Iron transfer from transferrin to ferritin mediated by pyrophosphate

There is no significant iron exchange from transferrin to ferritin in the absence of reducing and chelating agents. Pyrophosphate can release iron from transferrin and can be isolated as a ferric pyrophosphate complex by ion exchange chromatography. We have established that pyrophosphate alone can mediate iron exchange from transferrin to ferritin. Under these conditions, iron is incorporated directly into ferritin as Fe(III).     

Assimilatory ferric reductases in enterococci

Enterococci produced assimilatory ferric reductases which are surface-associated enzymes. This is the first report of the intracellular enzymic reduction of iron by enterococci. A correlation between ferric reductases activity and species affiliation and origin of strains was found. The expression of ferric reductases has not affected by the presence or absence of iron, hemin and hemoglobin in the growth medium. Enterococcal ferric reductases exhibit a very broad specificity. A number of different ferric organic and inorganic compounds, natural and synthetic iron chelators and iron body sources including lactoferrin, transferin, ferritin, haemoglobin, could be reduced. A surface-associated ferric reductases may be one component of a general iron scavenging mechanism which can be used by enterococci growing in a variety of environments.     

The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2–5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe²⁺ and phosphate. In the absence of ferritin, phosphate catalyzed Fe²⁺ oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10–20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe²⁺. Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe²⁺ and facilitate the transfer of the Fe³⁺ into apo transferrin in the presence of phosphate.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

The identification and biosynthesis of siderochromes formed by Micrococcus denitrificans.

1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.     

Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.

The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.     

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.     

Iron Oxidation and Deposition in the Biofilm Covering Montacuta ferruginosa (Mollusca,Bivalvia)

The shell of the bivalve Montacuta ferruginosa is covered with a rust-colored biofilm. This biofilm includes filamentous bacteria and protozoa encrusted with a mineral, rich in ferric ion and phosphate. The aim of this research was to study two possible microbial iron precipitation pathways in the biofilm, namely, microbial iron oxidation and microbial degradation of organic Fe(III) complexes. The iron-oxidizing activity was assayed spectrophotometrically by monitoring the formation of the dye Wurster blue in biofilm extracts. Iron-oxidizing activity was effectively detected in extracts obtained by oxalic acid treatment of biofilm fragments. Extracts obtained without oxalic acid treatment, heated extracts, or extracts supplemented with HgCl 2 did not show any activity. This suggests that an iron-oxidizing factor (IOF), possibly an enzyme, coprecipitated with the mineral. Additional information gathered by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel-filtration chromatography, and UV spectrophotometry indicate that the IOF would be a small peptide or glycopeptide (1,350 Da). Microbial degradation of organic Fe(III) complexes was assayed with biofilm fragments incubated in a medium containing ferric citrate. Analysis of the supernatants after various intervals revealed that the complex was degraded by living microorganisms much faster than in the heat-killed negative controls. We conclude that ferric iron precipitation in the biofilm may proceed by way of microbial Fe(II) oxidation as well as microbial degradation of organic Fe(III) complexes.     

The relationship between iron release, ferritin synthesis and intracellular iron distribution in mouse peritoneal macrophages. Evidence for a reduced level of metabolically available iron in elicited macrophages

The rate of iron release from thioglycollate-elicited mouse peritoneal macrophages pulsed with 59Fe-labelled transferrin-antitransferrin immune complexes was lower than that from resident or Corynebacterium parvum-activated macrophages. Anaerobic conditions increased the rate of iron release by thioglycollate-elicited macrophages but had no effect on resident or C. parvum-activated macrophages. Thioglycollate-elicited macrophages also contained less ferritin and were deficient in their ability to synthesis ferritin. Incubation of these cells in medium containing 100 microM iron caused some increase in ferritin synthesis, but the response to iron was much less pronounced than that by resident or C. parvum-activated macrophages. In the thioglycollate-elicited macrophages, relatively less iron was incorporated into ferritin, and more into other soluble macromolecules and insoluble haemosiderin-like compounds than in the other types of macrophages. It is proposed that thioglycollate-elicited macrophages tend to divert iron to a relatively inert intracellular pool, and that this could account for their reduced ability to release iron. Such a mechanism might help to explain the reduced release of iron by liver and spleen macrophages occurring during inflammation.     

Siderophore-mediated iron (III) transport in the mycelia of the cultivated fungus, Agaricus bisporus.

