Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria.

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions. The release process under optimized conditions (approx. 50 mumol/1 FMN, 1 mmol/l succinate, 0.35 mmol/1 Fe(III) (as ferritin iron), 37 degrees C and pH 7.40) amounts to 0.9--1.2 nmol iron/mg protein per min. The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

Studies on the utilization of ferritin iron in the ferrochelatase reaction of isolated rat liver mitochondria.

The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found that isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 micromol/1 FMN, 37 degrees C, pH 7.4 and 0.5 mmol/l Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, amounted to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochrondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.     

Mobilization of iron from ferritin by isolated mitochondria effects of species compatibility between ferritin and mitochondria and iron content of ferritin

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.     

Reduction of exogenous flavins and mobilization of iron from ferritin by isolated mitochondria

The release mechanism for ferritin iron and the nature of the compound(s) which donate iron to the mitochondria are two important problems of intracellular iron metabolism which still await their solution. We have previously shown that isolated mitochondria reduce exogenously added flavins in a ubiquinol-flavin oxidoreductase reaction at the C-side of the inner membrane and that the resulting dihydroflavins function as reductants in mitochondrial mobilization of iron from ferritin (Ulvik, R. J., and Romslo, I. (1981). Biochim. Biophys. Acta 635, 457-469). In the present study it is shown that the rate at which iron is removed from ferritin depends on the capability of the flavins to penetrate (1) the mitochondrial outer membrane and (2) the intersubunit channels of the ferritin protein shell. Intact mitochondria reduce flavins at rates which decrease in the following order: riboflavin > FAD > FMN. The ferritin iron mobilization rates decrease in the order of riboflavin > FMN > FAD. The results are further support for the operation of a flavin-dependent mitochondrial ferrireductase, and strengthen the suggested role for ferritin as a donor of iron to the mitochondria.     

NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E.

The role of iron in allyl alcohol-induced lipid peroxidation and hepatic necrosis was investigated in male NMRI mice in vivo. Ferrous sulfate (0.36 mmol/kg) or a low dose of ally alcohol (0.6 mmol/kg) itself caused only minor lipid peroxidation and injury to the liver within 1 h. When FeSO4 was administered before allyl alcohol, lipid peroxidation and liver injury were potentiated 50-100-fold. Pretreatment with DL-tocopherol acetate 5 h before allyl alcohol protected dose-dependently against allyl alcohol-induced lipid peroxidation and liver injury in vivo. Products of allyl alcohol metabolism, i.e. NADH and acrolein, both mobilized trace amounts of iron from ferritin in vitro. Catalytic concentrations of FMN greatly facilitated the NADH-induced reductive release of ferritin-bound iron. NADH effectively reduced ferric iron in solution. Consequently, a mixture of NADH and Fe3+ or NADH and ferritin induced lipid peroxidation in mouse liver microsomes in vitro. Our results suggest that the reductive stress (excessive NADH formation) during allyl alcohol metabolism can release ferrous iron from ferritin and can reduce chelated ferric iron. These findings provide a rationale for the strict iron-dependency of allyl alcohol-induced lipid peroxidation and hepatotoxicity in mice in vivo and document iron mobilization and reduction as one of several essential steps in the pathogenesis.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

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.     

A Chelator is Required for Microsomal LIPID Peroxidation Following Reductive Ferritin-Iron Mobilisation

In the past, antioxidant and chelator studies have implicated a role for iron-dependent oxidative damage in tissues subjected to ischaemia followed by reperfusion. As ferritin is a major source of iron in non-muscular organs and therefore a potential source of the iron required for oxygen radical chemistry, we have determined conditions under which ferritin iron reduction leads to the formation of a pool of iron which is capable of catalysing lipid peroxidation. Under anaerobic conditions and in the presence of rat liver microsomes, flavin mononucleotide (FMN) catalysed the reduction of ferritin iron as shown by both continuous spectrophotometric measurements of tris ferrozine-Fe(II) complex formation and post-reaction Fe(II) determination. The presence of either ferrozine or citrate was not found to alter the time course or extent of ferritin reduction. In contrast, the addition of air to the reactants after a 20 min period of anaerobic reduction resulted in peroxidation of the microsome suspension (as determined with the 2-thiobarbituric acid test) only in the presence of a chelator such as citrate, ADP or nitrilotriacetic acid. These results support the concept that reduced ferritin iron can mediate oxidative damage during reperfusion of previously ischaemic tissues, provided that chelating agents such as citrate or ADP are present.     

