, pentafluoromenadione, monohalogenated naphthoquinones, α-tetralone and 1,4-naphthoquinone. All of these compounds were inactive except for 1,4-naphthoquinone. Menadione activated full-length recombinant human heme oxygenase-2 (FL-hHO-2) as effectively as rat brain enzyme, but it did not activate rat spleen heme oxygenase. These observations are consistent with the idea that naphthoquinones such as menadione bind to a receptor in HO-2 and activate the enzyme through a mechanism that may involve redox properties.

Hal Is a Bacillus anthracis Heme Acquisition Protein

PubMed Central

Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

2012-01-01

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

Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

PubMed

Turlin, Evelyne; Débarbouillé, Michel; Augustyniak, Katarzyna; Gilles, Anne-Marie; Wandersman, Cécile

2013-01-01

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

Time-resolved single-turnover of caa(3) oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O(H) into E(H) state.

PubMed

Siletsky, Sergey A; Belevich, Ilya; Belevich, Nikolai P; Soulimane, Tewfik; Verkhovsky, Michael I

2011-09-01

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site. 2011 Elsevier B.V. All rights reserved.

Artemisinin dimer anti-cancer activity correlates with heme-catalyzed ROS generation and ER stress induction

PubMed Central

Stockwin, Luke H.; Han, Bingnan; Yu, Sherry X.; Hollingshead, Melinda G.; ElSohly, Mahmoud A.; Gul, Waseem; Slade, Desmond; Galal, Ahmed M.; Newton, Dianne L.

2009-01-01

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anti-cancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the anti-oxidant L-NAC, the iron chelator desferroxamine, and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity while inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage; iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian ER-resident SERCA Ca2+-ATPases. A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity. PMID:19533749

The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

PubMed Central

Laberge, Monique; Huang, Qing; Schweitzer-Stenner, Reinhard; Fidy, Judit

2003-01-01

Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca2+-bound and Ca2+-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B1g- and B2g-type distortions in the Ca2+-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca2+-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion. PMID:12668462

Clotrimazole enhances lysis of human erythrocytes induced by t-BHP.

PubMed

Lisovskaya, Irene L; Shcherbachenko, Irina M; Volkova, Rimma I; Ataullakhanov, Fazoil I

2009-08-14

Clotrimazole (CLT) is an antifungal and antimalarial agent also effective as a Gardos channel inhibitor. In addition, CLT possesses antitumor properties. Recent data provide evidence that CLT forms a complex with heme (hemin), which produces a more potent lytic effect than heme alone. This study addressed the effect of CLT on the lysis of normal human erythrocytes induced by tert-butyl hydroperoxide (t-BHP). For the first time, it was shown that 10 microM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes in both Ca(2+)-containing and Ca(2+)-free media, suggesting that the effect is not related to Gardos channels. CLT did not affect the rate of free radical generation, the kinetics of GSH degradation, methemoglobin formation and TBARS generation; therefore, we concluded that CLT does not cause additional oxidative damage to erythrocytes treated with t-BHP. It is tempted to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes: the CLT-hemin complex destabilizes the cell membrane more potently than hemin alone. If so, the effect of CLT on cell membrane damage during free-radical oxidation may be used to increase the efficacy of antitumor therapy.

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

PubMed

Bergmann, D J; Arciero, D M; Hooper, A B

1994-06-01

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?

PubMed

Crofts, Antony R; Holland, J Todd; Victoria, Doreen; Kolling, Derrick R J; Dikanov, Sergei A; Gilbreth, Ryan; Lhee, Sangmoon; Kuras, Richard; Kuras, Mariana Guergova

2008-01-01

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

Valence tautomerism in synthetic models of cytochrome P450

PubMed Central

Das, Pradip Kumar; Samanta, Subhra; McQuarters, Ashley B.; Lehnert, Nicolai

2016-01-01

CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs. PMID:27302948

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding.

PubMed

Fayadat, L; Niccoli-Sire, P; Lanet, J; Franc, J L

1999-04-09

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.

Molecular dynamics simulation of cytochrome c3: studying the reduction processes using free energy calculations.

PubMed Central

Soares, C M; Martel, P J; Mendes, J; Carrondo, M A

1998-01-01

The tetraheme cytochrome c3 from Desulfovibrio vulgaris Hildenborough is studied using molecular dynamics simulation studies in explicit solvent. The high heme content of the protein, which has its core almost entirely made up of c-type heme, presents specific problems in the simulation. Instability in the structure is observed in long simulations above 1 ns, something that does not occur in a monoheme cytochrome, suggesting problems in heme parametrization. Given these stability problems, a partially restrained model, which avoids destruction of the structure, was created with the objective of performing free energy calculations of heme reduction, studies that require long simulations. With this model, the free energy of reduction of each individual heme was calculated. A correction in the long-range electrostatic interactions of charge groups belonging to the redox centers had to be made in order to make the system physically meaningful. Correlation is obtained between the calculated free energies and the experimental data for three of four hemes. However, the relative scale of the calculated energies is different from the scale of the experimental free energies. Reasons for this are discussed. In addition to the free energy calculations, this model allows the study of conformational changes upon reduction. Even if the precise details of the structural changes that take place in this system upon individual heme reduction are probably out of the reach of this study, it appears that these structural changes are small, similarly to what is observed for other redox proteins. This does not mean that their effect is minor, and one example is the conformational change observed in propionate D from heme I when heme II becomes reduced. A motion of this kind could be the basis of the experimentally observed cooperativity effects between heme reduction, namely positive cooperativity. PMID:9545034

Abundance of the iron containing biomolecule, heme b, during the progression of a spring phytoplankton bloom in a mesocosm experiment

PubMed Central

Bellworthy, Jessica; Esposito, Mario; Achterberg, Eric P.

2017-01-01

Concentrations of heme b were determined in a mesocosm experiment situated in Gullmar Fjord off Sweden. The mesocosm experiment lasted for ca. one hundred days and was characterised by the growth of a primary nutrient replete and a secondary nutrient deplete phytoplankton bloom. Heme b varied between 40 ± 10 pmol L-1 in the prebloom period up to a maximum of 700 ± 400 pmol L-1 just prior to the time of the primary chlorophyll a maximum. Thereafter, heme b concentrations decreased again to an average of 120 ± 60 pmol L-1. When normalised to total particulate carbon, heme b was most abundant during the initiation of the nutrient replete spring bloom, when ratios reached 52 ± 24 μmol mol-1; ten times higher than values observed both pre and post the primary bloom. Concentrations of heme b correlated with those of chlorophyll a. Nevertheless, differences were observed in the relative concentrations of the two parameters, with heme b concentrations increasing relative to chlorophyll a during the growth of the primary bloom, decreasing over the period of the secondary bloom and increasing again through the latter period of the experiment. Heme b abundance was therefore influenced by nutrient concentrations and also likely by changing community composition. In half of the mesocosms, pCO2 was elevated and maintained at ca.1000 μatm, however we observed no significant differences between heme b in plus or ambient pCO2 mesocosms, either in absolute terms, or relative to total particulate carbon and chlorophyll a. The results obtained in this study contribute to our understanding of the distribution of this significant component of the biogenic iron pool, and provide an iron replete coastal water end member that aids the interpretation of the distributions of heme b in more iron deplete open ocean waters. PMID:28426768

Comparison of ligand migration and binding in heme proteins of the globin family

NASA Astrophysics Data System (ADS)

Karin, Nienhaus; Ulrich Nienhaus, G.

2015-12-01

The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins (via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.

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...

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Zhao-Hua; Kumari, Namita; Nekhai, Sergei

2013-06-07

Highlights: •Lipopolysaccharide stimulation of heme oxygenase-1 (HO-1) ameliorated HIV-1 infection of primary human macrophages. •The partial protection by HO-1 against HIV infection was associated with induction of chemokines such as MIP1α and MIP1β. •This mechanism explains lipopolysaccharide-stimulated HO-1-mediated inhibition of HIV-1 infection of macrophages. -- Abstract: We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed bymore » transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1β) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1β, and LD78β chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM.« less

Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle*

PubMed Central

Maalcke, Wouter J.; Reimann, Joachim; de Vries, Simon; Butt, Julea N.; Dietl, Andreas; Kip, Nardy; Mersdorf, Ulrike; Barends, Thomas R. M.; Jetten, Mike S. M.; Keltjens, Jan T.; Kartal, Boran

2016-01-01

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis. Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms. PMID:27317665

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

PubMed Central

Ruetz, Markus; Kumutima, Jacques; Lewis, Brianne E.; Filipovic, Milos R.; Lehnert, Nicolai; Stemmler, Timothy L.; Banerjee, Ruma

2017-01-01

Hydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H2S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H2S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H2S tightly and was postulated to play a role in modulating neuronal sensitivity to H2S in conditions such as stroke. However, the H2S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H2S. Ferric neuroglobin is slowly reduced by H2S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His64 residue to alanine promotes rapid binding of H2S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H2S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2–6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His64 ligand. PMID:28246171

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

Role of the heme oxygenases in abnormalities of the mesenteric circulation in cirrhotic rats.

PubMed

Sacerdoti, David; Abraham, Nader G; Oyekan, Adebayo O; Yang, Liming; Gatta, Angelo; McGiff, John C

2004-02-01

Carbon monoxide (CO), a product of heme metabolism by heme-oxygenase (HO), has biological actions similar to those of nitric oxide (NO). The role of CO in decreasing vascular responses to constrictor agents produced by experimental cirrhosis induced by carbon tetrachloride was evaluated before and after inhibition of HO with tin-mesoporphyrin (SnMP) in the perfused superior mesenteric vasculature (SMV) of cirrhotic and normal rats and in normal rats transfected with the human HO-1 (HHO-1) gene. Perfusion pressure and vasoconstrictor responses of the SMV to KCl, phenylephrine (PE), and endothelin-1 (ET-1) were decreased in cirrhotic rats. SnMP increased SMV perfusion pressure and restored the constrictor responses of the SMV to KCl, PE, and ET-1 in cirrhotic rats. The relative roles of NO and CO in producing hyporeactivity of the SMV to PE in cirrhotic rats were examined. Vasoconstrictor responses to PE were successively augmented by stepwise inhibition of CO and NO production, suggesting a complementary role for these gases in the regulation of reactivity of the SMV. Expression of constitutive but not of inducible HO (HO-1) was increased in the SMV of cirrhotic rats as was HO activity. Administration of adenovirus containing HHO-1 gene produced detection of HHO-1 RNA and increased HO activity in the SMV within 7 days. Rats transfected with HO-1 demonstrated reduction in both perfusion pressure and vasoconstrictor responses to PE in the SMV. We propose that HO is an essential component in mechanisms that modulate reactivity of the mesenteric circulation in experimental hepatic cirrhosis in rats.