Three structurally diverse iron (III) sequestering compounds (siderophores) were isolated from the supernatants of early stationary phase iron-deficient cultures of vegetative mycelia of the cultivated mushroom, Agaricus bisporus (ATCC 36416). The compounds were purified as their ferric chelates to homogeneity by gel permeation, cation exchange, and low-pressure reversed phase C18 chromatographies, and characterized as trihydroxamic acids. The chelates were identified as ferrichrome, ferric fusarinine C, and an unusual compound, des (diserylglycyl) ferrirhodin (DDF) by HPTLC cochromatography and electrophoresis against authentic samples, hydrolysis and amino acid analysis, and FAB-MS and 1H NMR spectroscopy. The iron transport activities of the three compounds (and of some structurally similar exogenous compounds) in young mycelial cells were determined by time- and concentration-dependent kinetic assays and inhibition experiments (CN-, N3-) using 55Fe(3+)-labeled chelates. 55Iron (III) uptake mediated by all three compounds was found to be via high affinity, energy-dependent processes; transport effectiveness was in the order: ferrichrome > DDF > ferric fusarinine C. The relative uptake of iron by lambda-cis ferrichromes was: ferrichrome > ferrirhodin > ferrichrome A; transport activity by the delta-cis fusarinines was: ferric fusarinine C > tris cis-(and trans-) fusarinine iron (III) > ferric N1-triacetylfusarinine C.     

Iron gathering by zoopathogenic fungi

Iron is a metal required by most microorganisms and is prominently used in the transfer of electrons during metabolism. The gathering of iron is, then, an essential process and its fulfillment becomes a crucial pathogenetic event for zoopathogenic fungi. Iron is rather unavailable because it occurs on the earth's surface in its insoluble ferric form in oxides and hydroxides. In the infected host iron is bound to proteins such as transferrin and ferritin. Solubilization of ferric iron is the major problem confronting microorganisms. This process is achieved by two major mechanisms: ferric reduction and siderophore utilization. Ferric reductase is frequently accompanied by a copper oxidase transport system. There is one example of direct ferric iron transport apparently without prior reduction. Ferric reduction may also be accomplished by low molecular mass compounds. Some fungi have evolved a process of iron acquisition involving the synthesis of iron-gathering compounds called siderophores. Even those fungi that do not synthesize siderophores have developed permeases for transport of such compounds formed by other organisms. Fungi can also reductively release iron from siderophores and transport the ferrous iron often by the copper oxidase transport system. There is a great diversity of iron-gathering mechanisms expressed by pathogenic fungi and such diversity may be found even in a single species.     

Hypoxia Alters Iron Homeostasis and Induces Ferritin Synthesis in Oligodendrocytes

Abstract: Both iron and the major iron-binding protein ferritin are enriched in oligodendrocytes compared with astrocytes and neurons, but their functional role remains to be determined. Progressive hypoxia dramatically induces the synthesis of ferritin in both neonatal rat oligodendrocytes and a human oligodendroglioma cell line. We now report that the release of iron from either transferrin or ferritin-bound iron, after a decrease in intracellular pH, also leads to the induction of ferritin synthesis. The hypoxic induction of ferritin synthesis can be blocked either with iron chelators (deferoxamine or phenanthroline) or by preventing intracellular acidification (which is required for the release of transferrin-bound iron) with weak base treatment (ammonium chloride and amantadine). Two sources of exogenous iron (hemin and ferric ammonium citrate) were able to stimulate ferritin synthesis in both oligodendrocytes and HOG in the absence of hypoxia. This was not additive to the hypoxic stimulation, suggesting a common mechanism. We also show that ferritin induction may require intracellular free radical formation because hypoxia-mediated ferritin synthesis can be further enhanced by cotreatment with hydrogen peroxide. This in turn was blocked by the addition of exogenous catalase to the culture medium. Our data suggest that disruption of intracellular free iron homeostasis is an early event in hypoxic oligodendrocytes and that ferritin may serve as an iron sequestrator and antioxidant to protect cells from subsequent iron-catalyzed lipid peroxidation injury.     

Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane

In high-affinity iron uptake in the yeast Saccharomyces cerevisiae, Fe(II) is oxidized to Fe(III) by the multicopper oxidase, Fet3p, and the Fe(III) produced is transported into the cell via the iron permease, Ftr1p. These two proteins are likely part of a heterodimeric or higher order complex in the yeast plasma membrane. We provide kinetic evidence that the Fet3p-produced Fe(III) is trafficked to Ftr1p for permeation by a classic metabolite channeling mechanism. We examine the (59)Fe uptake kinetics for a number of complexes containing mutant forms of both Fet3p and Ftr1p and demonstrate that a residue in one protein interacts with one in the other protein along the iron trafficking pathway as would be expected in a channeling process. We show that, as a result of some of these mutations, iron trafficking becomes sensitive to an added Fe(III) chelator that inhibits uptake in a strictly competitive manner. This inhibition is not strongly dependent on the chelator strength, however, suggesting that Fe(III) dissociation from the iron uptake complex, if it occurs, is kinetically slow relative to iron permeation. Metabolite channeling is a common feature of multifunctional enzymes. We constructed the analogous ferroxidase, permease chimera and demonstrate that it supports iron uptake with a kinetic pattern consistent with a channeling mechanism. By analogy to the Fe(III) trafficking that leads to the mineralization of the ferritin core, we propose that ferric iron channeling is a conserved feature of iron homeostasis in aerobic organisms.     

