Reduction and release of ferritin iron by plant phenolics

The reductive release of ferritin iron by several naturally occurring o-diphenols was studied. The initial rate of iron release was quantified by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex, which absorbs maximally at 562 nm. The initial rate of iron release was dependent upon o-diphenol concentration, but not on the concentration of the chromophoric chelating agent, ferrozine, Stoichiometric measurements resulted in a ratio of 2Fe(II) released per molecule of o-diphenol. The series of o-diphenols studied included, caffeic acid, chlorogenic acid, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, and several analogs. These reductants represent an oxidation reduction potential range of 0.38 volts. A direct correlation between reducing power of the o-diphenols and rate of ferritin iron release was observed. Superoxide dismutase, catalase, mannitol, or general radical traps had no effect on the rate of iron removal; however, EDTA and oxalate inhibited iron release. A mechanism for ferritin iron reduction and release by o-diphenols consistent with the experimental observations is discussed.     

Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The initial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with Vmax = 2.0 X 10(-7) M.min-1 and KM = 1.3 X 10(-3) M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Iron mobilization from ferritin by chelating agents

The release of iron from horse spleen ferritin by the chelating agents desferrioxamine B, rhodotorulic acid, 2,3-dihydroxybenzoate, 2,2′-bipyridyl and pyridine-2-aldehyde-2-pyridyl hydrazone (Paphy) has been studied in vitro. Ferritin prepared by classical procedures involving thermal denaturation releases its iron less effectively than ferritin isolated by a modified procedure that avoids this step. Desferrioxamine B and rhodotorulic acid are the most effective in releasing iron from both preparations of ferritin. When FMN is added, iron release by desferrioxamine B, rhodotorulic acid, and 2,3-dihydroxybenzoate was effectively blocked, whereas both bipyridyl and Paphy showed a marked simulation. A substantial increase in iron release was also observed for bipyridyl and Paphy with ascorbate; a less important increase was noted for rhodotorulic acid. EDTA exerted a marked inhibition of iron release from ferritin with rhodotorulic acid, 2,3-dihydroxybenzoate, bipyridyl, and Paphy. The effects of citrate and oxalate on iron release by the chelators was small. The effect of the concentration of flavin on iron release from ferritin by bipyridyl and desferrioxamine B have been studied. Desferrioxamine is unable to mobilize FeII from ferritin following reduction by reduced FMN, whereas bipyridyl can rapidly complex the ferrous iron. The results are discussed in the context of our current concepts of storage iron mobilization in the treatment of iron overload.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals.

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2--. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2-- is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.     

Commentary: The Role of Iron-Sulfur Clusters in in vivo Hydroxyl Radical Production

The in vivo production of HO- requires iron ions, H₂O₂ and O₂- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O₂-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H₂O₂, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO- production to a minimum.

O₂- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction.

In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the “free” iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors.

The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.     

Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dps(tery)) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dps(tery) at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dps(tery) and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dps(tery) brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.     

Haem binding to ferritin and possible mechanisms of physiological iron uptake and release by ferritin.

Haem binding to horse spleen ferritin and Pseudomonas aeruginosa bacterioferritin has been studied by spectroscopic methods. A maximum of 16 haems per ferritin molecule, and 24 haems per bacterioferritin molecule, has been shown to bind. The influence of the bound haem on the rate of reductive iron release has been investigated. With a range of reductants and in the absence of haem the rate of release varied with the reductant, but in the presence of haem the rate was both independent of the reductant and faster than with any of the reductants alone. This indicates the rate-limiting step for iron release in the absence of haem was electron-transfer across the protein shell. Based on the results obtained with the in vitro assay system and from a consideration of data currently in the literature, plausible schemes for ferritin and bacterioferritin iron uptake and release are described.     

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.     

The effects of ascorbic acid on the intracellular metabolism of iron and ferritin

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.     