Design and engineering of a man-made diffusive electron-transport protein.

PubMed

Fry, Bryan A; Solomon, Lee A; Leslie Dutton, P; Moser, Christopher C

2016-05-01

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7×10(6) M(-1) s(-1) to 1.2×10(9) M(-1) s(-1) follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and -19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanisms of the antihypertensive effects of Nigella sativa oil in L-NAME-induced hypertensive rats

PubMed Central

Jaarin, Kamsiah; Foong, Wai Dic; Yeoh, Min Hui; Kamarul, Zaman Yusoff Nik; Qodriyah, Haji Mohd Saad; Azman, Abdullah; Zuhair, Japar Sidik Fadhlullah; Juliana, Abdul Hamid; Kamisah, Yusof

2015-01-01

OBJECTIVES This study was conducted to determine whether the blood pressure-lowering effect of Nigella sativa might be mediated by its effects on nitric oxide, angiotensin-converting enzyme, heme oxygenase and oxidative stress markers. METHODS: Twenty-four adult male Sprague-Dawley rats were divided equally into 4 groups. One group served as the control (group 1), whereas the other three groups (groups 2-4) were administered L-NAME (25 mg/kg, intraperitoneally). Groups 3 and 4 were given oral nicardipine daily at a dose of 3 mg/kg and Nigella sativa oil at a dose of 2.5 mg/kg for 8 weeks, respectively, concomitantly with L-NAME administration. RESULTS Nigella sativa oil prevented the increase in systolic blood pressure in the L-NAME-treated rats. The blood pressure reduction was associated with a reduction in cardiac lipid peroxidation product, NADPH oxidase, angiotensin-converting enzyme activity and plasma nitric oxide, as well as with an increase in heme oxygenase-1 activity in the heart. The effects of Nigella sativa on blood pressure, lipid peroxidation product, nicotinamide adenine dinucleotide phosphate oxidase and angiotensin-converting enzyme were similar to those of nicardipine. In contrast, L-NAME had opposite effects on lipid peroxidation, angiotensin-converting enzyme and NO. CONCLUSION: The antihypertensive effect of Nigella sativa oil appears to be mediated by a reduction in cardiac oxidative stress and angiotensin-converting enzyme activity, an increase in cardiac heme oxygenase-1 activity and a prevention of plasma nitric oxide loss. Thus, Nigella sativa oil might be beneficial for controlling hypertension. PMID:26602523

Quantum Computational Studies of Electron Transfer in Respiratory Complex III and its Application for Designing New Mitocan Drugs

NASA Astrophysics Data System (ADS)

Hagras, Muhammad Ahmed

Electron transfer occurs in many biological systems which are imperative to sustain life; oxidative phosphorylation in prokaryotes and eukaryotes, and photophosphorylation in photosynthetic and plant cells are well-balanced and complementary processes. Investigating electron transfer in those natural systems provides detailed knowledge of the atomistic events that lead eventually to production of ATP, or harvesting light energy. Ubiquinol:cytochrome c oxidoreductase complex (also known as bc 1 complex, or respiratory complex III) is a middle player in the electron transport proton pumping orchestra, located in the inner-mitochondrial membrane in eukaryotes or plasma membrane in prokaryotes, which converts the free energy of redox reactions to electrochemical proton gradient across the membrane, following the fundamental chemiosmotic principle discovered by Peter Mitchell 1. In humans, the malfunctioned bc1 complex plays a major role in many neurodegenerative diseases, stress-induced aging, and cancer development, because it produces most of the reactive oxygen species, which are also involved in cellular signaling 2. The mitochondrial bc1 complex has an intertwined dimeric structure comprised of 11 subunits in each monomer, but only three of them have catalytic function, and those are the only domains found in bacterial bc1 complex. The core subunits include: Rieske domain, which incorporates iron-sulfur cluster [2Fe-2S]; trans-membrane cytochrome b domain, incorporating low-potential heme group (heme b L) and high-potential heme group (heme b H); and cytochrome c1 domain, containing heme c1 group and two separate binding sites, Qo (or QP) site where the hydrophobic electron carrier ubihydroquinol QH2 is oxidized, and Qi (or QN) site where ubiquinone molecule Q is reduced 3. Electrons and protons in the bc1 complex flow according to the proton-motive Q-cycle proposed by Mitchell, which includes a unique electron flow bifurcation at the Qo site. At this site, one electron of a bound QH2 molecule transfers to [2Fe-2S] cluster of the Rieske domain, docked at the proximal docking site, and another electron transfers to heme b L , which subsequently passes it to heme bH , and finally to Q or SQ molecule bound at the Qi-site 4. Rieske domain undergoes a domain movement 22 A to bind at the distal docking site, where [2Fe-2S] cluster passes its electron to heme c1, which in turn passes it to heme c of the water-soluble cytochrome c carrier 3c, 5 (which shuttles it to cytochrome c oxidase, complex IV). In the current compiled work presented in the subsequent chapters, we deployed a stacking tiers hierarchy where each chapter's work presents a foundation for the next one. In chapter 1, we first present different methods to calculate tunneling currents in proteins including a new derivation method for the inter-atomic tunneling current method. In addition, we show the results of the inter-atomic tunneling current theory on models based on heme bL-heme bH redox pair system in bc1 complex. Afterwards, in chapter 2, we examine the electron tunneling pathways 6 between different intra-monomeric and inter-monomeric redox centers of bc1 complex, including its electron carriers - ubiquinol, ubiquinone, and cytochrome c molecules, using the well-studied coarse-grained interatomic method of the tunneling current theory 7. Going through the different tunneling pathways in bc1 complex, we discovered a pair of internal switches that modulate the electron transfer rate which we discuss in full details in chapter 3. Motivated by the discovery of those internal switches, we discuss in chapter 4 the discovery of a new binding pocket (designated as NonQ-site or NQ-site for short) in bc 1 complex which is located at the opposite side of the enzyme with respect to Qo site. In contrast to Qo site, however, the NQ-site penetrates deeply in the cytochrome b domain and reaches very closely the LH region. Hence the NQ-site provides a suitable binding pocket for ligands that can influence the orientation of Phe90 residue, and hence modulate the corresponding ET rate between heme b L and heme bH. Finally we present in chapter 5 our unique integrated software package (called Electron Tunneling in Proteins Program or ETP) which provides an environment with different capabilities such as tunneling current calculation, semi-empirical quantum mechanical calculation and molecular modeling simulation for calculation and analysis of electron transfer reactions in proteins.

Time-resolved generation of membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH states.

PubMed

Siletsky, Sergey A; Belevich, Ilya; Belevich, Nikolai P; Soulimane, Tewfik; Wikström, Mårten

2017-11-01

Two electrogenic phases with characteristic times of ~14μs and ~290μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba 3 oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between Cu A and heme b. The slow phase includes electron redistribution from both Cu A and heme b to heme a 3 , and electrogenic proton transfer coupled to reduction of heme a 3 . The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a 3 is reduced, but there is no proton pumping and no reduction of Cu B . Single-electron reduction of the oxidized "unrelaxed" state (O H →E H transition) is accompanied by electrogenic reduction of the heme b/heme a 3 pair by Cu A in a "fast" phase (~22μs) and transfer of protons in "middle" and "slow" electrogenic phases (~0.185ms and ~0.78ms) coupled to electron redistribution from the heme b/heme a 3 pair to the Cu B site. The "middle" and "slow" electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach Cu B the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~0.1 H + /e - , probably due to the formed membrane potential in the experiment. Copyright © 2017 Elsevier B.V. All rights reserved.

The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin.

PubMed

Bellei, Marzia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Borsari, Marco; Lancellotti, Lidia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio

2018-01-01

Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using UV-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the SS bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (ΔH°' rc ) and entropy (ΔS°' rc ), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate. Copyright © 2017 Elsevier Inc. All rights reserved.

Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation.

PubMed

Tran, Rosalie; Boon, Elizabeth M; Marletta, Michael A; Mathies, Richard A

2009-09-15

Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.

Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

PubMed Central

Mouriño, Susana; Rodríguez-Ares, Isabel; Osorio, Carlos R.; Lemos, Manuel L.

2005-01-01

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity. PMID:16332832

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.

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

2008-01-08

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.

Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

PubMed Central

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

2010-01-01

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 μM 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

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy.

PubMed

Kruglik, Sergei G; Jasaitis, Audrius; Hola, Klara; Yamashita, Taku; Liebl, Ursula; Martin, Jean-Louis; Vos, Marten H

2007-05-01

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

PubMed Central

Kruglik, Sergei G.; Jasaitis, Audrius; Hola, Klara; Yamashita, Taku; Liebl, Ursula; Martin, Jean-Louis; Vos, Marten H.

2007-01-01

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR3) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR3 measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron–ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron–histidine band around 217 cm−1 as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the “oxygen cage” properties of this oxygen sensor protein. PMID:17446273

Resonance Raman studies of Escherichia coli cytochrome bd oxidase. Selective enhancement of the three heme chromophores of the "as-isolated" enzyme and characterization of the cyanide adduct.

PubMed

Sun, J; Osborne, J P; Kahlow, M A; Kaysser, T M; Hil, J J; Gennis, R B; Loehr, T M

1995-09-26

Cytochrome bd oxidase is a terminal bacterial oxidase containing three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be at a dinuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinone. One of the unique functional features of this enzyme is its resistance to high concentrations of cyanide (Ki in the millimolar range). With the appropriate selection of laser lines, the ligation and spin states of the b558, b595, and d hemes can be probed selectively by resonance Raman (rR) spectroscopy. Wavelengths between 400 and 500 nm predominantly excite the rR spectra of the b558 and b595 chromophores. Spectra obtained within this interval show a mixed population of spin and ligation states arising from b558 and b595, with the former more strongly enhanced at higher energy. Red excitation wavelengths (590-650 nm) generate rR spectra characteristic of chlorins, indicating the selective enhancement of the d heme. These rR results reveal that cytochrome bd oxidase "as isolated" contains the b558 heme in a six-coordinate low-spin ferric state, the b595 heme in a five-coordinate high-spin (5cHS) ferric state, and the d heme in a mixture of oxygenated (FeIIO2 <--> FeIIIO2-; d650) and ferryl-oxo (FeIV = O; d680) states. However, the rR spectra of these two chlorin species indicate that they are both in the 5cHS state, suggesting that the d heme is lacking a strongly coordinated sixth ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidoreductases on their way to industrial biotransformations.