Changes in the characteristics and distribution of ferritin in iron-loaded cell cultures.

When Chang liver cells are grown in an iron-rich medium for up to 20 weeks, iron loading up to 50 times the normal cellular iron content may be obtained, although ferritin increases only to about 10 times normal. Ferritin has been isolated from such cells, and the isoferritin pattern found on elution from DEAE-Sephadex A-50 by increasing chloride concentrations has been used as a basis for studying changes in the properties of ferritin under conditions of cellular loading. A consistent shift of peak ferritin-elution position to higher chloride concentrations (lower pI) occurs when cells are loaded with ferric nitrilotriacetate for increasing lengths of time. A change in immunoreactivity also takes place on loading, the ratio of ferritin reacting with heart and spleen ferritin antibodies increasing at any particular value of pI. Cells were pulse-labelled with [59Fe]ferric nitrilotriacetate and [3H]leucine followed by non-radioactive iron in the same form. During the 72 h after the synthesis of new protein and its incorporation of iron, there is a slight acid shift in its isoelectric point. This effect is seen in both normal and loaded cells, with the whole spectrum being shifted towards lower pI in the loaded state. These findings suggest that the shift to more acidic ferritins on iron loading and the associated changes in antigenicity may be unrelated to subunit composition.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

N-hydroxyguanidines as new heme ligands: UV-visible, EPR, and resonance Raman studies of the interaction of various compounds bearing a C=NOH function with microperoxidase-8

Interaction between microperoxidase-8 (MP8), a water-soluble hemeprotein model, and a wide range of N-aryl and N-alkyl N'-hydroxyguanidines and related compounds has been investigated using UV-visible, EPR, and resonance Raman spectroscopies. All the N-hydroxyguanidines studied bind to the ferric form of MP8 with formation of stable low-spin iron(III) complexes characterized by absorption maxima at 405, 535, and 560 nm. The complex obtained with N-(4-methoxyphenyl) N'-hydroxyguanidine exhibits EPR g-values at 2.55, 2.26, and 1.86. The resonance Raman (RR) spectrum of this complex is also in agreement with an hexacoordinated low-spin iron(III) structure. The dissociation constants (K(s)) of the MP8 complexes with mono- and disubstituted N-hydroxyguanidines vary between 15 and 160 microM at pH 7.4. Amidoximes also form low-spin iron(III) complexes of MP8, although with much larger dissociation constants. Under the same conditions, ketoximes, aldoximes, methoxyguanidines, and guanidines completely fail to form such complexes with MP8. The K(s) values of the MP8-N-hydroxyguanidine complexes decrease as the pH of the solution is increased, and the affinity of the N-hydroxyguanidines toward MP8 increases with the pK(a) of these ligands. Altogether these results show that compounds involving a -C(NHR)=NOH moiety act as good ligands of MP8-Fe(III) with an affinity that depends on the electron-richness of this moiety. The analysis of the EPR spectrum of the MP8-N-hydroxyguanidine complexes according to Taylor's equations shows a strong axial distortion of the iron, typical of those observed for hexacoordinated heme-Fe(III) complexes with at least one pi donor axial ligand (HO(-), RO(-), or RS(-)). These data strongly suggest that N-hydroxyguanidines bind to MP8 iron via their oxygen atom after deprotonation or weakening of their O-H bond. It thus seems that N-hydroxyguanidines could constitute a new class of strong ligands for hemeproteins and iron(III)-porphyrins.     

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron.     

Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction

In order to identify an enzyme capable of Fenton reaction in Synechocystis, we purified an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol39, 751-755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the presence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage protein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism.     

Synthesis of ferritin by neuroblastoma

Ferritin is an iron-containing protein which is a normal component of serum. The levels of ferritin are increased in the sera of some children with neuroblastoma, and this increase appears to be a potent indicator of prognosis. To determine whether synthesis of ferritin by the tumor cells contributes to these increased serum levels, we examined incorporation of radiolabeled leucine by CHP 126, a neuroblastoma derived cell line, into ferritin. Using sequential immunoprecipitation and gel electrophoresis of sonicates from cells maintained in medium containing iron in amounts standard for tissue culture, incorporation of label into ferritin was 0.04% of that into total protein synthesized over the same time period. Addition of up to 40 micrograms of iron as ferric ammonium citrate increased ferritin synthesis to a maximum of 0.16% without altering synthesis of total protein. The pattern of iron-induced enhancement in the neuroblastoma cells was similar to that which was seen using Chang liver cells, a cell line well known to be capable of ferritin synthesis. These results confirm that neuroblastoma cells can synthesize ferritin and that synthesis is regulated by exogenous iron.