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.     

Iron release analyses from ferritin by visible light irradiation

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47°C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.     

Iron release from ferritin and lipid peroxidation by radiolytically generated reducing radicals

Iron is involved in the formation of oxidants capable of damaging membranes, protein, and DNA. Using 137Cs gamma radiation, we investigated the release of iron from ferritin and concomitant lipid peroxidation by radiolytically generated reducing radicals, superoxide and the carbon dioxide anion radical. Both radicals released iron from ferritin with similar efficiencies and iron mobilization from ferritin required an iron chelator. Radiolytically generated superoxide anion resulted in peroxidation of phospholipid liposomes as measured by malondialdehyde formation only when ferritin was included as an iron source and the released iron was found to be chelated by the phospholipid liposomes.     

Iron release from haemosiderin and ferritin by therapeutic and physiological chelators.

Iron release from both human and horse spleen haemosiderin to desferrioxamine was substantially less than that released from ferritin samples. This finding contradicts a previous report [Kontoghiorges, Chambers & Hoffbrand (1987) Biochem. J. 241, 87-92]. Differences in phosphate content of cores and in core size between haemosiderin and ferritin did not account for the different iron-release rates. Iron released to acetate was found to stimulate lipid peroxidation in liposomes, whereas that released to stronger chelators such as citrate and desferal did not. Absorption spectra and gel-filtration studies suggest that the acetate-solubilized iron was in the form of low-molecular-mass (less than 5 kDa) ferrihydrite fragments.     

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.     

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).     

Superoxide-mediated release of iron from ferritin by some flavoenzymes

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.     

Nitric oxide mediates iron release from ferritin

Nitric oxide (NO) synthesis by cytotoxic activated macrophages has been postulated to result in a progressive loss of iron from tumor target cells as well as inhibition of mitochondrial respiration and DNA synthesis. In the present study, the addition of an NO-generating agent, sodium nitroprusside, to the iron storage protein ferritin resulted in the release of iron from ferritin and the released iron-catalyzed lipid peroxidation. Hemoglobin, which binds NO, and superoxide anion, which reacts with NO, inhibited nitroprusside-dependent iron release from ferritin, thereby providing evidence that NO can mobilize iron from ferritin. These results suggest that NO generation in vivo could lead to the mobilization of iron from ferritin disrupting intracellular iron homeostasis and increasing the level of reactive oxygen species.     

The release of iron from horse spleen ferritin by reduced flavins

Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH₂, FADH₂ and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH₂. Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe³⁺ ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin–dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH₂ which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH₂–ferritin and dithionite–ferritin complex formation showed that FMNH₂ required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH₂ in ferritin–Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.     

Rapid release of Iron from Ferritin by Siderophores

The ability of the microbial Siderophores deferriferrichrome, deferriferrichrome A, and enterobactin to remove iron from ferritin has been investigated. In contrast to previously published data with other chelators, all three Siderophores rapidly released iron from the mammalian storage protein Enterobactin was found most efficient at removing ferritin-bound iron. Using this siderophore, the mechanism by which ferritin sequesters iron was studied The relative iron saturation level of ferritin influenced the rate of chelation by the microbial Siderophores.     

Investigation of the release of iron from ferritin by naturally occurring antioxidants

Ferritin is the main intracellular iron storage protein. The release of iron from ferritin in the presence of a number of phenolic based compounds of nutritional significance was studied at physiological pH. The release of iron was measured by monitoring the formation of the iron(II)-ferrozine complex. The kinetics of this process were studied in Hepes buffer (pH 7.00), at 37 degrees C. The order of ability to remove iron from ferritin is epigallocatechin>gallic acid methyl ester approximately equal to sinapic acid>ferulic acid. The presence of the oxyradical scavenger urea resulted in a slight inhibition in the release of iron from ferritin by both gallic acid methyl ester and epigallocatechin. The ability of each reagent to release iron is interpreted on the basis of their ability to (a) reduce the bound iron and (b) complex the iron with the oxidised form of the phenol, thus mobilising it from the protein. These studies indicate that some phenolic based compounds that have been epidemiologically associated with a negative effect on iron absorption in man, can individually mobilise and release iron from ferritin under suitable conditions.     