PubMed

Martínez, Angel T; Ruiz-Dueñas, Francisco J; Camarero, Susana; Serrano, Ana; Linde, Dolores; Lund, Henrik; Vind, Jesper; Tovborg, Morten; Herold-Majumdar, Owik M; Hofrichter, Martin; Liers, Christiane; Ullrich, René; Scheibner, Katrin; Sannia, Giovanni; Piscitelli, Alessandra; Pezzella, Cinzia; Sener, Mehmet E; Kılıç, Sibel; van Berkel, Willem J H; Guallar, Victor; Lucas, Maria Fátima; Zuhse, Ralf; Ludwig, Roland; Hollmann, Frank; Fernández-Fueyo, Elena; Record, Eric; Faulds, Craig B; Tortajada, Marta; Winckelmann, Ib; Rasmussen, Jo-Anne; Gelo-Pujic, Mirjana; Gutiérrez, Ana; Del Río, José C; Rencoret, Jorge; Alcalde, Miguel

2017-11-01

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H 2 O 2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H 2 O 2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H 2 O 2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D 3 , drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holowiecki, Andrew

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, mostmore » notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs. - Highlights: • hmox1a, hmox2a, hmox2b and bvrb are sexually dimorphic in expression. • hmox paralogs were induced in adult tissues by cadmium exposure. • hmox1a, hmox1b and bvrb were induced by multiple pro-oxidants zebrafish embryos. • Differential expression of zebrafish hmox paralogs suggest partitioning of function. • Nrf2a mediates the induction of hmox1b and bvrb by cadmium in zebrafish embryos.« less

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...

The intriguing enhancement of chloroperoxidase mediated one-electron oxidations by azide, a known active-site ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andrew, Daniel; Hager, Lowell; Manoj, Kelath Murali, E-mail: muralimanoj@vit.ac.in

2011-12-02

Highlights: Black-Right-Pointing-Pointer Azide is a well known heme-enzyme active site ligand and inhibitor. Black-Right-Pointing-Pointer Herein, azide is reported to enhance a set of heme-enzyme mediated reactions. Black-Right-Pointing-Pointer This effect is disconnected from native enzyme-azide binding. Black-Right-Pointing-Pointer Azide could enhance heme-enzyme reactions via a newly proposed mechanism. Black-Right-Pointing-Pointer Azide contained in reagents could impact reaction outcomes in redox biochemistry. -- Abstract: Azide is a well-known inhibitor of heme-enzymes. Herein, we report the counter-intuitive observation that at some concentration regimes, incorporation of azide in the reaction medium enhances chloroperoxidase (CPO, a heme-enzyme) mediated one-electron abstractions from several substrates. A diffusible azidyl radicalmore » based mechanism is proposed for explaining the phenomenon. Further, it is projected that the finding could have significant impact on routine in situ or in vitro biochemistry studies involving heme-enzyme systems and azide.« less

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

Splitting of the O–O bond at the heme-copper catalytic site of respiratory oxidases

PubMed Central

Poiana, Federica; von Ballmoos, Christoph; Gonska, Nathalie; Blomberg, Margareta R. A.; Ädelroth, Pia; Brzezinski, Peter

2017-01-01

Heme-copper oxidases catalyze the four-electron reduction of O2 to H2O at a catalytic site that is composed of a heme group, a copper ion (CuB), and a tyrosine residue. Results from earlier experimental studies have shown that the O–O bond is cleaved simultaneously with electron transfer from a low-spin heme (heme a/b), forming a ferryl state (PR; Fe4+=O2−, CuB2+–OH−). We show that with the Thermus thermophilus ba3 oxidase, at low temperature (10°C, pH 7), electron transfer from the low-spin heme b to the catalytic site is faster by a factor of ~10 (τ ≅ 11 μs) than the formation of the PR ferryl (τ ≅110 μs), which indicates that O2 is reduced before the splitting of the O–O bond. Application of density functional theory indicates that the electron acceptor at the catalytic site is a high-energy peroxy state [Fe3+–O−–O−(H+)], which is formed before the PR ferryl. The rates of heme b oxidation and PR ferryl formation were more similar at pH 10, indicating that the formation of the high-energy peroxy state involves proton transfer within the catalytic site, consistent with theory. The combined experimental and theoretical data suggest a general mechanism for O2 reduction by heme-copper oxidases. PMID:28630929

Regulation of human heme oxygenase-1 gene expression under thermal stress.

PubMed

Okinaga, S; Takahashi, K; Takeda, K; Yoshizawa, M; Fujita, H; Sasaki, H; Shibahara, S

1996-06-15

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Souza, C.F.; Carneiro, A.B.; Silveira, A.B.

2009-12-18

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasitemore » proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.« less

Iron-heme-Bach1 axis is involved in erythroblast adaptation to iron deficiency.

PubMed

Kobayashi, Masahiro; Kato, Hiroki; Hada, Hiroshi; Itoh-Nakadai, Ari; Fujiwara, Tohru; Muto, Akihiko; Inoguchi, Yukihiro; Ichiyanagi, Kenji; Hojo, Wataru; Tomosugi, Naohisa; Sasaki, Hiroyuki; Harigae, Hideo; Igarashi, Kazuhiko

2017-03-01

Iron plays the central role in oxygen transport by erythrocytes as a constituent of heme and hemoglobin. The importance of iron and heme is also to be found in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is deactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1 -/- mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses these genes in erythroblasts under iron deficiency to balance the levels of heme and globin. Moreover, an increase in genome-wide DNA methylation was observed in erythroblasts of Bach1 -/- mice under iron deficiency. These findings reveal the principle role of iron as a regulator of gene expression in erythroblast maturation and suggest that the iron-heme-Bach1 axis is important for a proper adaptation of erythroblast to iron deficiency to avoid toxic aggregates of non-heme globin. Copyright© Ferrata Storti Foundation.

The primary structure of the hemoglobin of spectacled bear (Tremarctos ornatus, Carnivora).

PubMed

Hofmann, O; Braunitzer, G

1987-08-01

The complete primary structure of the alpha- and beta-chains of the hemoglobin of Spectacled Bear (Tremarctos ornatus) is presented. Following cleavage of the heme-protein link and chain separation by RP-HPLC, their amino-acid sequences were determined by Edman degradation in liquid- and gas-phase sequenators. The hemoglobin of Spectacled Bear displays only five amino-acid exchanges to that of Polar Bear (Ursus maritimus, Ursinae) and Asiatic Black Bear (Ursus tibetanus, Ursinae) whereas 8 and 12 replacements, respectively, to Giant Panda (Ailuropoda melanoleuca) and Lesser Panda (Ailurus fulgens) can be found. This clearly demonstrates that the Spectacled Bear, the most aberrant bear of the Ursidae, is somewhat intermediate between Pandas and Ursinae.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

PubMed

Almeida, Ana S; Soares, Nuno L; Vieira, Melissa; Gramsbergen, Jan Bert; Vieira, Helena L A

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death

PubMed Central

Almeida, Ana S.; Soares, Nuno L.; Vieira, Melissa; Gramsbergen, Jan Bert

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO’s increasing number of differentiated neurons in OHSC. In conclusion, CO’s increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO’s effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies. PMID:27144388

Microbial Community Composition and Functional Capacity in a Terrestrial Ferruginous, Sulfate-Depleted Mud Volcano

PubMed Central

Tu, Tzu-Hsuan; Wu, Li-Wei; Lin, Yu-Shih; Imachi, Hiroyuki; Lin, Li-Hung; Wang, Pei-Ling

2017-01-01

Terrestrial mud volcanoes (MVs) are an important natural source of methane emission. The role of microbial processes in methane cycling and organic transformation in such environments remains largely unexplored. In this study, we aim to uncover functional potentials and community assemblages across geochemical transitions in a ferruginous, sulfate-depleted MV of eastern Taiwan. Geochemical profiles combined with 16S rRNA gene abundances indicated that anaerobic oxidation of methane (AOM) mediated by ANME-2a group coincided with iron/manganese reduction by Desulfuromonadales at shallow depths deprived of sulfate. The activity of AOM was stimulated either by methane alone or by methane and a range of electron acceptors, such as sulfate, ferrihydrite, and artificial humic acid. Metagenomic analyses revealed that functional genes for AOM and metal reduction were more abundant at shallow intervals. In particular, genes encoding pili expression and electron transport through multi-heme cytochromes were prevalent, suggesting potential intercellular interactions for electron transport involved in AOM. For comparison, genes responsible for methanogenesis and degradation of chitin and plant-derived molecules were more abundant at depth. The gene distribution combined with the enhanced proportions of 16S rRNA genes related to methanogens and heterotrophs, and geochemical characteristics suggest that particulate organic matter was degraded into various organic entities that could further fuel in situ methanogenesis. Finally, genes responsible for aerobic methane oxidation were more abundant in the bubbling pool and near-surface sediments. These methane oxidizers account for the ultimate attenuation of methane discharge into the atmosphere. Overall, our results demonstrated that various community members were compartmentalized into stratified niches along geochemical gradients. These community members form a metabolic network that cascades the carbon transformation from the upstream degradation of recalcitrant organic carbon with fermentative production of labile organic entities and methane to downstream methane oxidation and metal reduction near the surface. Such a metabolic architecture enables effective methane removal under ferruginous, sulfate-depleted conditions in terrestrial MVs. PMID:29163423

Microbial Community Composition and Functional Capacity in a Terrestrial Ferruginous, Sulfate-Depleted Mud Volcano.

PubMed

Tu, Tzu-Hsuan; Wu, Li-Wei; Lin, Yu-Shih; Imachi, Hiroyuki; Lin, Li-Hung; Wang, Pei-Ling

2017-01-01

Terrestrial mud volcanoes (MVs) are an important natural source of methane emission. The role of microbial processes in methane cycling and organic transformation in such environments remains largely unexplored. In this study, we aim to uncover functional potentials and community assemblages across geochemical transitions in a ferruginous, sulfate-depleted MV of eastern Taiwan. Geochemical profiles combined with 16S rRNA gene abundances indicated that anaerobic oxidation of methane (AOM) mediated by ANME-2a group coincided with iron/manganese reduction by Desulfuromonadales at shallow depths deprived of sulfate. The activity of AOM was stimulated either by methane alone or by methane and a range of electron acceptors, such as sulfate, ferrihydrite, and artificial humic acid. Metagenomic analyses revealed that functional genes for AOM and metal reduction were more abundant at shallow intervals. In particular, genes encoding pili expression and electron transport through multi-heme cytochromes were prevalent, suggesting potential intercellular interactions for electron transport involved in AOM. For comparison, genes responsible for methanogenesis and degradation of chitin and plant-derived molecules were more abundant at depth. The gene distribution combined with the enhanced proportions of 16S rRNA genes related to methanogens and heterotrophs, and geochemical characteristics suggest that particulate organic matter was degraded into various organic entities that could further fuel in situ methanogenesis. Finally, genes responsible for aerobic methane oxidation were more abundant in the bubbling pool and near-surface sediments. These methane oxidizers account for the ultimate attenuation of methane discharge into the atmosphere. Overall, our results demonstrated that various community members were compartmentalized into stratified niches along geochemical gradients. These community members form a metabolic network that cascades the carbon transformation from the upstream degradation of recalcitrant organic carbon with fermentative production of labile organic entities and methane to downstream methane oxidation and metal reduction near the surface. Such a metabolic architecture enables effective methane removal under ferruginous, sulfate-depleted conditions in terrestrial MVs.

