Ferric iron reductases and their contribution to unicellular ferrous iron uptake

ABSTRACTIron acquisition is critical to the growth and virulence ofLegionella pneumophila. Previously, we found thatL. pneumophilauses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted byL. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability ofL. pneumophilaand other species ofLegionellato take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing ofL. pneumophilaculture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis ofL. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

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Characterization and partial purification of a ferrireductase from human duodenal microvillus membranes

Biochemical Journal ◽

10.1042/bj3090745 ◽

1995 ◽

Vol 309 (3) ◽

pp. 745-748 ◽

Cited By ~ 58

Author(s):

H D Riedel ◽

A J Remus ◽

B A Fitscher ◽

W Stremmel

Keyword(s):

Ferrous Iron ◽

Iron Uptake ◽

Plasma Membranes ◽

Ferric Iron ◽

Reductase Activity ◽

Nitrilotriacetic Acid ◽

Iron Chelator ◽

Partial Purification ◽

Uptake System ◽

Ammonium Sulphate Precipitation

Reduction of ferric iron in the presence of HuTu 80 cells or duodenal microvillus membranes (MVMs) was investigated. With both systems, NADH-dependent reduction of Fe3+/NTA (nitrilotriacetic acid) was demonstrated, using the ferrous iron chelator ferrozine. Uptake of Fe3+ from Fe3+/NTA by HuTu 80 cells was strongly inhibited by addition of ferrozine, indicating that Fe2+ is the substrate for the iron uptake system. With isolated plasma membranes it is shown that the reductase activity is sensitive to trypsin and incubation at 65 degrees C. The reductase activity could be extracted from the plasma membrane and partially purified by ammonium sulphate precipitation and isoelectric focusing. From the purification and inhibition characteristics we conclude that reduction of ferric iron on the surface of duodenal plasma membranes is catalysed by a membrane protein.

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Role of copper in mitochondrial iron metabolism

Blood ◽

10.1182/blood.v48.1.77.77 ◽

1976 ◽

Vol 48 (1) ◽

pp. 77-85 ◽

Cited By ~ 66

Author(s):

DM Williams ◽

D Loukopoulos ◽

GR Lee ◽

GE Cartwright

Keyword(s):

Cytochrome Oxidase ◽

Iron Metabolism ◽

Oxidase Activity ◽

Ferrous Iron ◽

Iron Uptake ◽

Ferric Iron ◽

Intracellular Iron ◽

Cytochrome Oxidase Activity ◽

Heme Synthesis

Abstract Heme synthesis by copper-deficient cells was investigated to elucidate the nature of the defect in intracellular iron metabolism. Iron uptake from transferrin by copper-deficient reticulocytes was 52% of normal, and the rate of heme synthesis was 33% of normal. Hepatic mitochondria isolated from copper-deficient animals were deficient in cytochrome oxidase activity and failed to synthesize heme from ferric iron (Fe III) and protoporphyrin at the normal rate. The rate of heme synthesis correlated with the cytochrome oxidase activity. Heme synthesis from Fe(III) and protoporphyrin by normal mitochondria was enhanced by succinate and inhibited by malonate, antimycin A, azide, and cyanide. It is proposed that an intact electron transport system is required for the reduction of Fe(III), thereby providing a pool of ferrous iron (Fe II) for protoheme and heme a synthesis.

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Effect of iron chelators on placental uptake and transfer of iron in rat

AJP Cell Physiology ◽

10.1152/ajpcell.1987.252.5.c477 ◽

1987 ◽

Vol 252 (5) ◽

pp. C477-C482 ◽

Cited By ~ 14

Author(s):

C. T. Wong ◽

H. J. McArdle ◽

E. H. Morgan

Keyword(s):

Ferrous Iron ◽

Iron Uptake ◽

Iron Transport ◽

Ferric Iron ◽

Cultured Cells ◽

Iron Chelator ◽

Cell Matrix ◽

Intracellular Vesicles ◽

Iron Excretion ◽

Cycling Time

The uptake of radiolabelled transferrin and iron by the rat placenta has been studied using two approaches. The first involved injection of a ferrous or ferric iron chelator followed by injection of label. Neither chelator decreased the amount of labelled transferrin in the placenta after 2-h incubation and only bipyridine, a ferrous iron chelator, inhibited iron transport to the fetus. Deferoxamine (DFO), a ferric iron chelator, had no effect on iron transport to the fetus but reduced iron uptake by the liver. Both bipyridine and DFO increased iron excretion into the gut and by the urinary tract to the same degree into the gut, but there was a 10-fold greater urinary excretion with bipyridine than with DFO. Injection of iron attached to the chelators showed that neither bipyridine nor DFO could donate iron to the fetus as efficiently as transferrin. The mechanism involved was further investigated by studying the effect of the chelators on uptake of transferrin-bound iron by placental cells in culture. DFO inhibited iron accumulation more effectively than bipyridine in the cultured cells. The effect was not due to a decrease in the cycling time of the receptor. The results can be explained if the iron is released from the transferrin in intracellular vesicles in the ferrous form, where it may be chelated by bipyridine and prevented from passing to the fetus or converted to the ferric form once it is inside the cell matrix.