Relevance of ferritin-binding sites on isolated mitochondria to the mobilization of iron from ferritin

Iron can be released from ferritin and utilized by isolated rat liver mitochondria for the synthesis of heme. Mobilization of iron from ferritin is initiated by the binding of ferritin to the mitochondria in an manner compatible with binding sites or receptors for ferritin on the mitochondria. The binding completes rapidly, it is independent of temperature, saturable, reversible and enhanced by K+ and Mg2+. The amount of ferritin binding sites is approx. 0.8 pmol/mg mitochondrial protein, and the affinity constant is 6.4 . 10(6)M-1. The binding kinetics correlate well with the functional features of the ferritin-mitochondrial interaction: i.e. mobilization of iron from ferritin followed by insertion of the iron into heme. The results support the concept of ferritin as a possible donor of iron to the mitochondria.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bäckström, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162–175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04±0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37°C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Studies on the oxidation of isobutyrylcarnitine by beef and rat liver mitochondria.

Mitochondria from beef liver oxidize isobutyrylcarnitine at approximately 50% the rate of succinate in the presence of rotenone. However, the oxidation rate of isobutyryl coenzyme A in the presence of l(-)-carnitine is very low and can be negligible in both rat and beef liver mitochondria. The limited stimulation of isobutyryl-CoA oxidation by l(-)-carnitine appears to be due to inhibition of isobutyrylcarnitine translocation rather than lack of formation of isobutyrylcarnitine. This conclusion is supported by the fact that: 1) isobutyrylcarnitine oxidation is inhibited by l(-)-carnitine; 2) some oxidation of isobutyryl-CoA is obtained when a low concentration (50 microM) of l(-)-carnitine is used; and 3) under conditions of high isobutyryl-coenzyme A and l(-)-carnitine concentrations (1 mM), isobutyryl-carnitine is produced in near theoretical amounts by these rat liver mitochondria. Other studies demonstrated that less than 25% of the carnitine isobutyryl transferase activity of beef liver mitochondria and rat liver mitochondria is located on the cytosol side of the acylcoenzyme A barrier of these mitochondria.     

Releasing iron from ferritin protein nanocage by reductive method: The role of electron transfer mediator

Background

Ferritin detoxifies excess of free Fe(II) and concentrates it in the form of ferrihydrite (Fe₂O₃·xH₂O) mineral. When in need, ferritin iron is released for cellular metabolic activities. However, the low solubility of Fe(III) at neutral pH, its encapsulation by stable protein nanocage and presence of dissolved O₂ limits in vitro ferritin iron release.

Pyrophosphate as a ligand for delivery of iron to isolated rat-liver mitochondria

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The capacity of the mitochondria to accumulate iron is higher than the capacity of pyrophosphate to mobilize iron from transferrin: with ferric-iron-pyrophosphate as iron donor, iron uptake and heme synthesis are about 10-times that at corresponding concentrations of iron-transferrin plus pyrophosphate. Uptake of iron from ferric-iron-pyrophosphate depends on a functionary respiratory chain and involves reductive cleavage of the ferric-iron-pyrophosphate complex. Apotransferrin inhibits uptake of iron from ferric-iron-pyrophosphate by competing with the mitochondria for iron. The results focus on pyrophosphate as a possible candidate for intracellular iron transport.     

Oxalate modulates thiobarbituric acid reactive species (TBARS) production in supernatants of homogenates from rat brain, liver and kidney: effect of diphenyl diselenide and diphenyl ditelluride