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.

PubMed

Vallelian, Florence; Garcia-Rubio, Ines; Puglia, Michele; Kahraman, Abdullah; Deuel, Jeremy W; Engelsberger, Wolfgang R; Mason, Ronald P; Buehler, Paul W; Schaer, Dominik J

2015-08-01

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein. Copyright © 2015 Elsevier Inc. All rights reserved.

Heme inhibition of ferrisiderophore reductase in Bacillus subtilis.

PubMed

Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

1982-11-01

Heme was a noncompetitive inhibitor (apparent K(i) and K'(i) = 0.043 mM) of a ferrisiderophore reductase purified from Bacillus subtilis; protoporphyrin IX had no effect. The cellular level of heme may partly regulate the function of this reductase to yield a controlled flow of iron into metabolism.

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

Heme induces IL-1β secretion through activating NLRP3 in kidney inflammation.

PubMed

Li, Qianwei; Fu, Weihua; Yao, Jiwei; Ji, Zheng; Wang, Yongquan; Zhou, Zhansong; Yan, Junan; Li, Weibing

2014-07-01

To produce proinflammatory master cytokine IL-1β in macrophages, two stimulation pathways are needed including TLRs-NF-κB axis and NLRPs/ASC-caspase-1 axis. Different signals including exogenous and endogenous trigger inflammatory response distinctly. Among them, the role of endogenous stimulators of inflammation is poorly understood. As a component of hemoglobin, free heme is released when hemolysis or extensive cell damage occur which results in inflammatory response. Here, we find that heme induces IL-1β secretion through activating NLRP3 inflammasome in macrophages. Heme activates NLRP3 through P2X receptors, especially the P2X7R and P2X4R. Most importantly, significantly enhancement of heme level and activation of NLRPs/ASC-caspase-1 axis were observed in mice kidney after unilateral ureteral obstruction which could be inhibited by enforced expression of heme oxygenase-1 (HO-1). Our study proves that heme is a potential danger activator of NLRP3 inflammasome that plays an essential role in IL-1β secretion during kidney inflammation and provides new insight into the mechanism of innate immune initiation. Further investigation will be beneficial to develop new molecular target and molecular diagnosis indicator in therapy of kidney inflammation.

Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia*

PubMed Central

Lunetti, Paola; Damiano, Fabrizio; De Benedetto, Giuseppe; Siculella, Luisa; Pennetta, Antonio; Muto, Luigina; Paradies, Eleonora; Marobbio, Carlo Marya Thomas; Dolce, Vincenza

2016-01-01

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. PMID:27476175

Inhibition of heme biosynthesis prevents transcription of iron uptake genes in yeast.

PubMed

Crisp, Robert J; Pollington, Annette; Galea, Charles; Jaron, Shulamit; Yamaguchi-Iwai, Yuko; Kaplan, Jerry

2003-11-14

Yeast are capable of modifying their metabolism in response to environmental changes. We investigated the activity of the oxygen-dependent high-affinity iron uptake system of Saccharomyces cerevisiae under conditions of heme depletion. We found that the absence of heme, due to a deletion in the gene that encodes delta-aminolevulinic acid synthase (HEM1), resulted in decreased transcription of genes belonging to both the iron and copper regulons, but not the zinc regulon. Decreased transcription of the iron regulon was not due to decreased expression of the iron sensitive transcriptional activator Aft1p. Expression of the constitutively active allele AFT1-1up was unable to induce transcription of the high affinity iron uptake system in heme-depleted cells. We demonstrated that under heme-depleted conditions, Aft1p-GFP was able to cycle normally between the nucleus and cytosol in response to cytosolic iron. Despite the inability to induce transcription under low iron conditions, chromatin immunoprecipitation demonstrated that Aft1p binds to the FET3 promoter in the absence of heme. Finally, we provide evidence that under heme-depleted conditions, yeast are able to regulate mitochondrial iron uptake and do not accumulate pathologic iron concentrations, as is seen when iron-sulfur cluster synthesis is disrupted.

O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7.

PubMed

Shadrina, Maria S; Peslherbe, Gilles H; English, Ann M

2015-09-01

Hemoglobin transports O2 by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O2 significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O2 to the solvent via a transient E7 channel. To reveal the O2 escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O2 diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7's conformation, we observe the same four or five escape routes leading directly from the α- or β-distal heme pockets to the solvent. Only 21-53% of O2 escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O2 from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O2 escapes to the solvent directly from the α- or β-distal pockets of the mutant increases to 70-88%. In contrast to O2, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes.

Reaction with cyanide of hydroxylamine oxidoreductase of Nitrosomonas europaea.

PubMed

Logan, M S; Balny, C; Hooper, A B

1995-07-18

Hydroxylamine oxidoreductase (HAO) catalyzes the reaction NH2OH+H2O-->HNO2+4e- + 4H+, a step in the energy-generating oxidation of ammonia to nitrite by the bacterium Nitrosomonas europaea. Each subunit of HAO contains 7 c-hemes and 1 heme P460. The latter, c-heme cross-linked from a methylene carbon to the ring of a protein tyrosine, forms part of the active site. The iron of heme P460 is probably linked by a bridging ligand to the iron of a c-heme. Here, the reaction of cyanide with ferric HAO was studied by optical, transient, and steady state kinetic techniques. The molecules, F-, Cl-, Br-, N3-, SCN-, and OCN- did not react with HAO. A single molecule of cyanide bound with high affinity to heme P460 of HAO. The optical and kinetic characteristics of formation of the monocyano complex of HAO resembled those of cyanide derivatives of other heme proteins. Cyanide, in the monocyano complex, was a noncompetitive inhibitor and remained bound during turnover. HAO was found in two forms. The most common form, HAO-A, formed only the monocyano derivative of heme P460, whereas the other, HAO-B, formed a mono- and dicyano complex. The optical properties and kinetics of formation of the mono- and dicyano complexes were different enough to easily allow independent analysis. The optical and kinetic characteristics of formation of the monocyano complex of heme P460 of HAO A and B were very similar. The dicyano complex of HAO-B appeared to result from the addition of a second molecule of cyanide to heme P460. The rate of conversion of the monocyano to the dicyano complex was stimulated 100-fold by the binding of substrate. Formation of the monoheme complex inhibited enzyme activity. The kinetic constants for the first-order formation of the monocyano derivative and the inhibition of substrate oxidation (under either transient or steady-state conditions) were different. The apparent discrepancy could be resolved by the hypothesis that HAO is functionally a dimer in which electrons rapidly equilibrate between the c-hemes of each subunit but not between oligomers. The results form the basis for the use of cyanide as a probe of the active site of HAO.

Protein kinase G–regulated production of H2S governs oxygen sensing

PubMed Central

Yuan, Guoxiang; Vasavda, Chirag; Peng, Ying-Jie; Makarenko, Vladislav V.; Raghuraman, Gayatri; Nanduri, Jayasri; Gadalla, Moataz M.; Semenza, Gregg L.; Kumar, Ganesh K.; Snyder, Solomon H.; Prabhakar, Nanduri R.

2015-01-01

Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardio-respiratory homeostasis during hypoxia. O2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H2S) synthesis by cystathionine-γ-lyase (CSE). We report that O2 stimulated the generation of CO, but not that of H2S, and required two cysteine residues in the heme regulatory motif (Cys265 and Cys282) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser377 of CSE, inhibiting the production of H2S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H2S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O2 sensing through PKG-dependent regulation of H2S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing. PMID:25900831

Pilot-scale tests of HEME and HEPA dissolution process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qureshi, Z.H.; Strege, D.K.

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 rinsedmore » 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.« less

Protein oxidative damage and heme oxygenase in sunlight-exposed human skin: roles of MAPK responses to oxidative stress.

PubMed

Akasaka, Emiko; Takekoshi, Susumu; Horikoshi, Yosuke; Toriumi, Kentarou; Ikoma, Norihiro; Mabuchi, Tomotaka; Tamiya, Shiho; Matsuyama, Takashi; Ozawa, Akira

2010-12-20

Oxidative stress derived from ultraviolet (UV) light in sunlight induces different hazardous effects in the skin, including sunburn, photo-aging and DNA mutagenesis. In this study, the protein-bound lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8OHdG) were investigated in chronically sun-exposed and sun-protected human skins using immunohistochemistry. The levels of antioxidative enzymes, such as heme oxygenase 1 and 2, Cu/Zn-SOD, Mn-SOD and catalase, were also examined. Oxidative stress is also implicated in the activation of signal transduction pathways, such as mitogen-activated protein kinase (MAPK). Therefore, the expression and distribution of phosphorylated p38 MAPK, phosphorylated Jun N-terminal kinase (JNK) and phosphorylated extracellular signal-regulated kinase (ERK) were observed. Skin specimens were obtained from the surgical margins. Chronically sunlight-exposed skin samples were taken from the ante-auricular (n = 10) and sunlight-protected skin samples were taken from the post-auricular (n = 10). HNE was increased in the chronically sunlight-exposed skin but not in the sunlight-protected skin. The expression of heme oxygenase-2 was markedly increased in the sunlight-exposed skin compared with the sun-protected skin. In contrast, the intensity of immunostaining of Cu/Zn-SOD, Mn-SOD and catalase was not different between the two areas. Phosphorylated p38 MAPK and phosphorylated JNK accumulated in the ante-auricular dermis and epidermis, respectively. These data show that particular anti-oxidative enzymes function as protective factors in chronically sunlight-exposed human skin. Taken together, our results suggest (1) antioxidative effects of heme oxygenase-2 in chronically sunlight-exposed human skin, and that (2) activation of p38 MAPK may be responsible for oxidative stress.

Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

PubMed

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.

The dual effects of nitrite on hemoglobin-dependent redox reactions.