Iron mobilization from ferritin using alpha-oxohydroxy heteroaromatic chelators.

Several alpha-oxohydroxy heteroaromatic chelators have been shown to mobilize iron from horse spleen ferritin. Although the reactions were slow, taking up to 3 days to reach completion, the amounts of iron mobilized were higher than those reported for other chelators. These results increase the prospects for the clinical use of alpha-oxohydroxy chelators in the treatment of iron overload.     

Superoxide dismutase-inhibitible reduction of cytochrome c by the alloxan radical. Implications for alloxan cytotoxicity.

Cytochrome c was reduced when superoxide was generated from xanthine oxidase in the presence of alloxan, and by the reaction of alloxan and with reduced glutathione. In each case, most of the reduction was inhibited by superoxide dismutase, but considerably more enzyme was required than with superoxide alone. This indicates that the superoxide dismutase-inhibitible cytochrome c reduction was mainly due to a direct reaction with the alloxan radical, and implies that other reactions that are inhibited by superoxide dismutase could be due to either alloxan radicals or superoxide.     

Superoxide dependent iron release from ferritin in inflammatory diseases

Convincing evidence is presented that oxygen free radicals are involved in the pathogenesis of rheumatoid arthritis (RA). Superoxide is produced by polymorphonuclear leucocytes (PMN) in synovial fluid and by macrophages in the synovial membrane. Tissue damage typical for free radical attack is detected in RA. No absolute deficiency of protective factors is found in RA compared to controls, but the available protection is insufficient to cope with all radicals formed. The toxicity of superoxide is increased by iron. It is doubtful whether a low molecular weight iron pool is present. Superoxide is able to release iron from ferritin, providing a suitable source of iron, for the formation of hydroxyl radicals. This new pathogenetic mechanism stimulates to the application of iron chelators in the treatment of RA. Preliminary results with desferrioxamine were disappointing because of serious side-effects. Hopefully in the future intra-articular injection of iron chelators with better pharmacodynamics will be possible. The interaction of free radicals and ferritin is probably also involved in the pathogenesis of other inflammatory diseases such as systemic lupus erythematosus, hepatitis, and haemochromatosus.     

Nitric oxide does not promote iron release from ferritin

It has been previously reported that iron release from ferritin could be promoted by nitric oxide (NO) generated from sodium nitroprusside. It was thus proposed that some of the toxic effects of NO could be related to its ability to increase intracellular free iron concentrations and generate an oxidative stress. On the contrary, the iron exchange experiments reported here show that NO from S-nitrosothiols is unable to promote iron release from ferritin. The discrepancy may be explained by the disregarded ability of ferrozine, the ferrous trap used in the previous report, to mobilize iron both from ferritin and from sodium nitroprusside spontaneously.     

Redox reactions associated with iron release from mammalian ferritin

The early redox events involved in iron reduction and mobilization in mammalian ferritin have been investigated by several techniques. Sedimentation velocity measurements of ferritin samples with altered core sizes, prepared by partial reduction and Fe2+ chelation, suggest two different events occur during iron loss from the ferritin core. Reductive optical titrations confirm this biphasic behavior by showing that the first 20-30% of core reduction has different optical properties than the latter 70-80%. Proton uptake during initial core reduction is near zero, but as the percent core reduction increases, the proton uptake (H+/e) values increase to 2 H+/e (2 H+/Fe3+ reduced) as core reduction approaches 1 e/Fe3+. Coulometric reduction of ferritin by mediators of different redox potential and different cross-sectional areas show a two-phase sigmoidal reaction pattern in which initial core reduction occurs at a slower rate than later core reduction. The above experiments were all conducted in the absence of iron chelators so that the observed results were all attributed to core reduction rather than the combined effects of core reduction accompanied by Fe2+ chelation. The coulometric reduction of ferritin by various mediators shows a correlation more with reduction potential than with molecular cross-sectional area. The role of the ferritin channels in core reduction is considered in terms of the reported results.     

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.     

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.