The aim of this paper was to investigate the mechanism(s) involved in the sodium oxalate pro-oxidative activity in vitro and the potential protection by diphenyl diselenide ((PhSe)(2)) and diphenyl ditelluride ((PhTe)(2)) using supernatants of homogenates from brain, liver and kidney. Oxalate causes a significant increase in the TBARS (thiobarbituric acid reactive species) production up to 4mmol/l and it had antioxidant activity from 8 to 16mmol/l in the brain and liver. Oxalate had no effect in kidney homogenates. The difference among tissues may be related to the formation of insoluble crystal of oxalate in kidney, but not in liver and brain homogenates. (PhSe)(2) and (PhTe)(2) reduced both basal and oxalate-induced TBARS in rat brain homogenates, whereas in liver homogenates they were antioxidant only on oxalate-induced TBARS production. (PhSe)(2) showed a modest effect on renal TBARS production, whereas (PhTe)(2) did not modulate TBARS in kidney preparations. Oxalate at 2mmol/l did not change deoxyribose degradation induced by Fe(2+) plus H(2)O(2), whereas at 20mmol/l it significantly prevents its degradation. Oxalate (up to 4mmol/l) did not alter iron (10micromol/l)-induced TBARS production in the brain preparations, whereas at 8mmol/l onwards it prevents iron effect. In liver preparations, oxalate amplifies iron pro-oxidant activity up to 4mmol/l, preventing iron-induced TBARS production at 16mmol/l onwards. These results support the antioxidant effect of organochalcogens against oxalate-induced TBARS production. In addition, our results suggest that oxalate pro- and antioxidant activity in vitro could be related to its interactions with iron ions.     

Hispidulin: antioxidant properties and effect on mitochondrial energy metabolism

Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3',4'-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05-0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H₂O₂ in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.     

On the non-ferritin depot iron fraction in the rat liver

Liver depot iron can be divided into two fractions: ferritin iron and non-ferritin depot iron. Three methods intended to measure the non-ferritin depot iron in the rat liver were compared using livers of normal rats and livers of rats loaded with iron by transfusion of erythrocytes. Liver depot iron varied between 75 and 850 μg Fe/g liver. Non-ferritin depot iron, measured as the iron fraction sedimentable at 10 000 × g, was in the range 4–22 μg Fe/g liver. This fraction did contain ferritin. When measured as the difference between total liver depot iron and heat-stable iron (ferritin iron), the range was 10–270 μg Fe/g liver but this fraction also includes some ferritin iron.The values derived with both methods were linearly proportional to the total liver depot iron values.Non-ferritin depot iron, when measured as the difference between total liver depot iron and total ferritin iron, ranged from 0 to 190 μg Fe/g liver. In this last method no ferritin iron is included. This method provides the best estimate of the non-ferritin depot iron fraction. The concentrations obtained with this method were not always linearly proportional to the total liver depot iron concentration. Intravenous injection of rat liver ferritin resulted in a rapid accumulation of ferritin iron in the liver, together with an increase of the non-ferritin depot iron fraction from 18 μg Fe/g liver to 55 μg Ge/g liver. This confirms a relationship between ferritin catabolism and the non-ferritin depot iron fraction.     

Effects of hexachlorobenzene and iron loading on rat liver mitochondria

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occuring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a striking increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.     

The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates.

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.     

Arsenic species that cause release of iron from ferritin and generation of activated oxygen

The in vitro effects of four different species of arsenic (arsenate, arsenite, monomethylarsonic acid, and dimethylarsinic acid) in mobilizing iron from horse spleen ferritin under aerobic and anaerobic conditions were investigated. Dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) significantly released iron from horse spleen ferritin either with or without the presence of ascorbic acid, a strong synergistic agent. Ascorbic acid-mediated iron release was time-dependent as well as both DMA(III) and ferritin concentration-dependent. Iron release from ferritin by DMA(III)) alone or with ascorbic acid was not significantly inhibited by superoxide dismutase (150 or 300 units/ml). However, the iron release was greater under anaerobic conditions (nitrogen gas), which indicates direct chemical reduction of iron from ferritin by DMA(III), with or without ascorbic acid. Both DMA(V) and DMA(III)) released iron from both horse spleen and human liver ferritin. Further, the release of ferritin iron by DMA(III)) with ascorbic acid catalyzed bleomycin-dependent degradation of calf thymus DNA. These results indicate that exogenous methylated arsenic species and endogenous ascorbic acid can cause (a) the release of iron from ferritin, (b) the iron-dependent formation of reactive oxygen species, and (c) DNA damage. This reactive oxygen species pathway could be a mechanism of action of arsenic carcinogenesis in man.     

Effect of chaotropes on the kinetics of iron release from ferritin by flavin nucleotides

BackgroundFerritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied, but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH₂ in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed.MethodsThe iron release kinetics from horse and human ferritins by FMNH₂ were monitored at 522 nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton.Results and conclusionsUnder our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH₂ and is independent on the type or amount of chaotropes present. Diffusion of FMNH₂ through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed.General significanceCaution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.