PubMed

Lu, Naihao; Chen, Chao; He, Yingjie; Tian, Rong; Xiao, Qiang; Peng, Yi-Yuan

2014-08-31

Evidence to support the role of heme proteins-dependent reactions as major inducers of oxidative damage is increasingly present. Nitrite (NO2(-)) is one of the major end products of NO metabolism, and from the daily consumption. Although the biological significance of heme proteins/NO2(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO2(-) on heme proteins-dependent redox reactions have been greatly underestimated. In this study, we investigated the influence of NO2(-) on met-hemoglobin (Hb)-dependent oxidative and nitrative stress. It was found that NO2(-) effectively reduced cytotoxic ferryl intermediate back to ferric Hb in a biphasic kinetic reaction. However, the presence of NO2(-) surprisingly exerted pro-oxidant effect on Hb-H2O2-induced protein (bovine serum albumin, enolase) oxidation at low concentrations and enhanced the loss of HepG2 cell viability. In the reduction of ferryl Hb to ferric state, NO2(-) was decreased and oxidized to a nitrating agent NO2, Tyr12 and Tyr191 in enolase were subsequently nitrated. In contrast to the frequently inhibitive effect of nitrotyrosine, NO2(-)-triggered tyrosine nitration might play an important role in enolase activation. These data provided novel evidence that the dietary intake and potential therapeutic application of NO2(-) would possess anti- and pro-oxidant activities through interfering in hemoglobin-dependent redox reactions. Besides the classic role in protein tyrosine nitration, the dual effects on hemoglobin-triggered oxidative stress may provide new insights into the physiological and toxicological implications of NO2(-) with heme proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Possible Dynamically Gated Conductance along Heme Wires in Bacterial Multiheme Cytochromes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle MA; Rosso, Kevin M.

2014-07-24

The staggered cross decaheme configuration of electron transfer co-factors in the outer-membrane cytochrome MtrF may serve as a prototype for conformationally-gated multi-heme electron transport. Derived from the bacterium Shewanella oneidensis, the staggered cross configuration reveals intersecting c-type octaheme and tetraheme “wires” containing thermodynamic “hills” and “valleys”, suggesting that the protein structure may include a dynamical mechanism for conductance and pathway switching depending on enzymatic functional need. Recent molecular simulations have established the pair-wise electronic couplings, redox potentials, and reorganization energies to predict the maximum conductance along the various heme wire pathways by sequential hopping of a single electron (PNAS (2014)more » 11,611-616). Here, we expand this information with classical molecular and statistical mechanics calculations of large-amplitude protein dynamics in MtrF, to address its potential to modulate pathway conductance, including assessment of the effect of the total charge state. Explicit solvent molecular dynamics simulations of fully oxidized and fully reduced MtrF employing ten independent 50-ns simulations at 300 K and 1 atm showed that reduced MtrF is more expanded and explores more conformational space than oxidized MtrF, and that heme reduction leads to increased heme solvent exposure. The slowest mode of collective decaheme motion is 90% similar between the oxidized and reduced states, and consists primarily of inter-heme separation with minor rotational contributions. The frequency of this motion is 1.7×107 s 1 for fully-oxidized and fully-reduced MtrF, respectively, slower than the downhill electron transfer rates between stacked heme pairs at the octaheme termini and faster than the electron transfer rates between parallel hemes in the tetraheme chain. This implies that MtrF uses slow conformational fluctuations to modulate electron flow along the octaheme pathway, apparently for the purpose of increasing the residence time of electrons on lowest potential hemes 4 and 9. This apparent gating mechanism should increase the success rate of electron transfer from MtrF to low potential environmental acceptors via these two solvent-exposed hemes.« less

Growth promotion of the opportunistic human pathogen, Staphylococcus lugdunensis, by heme, hemoglobin, and coculture with Staphylococcus aureus

PubMed Central

Brozyna, Jeremy R; Sheldon, Jessica R; Heinrichs, David E

2014-01-01

Staphylococcus lugdunensis is both a commensal of humans and an opportunistic pathogen. Little is currently known about the molecular mechanisms underpinning the virulence of this bacterium. Here, we demonstrate that in contrast to S. aureus,S. lugdunensis makes neither staphyloferrin A (SA) nor staphyloferrin B (SB) in response to iron deprivation, owing to the absence of the SB gene cluster, and a large deletion in the SA biosynthetic gene cluster. As a result, the species grows poorly in serum-containing media, and this defect was complemented by introduction of the S. aureusSA gene cluster into S. lugdunensis. S. lugdunensis expresses the HtsABC and SirABC transporters for SA and SB, respectively; the latter gene set is found within the isd (heme acquisition) gene cluster. An isd deletion strain was significantly debilitated for iron acquisition from both heme and hemoglobin, and was also incapable of utilizing ferric-SB as an iron source, while an hts mutant could not grow on ferric-SA as an iron source. In iron-restricted coculture experiments, S. aureus significantly enhanced the growth of S. lugdunensis, in a manner dependent on staphyloferrin production by S. aureus, and the expression of the cognate transporters by S. lugdunensis. PMID:24515974

Arterial carboxyhemoglobin level and outcome in critically ill patients.

PubMed

Melley, Daniel D; Finney, Simon J; Elia, Androula; Lagan, Anna L; Quinlan, Gregory J; Evans, Timothy W

2007-08-01

Arterial carboxyhemoglobin is elevated in patients with critical illness. It is an indicator of the endogenous production of carbon monoxide by the enzyme heme oxygenase, which modulates the response to oxidant stress. The objective was to explore the hypothesis that arterial carboxyhemoglobin level is associated with inflammation and survival in patients requiring cardiothoracic intensive care. Prospective, observational study. A cardiothoracic intensive care unit. All patients admitted over a 15-month period. None. Arterial carboxyhemoglobin, bilirubin, and standard biochemical, hematologic, and physiologic markers of inflammation were measured in 1,267 patients. Associations were sought between levels of arterial carboxyhemoglobin, markers of the inflammatory response, and clinical outcome. Intensive care unit mortality was associated with lower minimum and greater maximal carboxyhemoglobin levels (p < .0001 and p < .001, respectively). After adjustment for age, gender, illness severity, and other relevant variables, a lower minimum arterial carboxyhemoglobin was associated with an increased risk of death from all causes (odds risk of death, 0.391; 95% confidence interval, 0.190-0.807; p = .011). Arterial carboxyhemoglobin correlated with markers of the inflammatory response. Both low minimum and high maximum levels of arterial carboxyhemoglobin were associated with increased intensive care mortality. Although the heme oxygenase system is protective, excessive induction may be deleterious. This suggests that there may be an optimal range for heme oxygenase-1 induction.

DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

PubMed

Hemeryck, Lieselot Y; Rombouts, Caroline; De Paepe, Ellen; Vanhaecke, Lynn

2018-05-01

The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O 6 -carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N 4 -etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans*

PubMed Central

Molitor, Bastian; Stassen, Marc; Modi, Anuja; El-Mashtoly, Samir F.; Laurich, Christoph; Lubitz, Wolfgang; Dawson, John H.; Rother, Michael; Frankenberg-Dinkel, Nicole

2013-01-01

Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor). PMID:23661702

Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3β pathway.

PubMed

Dal-Cim, Tharine; Molz, Simone; Egea, Javier; Parada, Esther; Romero, Alejandro; Budni, Josiane; Martín de Saavedra, Maria D; del Barrio, Laura; Tasca, Carla I; López, Manuela G

2012-08-01

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligo-induced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A(1) or A(2A) receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca(2+)-activated K(+) channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38(MAPK) inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3β phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3β proteins and induction of the antioxidant enzyme HO-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

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…

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...

On the Monoheme Character of Cytochromes c′

PubMed Central

Kennel, S. J.; Meyer, T. E.; Kamen, M. D.; Bartsch, R. G.

1972-01-01

Interpretations of data bearing on structures of cytochromes cc′—a class of variant c-type heme proteins from bacteria—in support of a diheme-bearing single chain as a basic structural unit, appear to be invalid in the light of recent studies. These reveal that nearly all members of this class exist as dimers that can be dissociated into, if they do not already exist as, monoheme-bearing monomers. The particular case of the Chromatium protein, held to be the source of a peptic-“core” peptide containing two covalently-bonded heme groups, has been re-examined by preparation of tryptic and chymotryptic peptides derived from the heme-bearing region of the protein, as well as by repetition of experiments on peptic digestion, with more rigorous purification of the resultant peptides than was previously done. It is shown that this protein can also be dissociated into identical subunits, bearing a single heme prosthetic group, and accounting essentially for all its heme content. Thus, the previous terminology—cytochromes cc′—based on supposition of heme group heterogeneity, is inconsistent with these findings and should be replaced by cytochromes c′. PMID:4343972

Thermodynamics of Electron Flow in the Bacterial Deca-heme Cytochrome MtrF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Breuer, Marian; Zarzycki, Piotr P.; Blumberger, Jochen

2012-07-01

Electron transporting multiheme cytochromes are essential to the metabolism of microbes that inhabit soils and carry out important biogeochemical processes. Recently the first crystal structure of a prototype bacterial deca-heme cytochrome (MtrF) has been resolved and its electrochemistry characterized. However, the molecular details of electron conductance along heme chains in the cytochrome are difficult to access via experiment due to the nearly identical chemical nature of the heme cofactors. Here we employ large-scale molecular dynamics simulations to compute the reduction potentials of the ten hemes of MtrF in aqueous solution. We find that as a whole they fall within amore » range of about 0.3 V in agreement with experiment. Individual reduction potentials give rise to a free energy profile for electron conduction that is approximately symmetric with respect to the center of the protein. Our calculations indicate that there is no significant potential bias along the orthogonal octa- and tetra-heme chains suggesting that under aqueous conditions MtrF is a nearly reversible two-dimensional conductor.« less

Upregulation of heme oxygenase-1 gene by turpentine oil-induced localized inflammation: involvement of interleukin-6.

PubMed

Tron, Kyrylo; Novosyadlyy, Ruslan; Dudas, Jozsef; Samoylenko, Anatoly; Kietzmann, Thomas; Ramadori, Giuliano

2005-03-01

Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4-6 h after TO administration. HO-1 protein levels in the liver were elevated starting from 6-12 h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Soon Won; Sohn, Eun Jeong; Kim, Dae Won

Highlights: {yields} Recombinant PEP-1 heme oxygenase-1 expression vector was constructed and overexpressed. {yields} We investigated transduction efficiency of PEP-1-HO-1 protein in Raw 264.7 cells. {yields} PEP-1-HO-1 was efficiently transduced into Raw 264.7 cells in a dose and time dependent manner. {yields} PEP-1-HO-1 exerted anti-inflammatory activity in Raw 264.7 cells and in a mice edema model. {yields} PEP-1-HO-1 could be used as a therapeutic drug against inflammatory diseases. -- Abstract: Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe{sup 2+}), is up-regulated by several cellular stress and cell injuries, including inflammation,more » ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60 h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.« less

Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.