Ferritins: iron/oxygen biominerals in protein nanocages

Ferritin protein nanocages that form iron oxy biominerals in the central nanometer cavity are nature’s answer to managing iron and oxygen; gene deletions are lethal in mammals and render bacteria more vulnerable to host release of antipathogen oxidants. The multifunctional, multisubunit proteins couple iron with oxygen (maxi-ferritins) or hydrogen peroxide (mini-ferritins) at catalytic sites that are related to di-iron sites oxidases, ribonucleotide reductase, methane monooxygenase and fatty acid desaturases, and synthesize mineral precursors. Gated pores, distributed symmetrically around the ferritin cages, control removal of iron by reductants and chelators. Gene regulation of ferritin, long known to depend on iron and, in animals, on a noncoding messenger RNA (mRNA) structure linked in a combinatorial array to functionally related mRNA of iron transport, has recently been shown to be linked to an array of proteins for antioxidant responses such as thioredoxin and quinone reductases. Ferritin DNA responds more to oxygen signals, and ferritin mRNA responds more to iron signals. Ferritin genes (DNA and RNA) and protein function at the intersection of iron and oxygen chemistry in biology.     

Potentiometric assessment of iron release during ferritin reduction by exogenous agents

This work studied the possibilities for quantitative determination of iron mobilization in connection with ferritin reduction by ascorbic acid (vitamin C) and sodium dithionite in vitro. The iron storage protein was incubated with an excess of reductant in aerobic conditions in the absence of complexing agents in the medium. The release of Fe²⁺ was let to go to completion, and the overall content of Fe²⁺ in the solution was evaluated with the aid of potentiometric titration using Ce⁴⁺ as an oxidizing titrant. Results suggest a moderate iron efflux under the influence of the chosen reducing agents. Although such a reduction of the protein mineral core by dihydroxyfumarate contributes greatly to the iron mobilization, ferritin behavior with vitamin C and dithionite seems to be different. Although redox properties of dihydroxyfumarate are determined by hydroxyl groups similar to those of ascorbic acid, the two compounds differ significantly in structure, and this could be the basis for an explanation of the specificities in their interaction with ferritin. As revealed by the study, potentiometric titration promises to be a reliable tool for evaluation of the amount of Fe²⁺ present in the solution as a result of the reduction of the ferritin’s mineral core.     

Iron release from ferritin by flavin nucleotides

Background

Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.

Methods

Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.

Results

The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.

Conclusions

Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.

General significance

There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.     

Release of iron from ferritin by semiquinone, anthracycline, bipyridyl, and nitroaromatic radicals

The cytotoxicity of many xenobiotics is related to their ability to undergo redox reactions and iron dependent free radical reactions. We have measured the ability of a number of redox active compounds to release iron from the cellular iron storage protein, ferritin. Compounds were reduced to their corresponding radicals with xanthine oxidase/hypoxanthine under N₂ and the release of Fe²⁺ was monitored by complexation with ferrozine. Ferritin iron was released by a number of bipyridyl radicals including those derived from diquat and paraquat, the anthracycline radicals of adriamycin, daunorubicin and epirubicin, the semiquinones of anthraquinone-2-sulphonate, 1,5 and 2,6-dihydroxyanthraquinone, 1-hydroxyanthraquinone, purpurin, and plumbagin, and the nitroaromatic radicals of nitrofurantoin and metronidazole. In each case, iron release was more efficient than with an equivalent flux of superoxide. Introduction of air decreased the rate of iron release, presumably because the organic radicals reacted with O₂ to form superoxide. In air, iron release was inhibited by superoxide dismutase. Semiquinones of menadione, benzoquinone, duroquinone, anthraquinone 1,5 and 2,6-disulphonate, 1,4 naphthoquinone-2-sulphonate and naphthoquinone, when formed under N₂, were unable to release ferrin iron. In air, these systems gave low rates of superoxide dismutase-inhibitible iron release. Of the compounds investigated, those with a single electron reduction potential less than that of ferritin were able to release ferritin iron.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Exchange of iron by gallium in siderophores

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.