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The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain

Microbiology ◽

10.1099/mic.0.072835-0 ◽

2014 ◽

Vol 160 (2) ◽

pp. 446-457 ◽

Cited By ~ 11

Author(s):

Girija Ramakrishnan ◽

Bhaswati Sen

Keyword(s):

Outer Membrane ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Ferrous Iron ◽

Iron Uptake ◽

Ferric Iron ◽

Live Vaccine ◽

Live Vaccine Strain ◽

High Affinity ◽

Schu S4

Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation.

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Role of copper in mitochondrial iron metabolism

Blood ◽

10.1182/blood.v48.1.77.bloodjournal48177 ◽

1976 ◽

Vol 48 (1) ◽

pp. 77-85 ◽

Cited By ~ 1

Author(s):

DM Williams ◽

D Loukopoulos ◽

GR Lee ◽

GE Cartwright

Keyword(s):

Cytochrome Oxidase ◽

Iron Metabolism ◽

Oxidase Activity ◽

Ferrous Iron ◽

Iron Uptake ◽

Ferric Iron ◽

Intracellular Iron ◽

Cytochrome Oxidase Activity ◽

Heme Synthesis

Heme synthesis by copper-deficient cells was investigated to elucidate the nature of the defect in intracellular iron metabolism. Iron uptake from transferrin by copper-deficient reticulocytes was 52% of normal, and the rate of heme synthesis was 33% of normal. Hepatic mitochondria isolated from copper-deficient animals were deficient in cytochrome oxidase activity and failed to synthesize heme from ferric iron (Fe III) and protoporphyrin at the normal rate. The rate of heme synthesis correlated with the cytochrome oxidase activity. Heme synthesis from Fe(III) and protoporphyrin by normal mitochondria was enhanced by succinate and inhibited by malonate, antimycin A, azide, and cyanide. It is proposed that an intact electron transport system is required for the reduction of Fe(III), thereby providing a pool of ferrous iron (Fe II) for protoheme and heme a synthesis.

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Olfactory ferric and ferrous iron absorption in iron-deficient rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00004.2012 ◽

2012 ◽

Vol 302 (12) ◽

pp. L1280-L1286 ◽

Cited By ~ 8

Author(s):

V. M. Ruvin Kumara ◽

Marianne Wessling-Resnick

Keyword(s):

Nasal Cavity ◽

Ferrous Iron ◽

Iron Uptake ◽

Ferric Iron ◽

Iron Absorption ◽

Iron Status ◽

Occupational Exposures ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter ◽

Iron Deficient

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood 59Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of 59Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade ( b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.

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Faculty Opinions recommendation of Vibrio cholerae VciB promotes iron uptake via ferrous iron transporters.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1120776.577025 ◽

2008 ◽

Author(s):

Simon Andrews

Keyword(s):

Vibrio Cholerae ◽

Ferrous Iron ◽

Iron Uptake

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Laser-Excited Fluorescence of Dityrosine

Applied Spectroscopy ◽

10.1366/0003702953965678 ◽

1995 ◽

Vol 49 (11) ◽

pp. 1669-1676 ◽

Cited By ~ 14

Author(s):

Sahar F. Mahmoud ◽

Stephen E. Bialkowski

Keyword(s):

Ferrous Iron ◽

Ferric Iron ◽

Iron Concentration ◽

Fluorescence Emission ◽

Fluorescence Yield ◽

Photomultiplier Tube ◽

Alkaline Solutions ◽

Array Detector ◽

Fluorescence Maximum ◽

Fluorescence Efficiency

In this research, laser-excited fluorescence was examined for sensitive detection of aqueous dityrosine. Samples were excited with a 6.3-mW, 325-nm helium-cadmium laser focused into a small volume-fluorescence cell with a 10-cm lens. The resulting fluorescence emission was collected perpendicular to the excitation and detected with two different schemes. An optical bandpass filter was used with a photomultiplier tube for sensitive quantitative measurement, while a photodiode array detector was used in conjunction with a spectrograph for qualitative characterization of fluorescence emission spectra. Dityrosine detection on the order of 2 × 10−11 M was obtained with the use of the photomultiplier tube with bandpass optical filter. The dityrosine fluorescence yield is found to vary with the solution pH, the relative concentrations of ferric and ferrous iron, and the amount of dissolved oxygen. A maximum fluorescence yield is observed for iron-free, oxygen-free alkaline solutions. Fluorescence quenching by oxygen is a cumulative photolysis effect that diminished fluorescence yield with increased irradiation time. Flowing the solutions minimized photolysis effects in oxygenated solutions. Quenching by ferrous and ferric iron is found to be due primarily to complex formation. The ferrous iron complex appears to have a fluorescence efficiency of ∼20% of the free dityrosine. The ferric iron dityrosine complex appears to have two ferric ions per molecule at low iron concentration. Other complexes may form at different concentrations. Solvent effects on dityrosine absorption and fluorescence spectra were also investigated. A red shift in dityrosine fluorescence maximum was observed in 1 M trichloroacetic acid and in N, N-dimethylformamide. The fluorescence emission maximum was shifted to the blue in acetonitrile and glacial acetic acid. These shifts were attributed to typical solvochromic behavior.

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