2005-10-14

Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substratemore » and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N {sup {omega}}-hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein from a pathogenic bacterium opens up avenues for further studies in understanding the importance of this protein in pathogenicity.« less

Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

PubMed

Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

2011-12-01

Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (<20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural characterization of a family of cytochromes c{sub 7} involved in Fe(III) respiration by Geobacter sulfurreducens.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pokkuluri, P. R.; Londer, Y. Y.; Yang, X.

2010-02-01

Periplasmic cytochromes c{sub 7} are important in electron transfer pathway(s) in Fe(III) respiration by Geobacter sulfurreducens. The genome of G. sulfurreducens encodes a family of five 10-kDa, three-heme cytochromes c{sub 7}. The sequence identity between the five proteins (designated PpcA, PpcB, PpcC, PpcD, and PpcE) varies between 45% and 77%. Here, we report the high-resolution structures of PpcC, PpcD, and PpcE determined by X-ray diffraction. This new information made it possible to compare the sequences and structures of the entire family. The triheme cores are largely conserved but are not identical. We observed changes, due to different crystal packing, inmore » the relative positions of the hemes between two molecules in the crystal. The overall protein fold of the cytochromes is similar. The structure of PpcD differs most from that of the other homologs, which is not obvious from the sequence comparisons of the family. Interestingly, PpcD is the only cytochrome c{sub 7} within the family that has higher abundance when G. sulfurreducens is grown on insoluble Fe(III) oxide compared to ferric citrate. The structures have the highest degree of conservation around 'heme IV'; the protein surface around this heme is positively charged in all of the proteins, and therefore all cytochromes c{sub 7} could interact with similar molecules involving this region. The structures and surface characteristics of the proteins near the other two hemes, 'heme I' and 'heme III', differ within the family. The above observations suggest that each of the five cytochromes c{sub 7} could interact with its own redox partner via an interface involving the regions of heme I and/or heme III; this provides a possible rationalization for the existence of five similar proteins in G. sulfurreducens.« less

Identification of iron and heme utilization genes in Aeromonas and their role in the colonization of the leech digestive tract

PubMed Central

Maltz, Michele; LeVarge, Barbara L.; Graf, Joerg

2015-01-01

It is known that many pathogens produce high-affinity iron uptake systems like siderophores and/or proteins for utilizing iron bound to heme-containing molecules, which facilitate iron-acquisition inside a host. In mutualistic digestive-tract associations, iron uptake systems have not been as well studied. We investigated the importance of two iron utilization systems within the beneficial digestive-tract association Aeromonas veronii and the medicinal leech, Hirudo verbana. Siderophores were detected in A. veronii using chrome azurol S. Using a mini Tn5, a transposon insertion in viuB generated a mutant unable to utilize iron using siderophores. The A. veronii genome was then searched for genes potentially involved in iron utilization bound to heme-containing molecules. A putative outer membrane heme receptor (hgpB) was identified with a transcriptional activator, termed hgpR, downstream. The hgpB gene was interrupted with an antibiotic resistance cassette in both the parent strain and the viuB mutant, yielding an hgpB mutant and a mutant with both iron uptake systems inactivated. In vitro assays indicated that hgpB is involved in utilizing iron bound to heme and that both iron utilization systems are important for A. veronii to grow in blood. In vivo colonization assays revealed that the ability to acquire iron from heme-containing molecules is critical for A. veronii to colonize the leech gut. Since iron and specifically heme utilization is important in this mutualistic relationship and has a potential role in virulence factor of other organisms, genomes from different Aeromonas strains (both clinical and environmental) were queried with iron utilization genes of A. veronii. This analysis revealed that in contrast to the siderophore utilization genes heme utilization genes are widely distributed among aeromonads. The importance of heme utilization in the colonization of the leech further confirms that symbiotic and pathogenic relationships possess similar mechanisms for interacting with animal hosts. PMID:26284048

Spectroscopic characterization of mononitrosyl complexes in heme-nonheme diiron centers within the myoglobin scaffold (FeBMbs): relevance to denitrifying NO reductase†

PubMed Central

Hayashi, Takahiro; Miner, Kyle D.; Yeung, Natasha; Lin, Ying-Wu; Lu, Yi; Moënne-Loccoz, Pierre

2011-01-01

Denitrifying NO reductases are evolutionarily related to the superfamily of heme-copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N2O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines, and Val-68 to a glutamic acid to create a nonheme FeB site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV-vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and CuI- or ZnII-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}7 species observable at liquid nitrogen temperature, the FeII-loaded proteins are EPR silent at ≥ 30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using 15NO and 15N18O. The apo- and CuI-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of FeII at the FeB site shifts the heme ν(FeNO) by +17 cm-1 and the ν(NO) by -50 cm-1 to 1549 cm-1. This low ν(NO) is without precedent for a six-coordinate heme {FeNO}7 species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme FeII. Detection of a similarly low ν(NO) in the ZnII-loaded protein supports this interpretation. PMID:21634416

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

PubMed

Stranava, Martin; Man, Petr; Skálová, Tereza; Kolenko, Petr; Blaha, Jan; Fojtikova, Veronika; Martínek, Václav; Dohnálek, Jan; Lengalova, Alzbeta; Rosůlek, Michal; Shimizu, Toru; Martínková, Markéta

2017-12-22

The heme-based oxygen sensor histidine kinase Af GcHK is part of a two-component signal transduction system in bacteria. O 2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His 183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH - and -CN - complexes of Af GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN - and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length Af GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of Af GcHK. We conclude that Af GcHK functions as an ensemble of molecules sampling at least two conformational states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product

PubMed Central

Kumano, Takuto; Tsujimura, Seiya; Kobayashi, Michihiko

2017-01-01

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate. PMID:28410434

New function of aldoxime dehydratase: Redox catalysis and the formation of an unexpected product.

PubMed

Yamada, Masatoshi; Hashimoto, Yoshiteru; Kumano, Takuto; Tsujimura, Seiya; Kobayashi, Michihiko

2017-01-01

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

Influence of production process design on inclusion bodies protein: the case of an Antarctic flavohemoglobin

PubMed Central

2010-01-01

Background Protein over-production in Escherichia coli often results in formation of inclusion bodies (IBs). Some recent reports have shown that the aggregation into IBs does not necessarily mean that the target protein is inactivated and that IBs may contain a high proportion of correctly folded protein. This proportion is variable depending on the protein itself, the genetic background of the producing cells and the expression temperature. In this paper we have evaluated the influence of other production process parameters on the quality of an inclusion bodies protein. Results The present paper describes the recombinant production in Escherichia coli of the flavohemoglobin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. Flavohemoglobins are multidomain proteins requiring FAD and heme cofactors. The production was carried out in several different experimental setups differing in bioreactor geometry, oxygen supply and the presence of a nitrosating compound. In all production processes, the recombinant protein accumulates in IBs, from which it was solubilized in non-denaturing conditions. Comparing structural properties of the solubilized flavohemoglobins, i.e. deriving from the different process designs, our data demonstrated that the protein preparations differ significantly in the presence of cofactors (heme and FAD) and as far as their secondary and tertiary structure content is concerned. Conclusions Data reported in this paper demonstrate that other production process parameters, besides growth temperature, can influence the structure of a recombinant product that accumulates in IBs. To the best of our knowledge, this is the first reported example in which the structural properties of a protein solubilized from inclusion bodies have been correlated to the production process design. PMID:20334669

Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Zuxu; de Serrano, Vesna; Betts, Laurie

2009-01-28

The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5 {angstrom} away from the heme. These conformationsmore » are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25 {angstrom} from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP.« less

Characterization of 2-Oxindole Forming Heme Enzyme MarE, Expanding the Functional Diversity of the Tryptophan Dioxygenase Superfamily.

PubMed

Zhang, Yuyang; Zou, Yi; Brock, Nelson L; Huang, Tingting; Lan, Yingxia; Wang, Xiaozheng; Deng, Zixin; Tang, Yi; Lin, Shuangjun

2017-08-30

3-Substituted 2-oxindoles are important structural motifs found in many biologically active natural products and pharmaceutical lead compounds. Here, we report an enzymatic formation of the 3-substituted 2-oxindoles catalyzed by MarE in the maremycin biosynthetic pathway in Streptomyces sp. B9173. MarE is a homologue of Fe II /heme-dependent tryptophan 2,3-dioxygenases (TDOs). Typical TDOs usually catalyze the insertion of two oxygen atoms from O 2 into an indole ring to generate N-formylkynurenine (NFK)-like products. In contrast, MarE catalyzes the insertion of a single oxygen atom from O 2 into an indole ring, to probably generate an epoxyindole intermediate that undergoes an unprecedented 2,3-hydride migration to form 2-oxindole structure. MarE shows substrate robustness to catalyze the conversion of a series of 3-substituted indoles into their corresponding 3-substituted 2-oxindoles. Although containing most key amino acid residues conserved in well-known TDO homologues, MarE falls into a separate new subgroup in the phylogenetic tree. The characterization of MarE and its homologue enriches the functional diversities of TDO superfamily and provides a new strategy for discovering novel natural products containing 3-substituted 2-oxindole pharmacophores by genome mining.

The effects of increased heme oxygenase-1 on the lymphoproliferative response in dogs with visceral leishmaniasis.

PubMed

Almeida, Breno Fernando Martins de; Silva, Kathlenn Liezbeth Oliveira; Chiku, Vanessa Marim; Leal, Aline Aparecida Correa; Venturin, Gabriela Lovizutto; Narciso, Luis Gustavo; Fink, Maria Fernanda Cereijido Bersni; Eugênio, Flavia de Rezende; Santos, Paulo Sergio Patto Dos; Ciarlini, Paulo Cesar; Lima, Valéria Marçal Felix de

2017-05-01

Canine visceral leishmaniasis (CVL) is known to affect the cellular immunity of infected dogs, through impairing lymphoproliferation and microbicidal mechanisms. This study examined heme oxygenase-1 (HO-1) and its metabolites, oxidative stress and IL-10 levels in CVL and investigated correlations between these parameters. Additionally, the effects of HO-1 inhibition on the lymphoproliferative response and cytokine production in lymph node cells (LNCs) from infected dogs were evaluated. Forty-four dogs, 24 controls and 20 dogs with CVL were selected. Plasma and splenic levels of HO-1, haptoglobin, soluble CD163 receptor, ferritin and IL-10 were determined using capture ELISA. The HO-1 levels and relative gene expression in peripheral blood and bone marrow mononuclear cells were also determined. LNCs proliferation was evaluated with an HO-1 activator and with an HO-1 inhibitor, in the presence of the Leishmania infantum soluble antigen (SAgL), using flow cytometry. HO-1, IL-2, IFN-gamma and IL-10 were also determined in these cultures using capture ELISA. Infected dogs presented oxidative stress and increased HO-1 levels and relative gene expression, with correlation between oxidative stress and HO-1. The substances from heme metabolism and IL-10 were also elevated in the plasma and spleens of infected dogs. IL-10 and HO-1 levels were positively correlated with one another. Inhibition of HO-1 increased LNCs proliferation and decreased IL-10 and IL-2 production in the presence of SAgL. The increased HO-1 metabolism observed in CVL is probably associated with oxidative stress and increased IL-10, which could be one of the mechanisms responsible for inhibition of the lymphoproliferative response in sick dogs. Copyright © 2016 Elsevier GmbH. All rights reserved.

4-Hydroxy-2(E)-nonenal metabolism differs in Apc(+/+) cells and in Apc(Min/+) cells: it may explain colon cancer promotion by heme iron.

PubMed

Baradat, Maryse; Jouanin, Isabelle; Dalleau, Sabine; Taché, Sylviane; Gieules, Mathilde; Debrauwer, Laurent; Canlet, Cécile; Huc, Laurence; Dupuy, Jacques; Pierre, Fabrice H F; Guéraud, Françoise

2011-11-21

Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.

Cupping therapy: An analysis of the effects of suction on skin and the possible influence on human health.

PubMed

Lowe, Duane T

2017-11-01

Cupping therapy is a traditional therapy that has been employed worldwide for thousands of years. Despite a lack of quality clinical studies evaluating the efficacy of cupping therapy, its long history and widespread use throughout the world suggests the commonly claimed health benefits should not be completely discounted as without merit. The goal of this paper is to present the research detailing what is known concerning the effects of suction on skin and underlying tissue, and the reaction of the body to that stimulus. Understanding the literature on the physiological effects of this mechanical force may help elaborate an explanation for the advertised local and systemic effects of cupping therapy. Negative pressure causes stretching of the skin and underlying tissue and dilation of the capillaries. This stimulates an increase in tissue blood flow, eventually leading to capillary rupture and ecchymosis. Macrophages phagocytize the erythrocytes in the extravascular space which stimulates the production of Heme Oxygenase-1 (HO-1) to metabolize the heme. Heme catalysis results in the production of carbon monoxide (CO), biliverdin(BV)/bilirubin(BR) and iron. HO-1, BV, BR, and CO has been shown to have antioxidant, anti-inflammatory, antiproliferative, and neuromodulatory effects in animal and human systems. These substances also stimulate a shift of macrophages to the anti-inflammatory M2 phenotype. There is evidence that the effects are both local and systemic. Besides the mechanical effect of cupping increasing the local blood flow and stretching underlying tissue, activation of the HO-1 system could account for many of cupping therapy's claimed local and systemic health benefits. Published by Elsevier Ltd.

Identification of the altered pyrrole in the isomeric sulfmyoglobins: hyperfine shift patterns as indicators of ring saturation in ferric chlorins.

PubMed

Chatfield, M J; La Mar, G N; Smith, K M; Leung, H K; Pandey, R K

1988-03-08

Analysis of the 1H NMR hyperfine shift patterns of isomeric sulfmyoglobins is carried out in the met-aquo and met-cyano states to determine the site of saturation in each protein. The utility of the patterns for structure elucidation is established by specific deuterium labeling of the heme methyls of the terminal base product. On the basis of the known saturation of ring B in this isomer [Chatfield, M.J., La Mar, G.N., Lecomte, J.T.J., Balch, A.L., Smith, K.M., & Langry, K.C. (1986) J. Am. Chem. Soc. 108, 7108-7110], the methyl resonance of the saturated ring is found to have strongly attenuated contact shift. Thus, the heme methyl contact shift pattern is diagnostic for the saturated pyrrole in the high-spin state. This rationale is then applied to analyze the assigned NMR spectra of the initial and terminal acid sulfmyoglobin products, revealing that the same ring B is saturated in each isomer. In contrast, the heme methyl contact shift pattern in low-spin ferric complexes reveals that the methyls both on the affected pyrrole and on the trans pyrrole are influenced similarly on sulfmyoglobin formation, precluding the use of this methyl shift pattern as a unique indicator of the site of saturation. Identification of exchangeable proximal histidine resonances for met-aquo sulfmyoglobin complexes with shifts similar to that in native myoglobin dictates inconsequential axial alterations in the sulfmyoglobins, while location of downfield meso proton resonances analogous to those of the native protein demonstrates the retention of the coordinate water in the active site of met-sulfmyoglobin.

An Ancient Relative of Cyclooxygenase in Cyanobacteria Is a Linoleate 10S-Dioxygenase That Works in Tandem with a Catalase-related Protein with Specific 10S-Hydroperoxide Lyase Activity*

PubMed Central

Brash, Alan R.; Niraula, Narayan P.; Boeglin, William E.; Mashhadi, Zahra

2014-01-01

In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as “cyclooxygenase-2,” appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate. PMID:24659780

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells.

PubMed

Foresti, Roberta; Bains, Sandip K; Pitchumony, Tamil Selvi; de Castro Brás, Lisandra E; Drago, Filippo; Dubois-Randé, Jean-Luc; Bucolo, Claudio; Motterlini, Roberto

2013-10-01

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Monomeric Yeast Frataxin is an Iron-Binding Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook,J.; Bencze, K.; Jankovic, A.

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly)more » share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Monomeric Yeast Frataxin is an Iron Binding Protein†

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, J.; Bencze, K; Jankovic, A

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) sharemore » requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

PubMed

Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

1989-12-08

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains.

PubMed

Lopes Ferreira, Nicolas; Mathis, Hugues; Labbé, Diane; Monot, Frédéric; Greer, Charles W; Fayolle-Guichard, Françoise

2007-06-01

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C2 to C16). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C1 to C16) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.

Exposing the Complex III Qo semiquinone radical

PubMed Central

Zhang, Haibo; Osyczka, Artur; Dutton, P. L.; Moser, Christopher C.

2012-01-01

Complex III Qo site semiquinone has been assigned pivotal roles in productive energy-conversion and destructive superoxide generation. After a 30 year search, a genetic heme bH knockout arrests this transient semiquinone EPR radical, revealing the natural engineering balance pitting energy-conserving, short-circuit minimizing, split electron transfer and catalytic speed against damaging oxygen reduction. PMID:17560537

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

PubMed Central

Mitra, Avishek; Speer, Alexander; Lin, Kan; Ehrt, Sabine

2017-01-01

ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. PMID:28119467

Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

PubMed

Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

2015-05-01

Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, Inc.

Defense mechanism of heme oxygenase-1 against cytotoxic and receptor activator of nuclear factor-kappaB ligand inducing effects of hydrogen peroxide in human periodontal ligament cells.

PubMed

Pi, S-H; Kim, S-C; Kim, H-T; Lee, H-J; Lee, S-K; Kim, E-C

2007-08-01

Although induction of heme oxygenase-1 by H2O2 has been reported, the protective role of heme oxygenase-1 against the cytotoxic and osteoclastogenic effects of H2O2 have not been elucidated in human periodontal ligament cells. The aim of this work was to investigate the defense mechanism of heme oxygenase-1 on H2O2-induced cytotoxicity and to analyze the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin as markers for osteoclast differentiation in periodontal ligament cells. Using human periodontal ligament cells, cytotoxicity was measured by the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay, and expression of heme oxygenase-1, RANKL, and osteoprotegerin mRNA was determined by reverse transcription-polymerase chain reaction. H2O2 produced a cytotoxic effect by reducing the cell viability and enhancing the expression of heme oxygenase-1 and RANKL mRNAs in a concentration- and time-dependent manner. Additional experiments revealed that heme oxygenase-1 inducer (hemin), a membrane-permeable cGMP analog (8-bromo-cGMP), carbon monoxide, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase inhibitor, protein kinase inhibitor (KT5823), and nuclear factor-kappaB inhibitor (pyrrolidine dithiocarbamate) also blocked the effects of H2O2 on cell viability and RANKL mRNA expression in periodontal ligament cells. These data suggest that heme oxygenase-1 induction plays a protective role in periodontal ligament cells against the cytotoxic and RANKL-inducing effects of H2O2, through multiple signaling pathways.

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

PubMed Central

Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

2014-01-01

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

Heme Derived from Corynebacterium glutamicum: A Potential Iron Additive for Swine and an Electron Carrier Additive for Lactic Acid Bacterial Culture.

PubMed

Choi, Su-In; Park, Jihoon; Kim, Pil

2017-03-28

To investigate the potential applications of bacterial heme, aminolevulinic acid synthase (HemA) was expressed in a Corynebacterium glutamicum HA strain that had been adaptively evolved against oxidative stress. The red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. To investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain in a 5 ton fermenter followed by spray-drying the biomass with flour as an excipient (biomass: flour = 1:10 (w/w)). The 10% prototype additive along with regular feed was supplied to a pig, resulting in a 1.1 kg greater increase in weight gain with no diarrhea in 3 weeks as compared with that in a control pig that was fed an additive containing only flour. To verify if C. glutamicum -synthesized heme is a potential electron carrier, lactic acid bacteria were cultured under aerobic conditions with the extracted heme. The biomasses of the aerobically grown Lactococcus lactis , Lactobacillus rhamosus , and Lactobacillus casei were 97%, 15%, and 4% greater, respectively, than those under fermentative growth conditions. As a potential preservative, cultures of the four strains of lactic acid bacteria were stored at 4°C with the extracted heme and living lactic acid bacterial cells were counted. There were more L. lactis and L. plantarum live cells when stored with heme, whereas L. rhamosus and L. casei showed no significant differences in live-cell numbers. The potential uses of the heme from C. glutamicum are further discussed.

Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

PubMed

Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

2009-10-15

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

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

PubMed Central

2016-01-01

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

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release*

PubMed Central

Caillet-Saguy, Célia; Piccioli, Mario; Turano, Paola; Lukat-Rodgers, Gudrun; Wolff, Nicolas; Rodgers, Kenton R.; Izadi-Pruneyre, Nadia; Delepierre, Muriel; Lecroisey, Anne

2012-01-01

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His32 and Tyr75, respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr75-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins. PMID:22700962

Oxygen binding to partially nitrosylated hemoglobin.

PubMed

Fago, Angela; Crumbliss, Alvin L; Hendrich, Michael P; Pearce, Linda L; Peterson, Jim; Henkens, Robert; Bonaventura, Celia

2013-09-01

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group*

PubMed Central

Auer, Markus; Gruber, Clemens; Bellei, Marzia; Pirker, Katharina F.; Zamocky, Marcel; Kroiss, Daniela; Teufer, Stefan A.; Hofbauer, Stefan; Soudi, Monika; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Obinger, Christian

2013-01-01

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role. PMID:23918925

The interaction of heme with plakortin and a synthetic endoperoxide analogue: new insights into the heme-activated antimalarial mechanism

NASA Astrophysics Data System (ADS)

Persico, Marco; Fattorusso, Roberto; Taglialatela-Scafati, Orazio; Chianese, Giuseppina; de Paola, Ivan; Zaccaro, Laura; Rondinelli, Francesca; Lombardo, Marco; Quintavalla, Arianna; Trombini, Claudio; Fattorusso, Ernesto; Fattorusso, Caterina; Farina, Biancamaria

2017-04-01

In the present work we performed a combined experimental and computational study on the interaction of the natural antimalarial endoperoxide plakortin and its synthetic analogue 4a with heme. Obtained results indicate that the studied compounds produce reactive carbon radical species after being reductively activated by heme. In particular, similarly to artemisinin, the formation of radicals prone to inter-molecular reactions should represent the key event responsible for Plasmodium death. To our knowledge this is the first experimental investigation on the reductive activation of simple antimalarial endoperoxides (1,2-dioxanes) by heme and results were compared to the ones previously obtained from the reaction with FeCl2. The obtained experimental data and the calculated molecular interaction models represent crucial tools for the rational optimization of our promising class of low-cost synthetic antimalarial endoperoxides.

Comparison of the Heme Iron Utilization Systems of Pathogenic Vibrios

PubMed Central

O’Malley, S. M.; Mouton, S. L.; Occhino, D. A.; Deanda, M. T.; Rashidi, J. R.; Fuson, K. L.; Rashidi, C. E.; Mora, M. Y.; Payne, S. M.; Henderson, D. P.

1999-01-01

Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae. PMID:10348876

Heme impairs the ball-and-chain inactivation of potassium channels.

PubMed

Sahoo, Nirakar; Goradia, Nishit; Ohlenschläger, Oliver; Schönherr, Roland; Friedrich, Manfred; Plass, Winfried; Kappl, Reinhard; Hoshi, Toshinori; Heinemann, Stefan H

2013-10-15

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Amomum tsao-ko suppresses lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages via Nrf2-dependent heme oxygenase-1 expression.

PubMed

Li, Bin; Choi, Hee-Jin; Lee, Dong-Sung; Oh, Hyuncheol; Kim, Youn-Chul; Moon, Jin-Young; Park, Won-Hwan; Park, Sun-Dong; Kim, Jai-Eun

2014-01-01

Amomum tsao-ko Crevost et Lemaire, used as a spice in Asia, is an important source of Chinese cuisine and traditional Chinese medicines. A. tsao-ko is reported to exert a variety of biological and pharmacological activities, including anti-proliferative, anti-oxidative and neuroprotective effects. In this study, NNMBS227, consisting of the ethanol extract of A. tsao-ko, exhibited potent anti-inflammatory activities in RAW264.7 macrophages. We investigated the effect of NNMBS227 in the suppression of pro-inflammatory mediators, including pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-1β) in LPS stimulated macrophages. NNMBS227 also inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of nuclear factor kappa B (NF-κB) p65 caused by stimulation with LPS. In addition, NNMBS227 induced heme oxygenase (HO)-1 expression through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in macrophages. Using tin protoporphyrin (SnPP), an HO activity inhibitor, we confirmed an association between the anti-inflammatory effects of NNMBS227 and the up-regulation of HO-1. These findings suggest that Nrf2-dependent increases in the expression of HO-1 induced by NNMBS227 conferred anti-inflammatory activities in LPS stimulated RAW264.7 macrophages.

Chlorite dismutases – a heme enzyme family for use in bioremediation and generation of molecular oxygen

PubMed Central

Hofbauer, Stefan; Schaffner, Irene; Furtmüller, Paul G; Obinger, Christian

2014-01-01

Chlorite is a serious environmental concern, as rising concentrations of this harmful anthropogenic compound have been detected in groundwater, drinking water, and soil. Chlorite dismutases (Clds) are therefore important molecules in bioremediation as Clds catalyze the degradation of chlorite to chloride and molecular oxygen. Clds are heme b-containing oxidoreductases present in numerous bacterial and archaeal phyla. This review presents the phylogeny of functional Clds and Cld-like proteins, and demonstrates the close relationship of this novel enzyme family to the recently discovered dye-decolorizing peroxidases. The available X-ray structures, biophysical and enzymatic properties, as well as a proposed reaction mechanism, are presented and critically discussed. Open questions about structure-function relationships are addressed, including the nature of the catalytically relevant redox and reaction intermediates and the mechanism of inactivation of Clds during turnover. Based on analysis of currently available data, chlorite dismutase from “Candidatus Nitrospira defluvii” is suggested as a model Cld for future application in biotechnology and bioremediation. Additionally, Clds can be used in various applications as local generators of molecular oxygen, a reactivity already exploited by microbes that must perform aerobic metabolic pathways in the absence of molecular oxygen. For biotechnologists in the field of chemical engineering and bioremediation, this review provides the biochemical and biophysical background of the Cld enzyme family as well as critically assesses Cld's technological potential. PMID:24519858

Carnosic acid attenuates cartilage degeneration through induction of heme oxygenase-1 in human articular chondrocytes.

PubMed

Ishitobi, Hiroyuki; Sanada, Yohei; Kato, Yoshio; Ikuta, Yasunari; Shibata, Sachi; Yamasaki, Satoshi; Lotz, Martin K; Matsubara, Kiminori; Miyaki, Shigeru; Adachi, Nobuo

2018-04-17

Osteoarthritis (OA) is common age-associated disease, and associated with joint pain, mobility limitations and compromised overall quality of life. OA treatment is currently limited to pain management and joint arthroplasty at end stage disease. Oxidative damage to cartilage extracellular matrix and cells is an important mechanism in joint aging and OA pathogenesis. Evidence from in vitro and in vivo models of OA suggests that pharmaceuticals and natural compounds with antioxidant properties reduce expression of mediators of OA pathogenesis and OA severity in animal models. Among the signaling pathways that control cellular protective mechanisms against oxygen radical damage is heme oxygenase-1 (HO-1). We recently report HO-1 reduced OA severity in a mouse model. This led to the hypothesis that compounds that increase HO-1 expression have therapeutic potential in OA. Carnosic acid (CA), a natural diterpene with oxidant activity, is prevents cartilage degeneration though induction of HO-1. CA induced HO-1 and miR-140 expression in human articular chondrocytes, and cartilage degeneration was attenuated by CA treatment. Induced HO-1 by CA was in part associated with downregulation via miR-140 binding to 3'UTR of BTB and CNC homology 1 (BACH1). These findings suggest that CA attenuates cartilage degradation through HO-1 upregulation and has potential as a supplement for OA prevention. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons.

PubMed

Singh, Neha; Bhatla, Satish C

2016-02-29

Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29 kDa) of HO-1. NaCl-modulated HO-1 activity correlates with endogenous NO content in the cotyledons. Increased NO accumulation by hemin treatment also correlates with enhanced activity of HO-1 in both control and NaCl stress conditions. Present work indicates that NO positively modulates HO-1 activity in sunflower seedling cotyledons. NaCl stress tends to antagonize NO action on HO-1 activity. NO (from sodium nitroprusside; SNP) is probably positively modulating HO-1 activity by way of its interaction/binding with heme group. Present work also shows enhanced NO accumulation in seedling cotyledons both in the absence or presence of iron in the growth medium, in response to NaCl stress. Thus, a probable link between endogenous NO, NaCl stress and iron-homeostasis by way of modulation of HO-1 activity at early stage of sunflower seedling growth has been proposed. Copyright © 2016 Elsevier Inc. All rights reserved.

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy

DOE PAGES

Levantino, M.; Lemke, H. T.; Schirò, G.; ...

2015-07-01

We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (~70 fs) relaxation preceding a slower (~400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

Direct observation of vibrational energy flow in cytochrome c.

PubMed

Fujii, Naoki; Mizuno, Misao; Mizutani, Yasuhisa

2011-11-10

Vibrational energy flow in ferric cytochrome c has been examined by picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) measurements. By taking advantage of the extremely short nonradiative excited state lifetime of heme in the protein (< ps), excess vibrational energy of 20000-25000 cm(-1) was optically deposited selectively at the heme site. Subsequent energy relaxation in the protein moiety was investigated by monitoring the anti-Stokes UVRR intensities of the Trp59 residue, which is a single tryptophan residue involved in the protein that is located close to the heme group. It was found from temporal changes of the anti-Stokes UVRR intensities that the energy flow from the heme to Trp59 and the energy release from Trp59 took place with the time constants of 1-3 and ~8 ps, respectively. These data are consistent with the time constants for the vibrational relaxation of the heme and heating of water reported for hemeproteins. The kinetics of the energy flow were not affected by the amount of excess energy deposited at the heme group. These results demonstrate that the present technique is a powerful tool for studying the vibrational energy flow in proteins.

Heme exporter FLVCR is required for T cell development and peripheral survival.

PubMed

Philip, Mary; Funkhouser, Scott A; Chiu, Edison Y; Phelps, Susan R; Delrow, Jeffrey J; Cox, James; Fink, Pamela J; Abkowitz, Janis L

2015-02-15

All aerobic cells and organisms must synthesize heme from the amino acid glycine and the tricarboxylic acid cycle intermediate succinyl CoA for incorporation into hemoproteins, such as the cytochromes needed for oxidative phosphorylation. Most studies on heme regulation have been done in erythroid cells or hepatocytes; however, much less is known about heme metabolism in other cell types. The feline leukemia virus subgroup C receptor (FLVCR) is a 12-transmembrane domain surface protein that exports heme from cells, and it was shown to be required for erythroid development. In this article, we show that deletion of Flvcr in murine hematopoietic precursors caused a complete block in αβ T cell development at the CD4(+)CD8(+) double-positive stage, although other lymphoid lineages were not affected. Moreover, FLVCR was required for the proliferation and survival of peripheral CD4(+) and CD8(+) T cells. These studies identify a novel and unexpected role for FLVCR, a major facilitator superfamily metabolite transporter, in T cell development and suggest that heme metabolism is particularly important in the T lineage. Copyright © 2015 by The American Association of Immunologists, Inc.

Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid.

PubMed

Pan, Yadi; Yang, Yufang; Pang, Yanjiao; Shi, Ying; Long, Yijuan; Zheng, Huzhi

2018-08-01

Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 μM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase. Copyright © 2018 Elsevier B.V. All rights reserved.