Aluminum accumulation in serum liver and spleen of fe-depleted and fe-adequate rats

The study described here was planned to test the hypothesis that Al absorption and accumulation in the body are inversely related to Fe status. Aluminum3+ and Fe3+ have similar ionic radii and charge densities, pH-solubility relationships, and affinities for ligands, such as citrate and transferrin. Male weanling Sprague-Dawley rats were pair fed an Fe-deficient or Fe-adequate (control) diet for 2 wk. Each diet group was then randomly assigned to receive for four more weeks the Fe-deficient or adequate diet with: 1. 2% AlCl3; 2. AlCl3 + 3.5% Na citrate; or 3. No Al or citrate. Iron depletion, confirmed by measurements of hemoglobin, hematocrit, serum Fe, and Fe binding capacity, increased concentrations of serum, liver, and spleen Al in all groups fed AlCl3. However, the increase owing to Fe deficiency was significant only when Al was fed with citrate. The data suggest that Fe deficiency enhances both Al absorption and accumulation in liver and spleen.     

Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate

The kinetics of release of Al3+ from human serum dialuminum transferrin (Al2Tf) to citrate were investigated at 37 degrees C, pH 7.4, mu = 0.7 M, by difference UV spectrophotometry. The two metal-binding sites are not identical but behave in a kinetically similar manner to give apparent second-order rate constants of 0.60 and 0.38 M-1 s-1, respectively, for release of the first Al3+ from Al2Tf. The rate constants for release of the second metal ion from the monoaluminum transferrins are 0.27 and 0.12 M-1 s-1. The kinetic scheme for release of A13+ from Al2Tf is therefore similar to that for release of Fe3+ from Fe2Tf, but the rate of constants for metal ion release are between two and four orders of magnitude larger.     

The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.     

Competition of Mn2+ and Zn2+ with59Fe2+ and59Fe3+ for the plasma membrane receptors from lactating mouse mammary gland

Nonlabeled MnCl₂ and ZnSO₄ compete with⁵⁹Fe²⁺-ascorbate and⁵⁹Fe ₂ ³⁺ O₃ for transport binding sites situated on the plasma membranes of lactating mouse mammary gland cells. The binding was found to be a process reaching saturation. The heterologous competition used here ruled out the participation of transferrin and to propose that Fe, Mn, and Zn are transported from blood to milk by a mechanism involving one receptor during lactation. Further experiments are necessary to establish the details of the transport mechanism.     

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.     

Effects of dietary supplementation with aluminum and citrate on iron metabolism in the rat

The metabolism of iron (Fe) has been shown to interact with that of aluminum (Al) in relation to intestinal absorption, transport in the blood plasma, and the induction of lipid peroxidation and cellular damage. Also, dietary supplementation with citrate has been shown to increase the absorption of both metals and, in the presence of high intakes of Fe and Al, leads to excessive accumulation of both metals in the body. In this study, the likely interaction between Al and internal Fe metabolism was investigated using rats fed diets that were either deficient, sufficient, or loaded with Fe, with or without the addition of Al and sodium citrate. These diets commenced when the rats were 4 wk old and were continued for 9–11 wk. At that time, Fe metabolism as assessed by measurement of organ uptake of⁵⁹Fe and¹²⁵I-transferrin, after iv injection of transferrin labeled with both isotopes, plus measurement of tissue concentrations of nonheme Fe and Al. The Fedeficient diet and Fe-loaded diet led to states of Fe deficiency and Fe overload in the rats, and supplementation of the diet with Al increased Al levels in the kidneys, liver, and femurs, but, generally, only when the diet also contained citrate. Neither Al nor citrate supplementation of the diet had any effect on nonheme Fe concentrations in the liver, kidney, or brain, or on the uptake of⁵⁹Fe or¹²⁵I-transferrin by liver, kidney, brain, or spleen. Only with the femurs was a significant effect observed: increased⁵⁹Fe uptake in association with increased Al intake. Therefore, using this animal model, there was little evidence for interaction between Fe and Al metabolism, and no support was obtained for the hypothesis that dietary supplementation with Fe and citrate can lead to excessive Fe absorption and deposition in the tissues.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.     

Binding of manganese in human and rat plasma

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.     

N-Carboxyalkyl derivatives of 3-hydroxy-4-pyridinones: synthesis,complexation with Fe(III), Al(III) and Ga(III) and in vivo evaluation

A set of three N-carboxyalkyl 3-hydroxy-4-pyridinones has been studied as bidentate M(III) chelators (M=Fe, Al, Ga), with potential for oral administration. After preparation of the ligands, their protonation constants (log K(i)) and the stability constants of their metal complexes have been determined. The distribution coefficients of these compounds, between 1-octanol and Tris buffer pH 7.4, were measured. The effect of these compounds on the biodistribution of 67Ga-citrate loaded rats was investigated and compared with that of the administered 67Ga-complexes. Results indicated that, among these chelating agents, the N-carboxyethyl derivative has the highest affinity towards this set of metal ions, irrespective of the metal, and that it could even compete with transferrin, the main Fe-plasma protein. The binding affinity and the hydrophilic character decrease with the increase in the size of the alkylic chain. The biological assays indicate that the complex formation in vivo is characterized by a high kinetics and thermodynamic stability, suggesting a competition with the transferrin. All the ligands were found to enhance the excretion of the gallium. Noteworthy is the observed Ga bone fixation, mostly with the ethyl derivative, thus suggesting the potential use of the complex as a bone seeking agent.     

Monocarboxylate Transporters are not Responsible for Cr3+ Transport from Endosomes

Cr(3+), similar to Fe(3+), is transported into cells primarily via endocytosis as the metal-transferrin complex. As Cr(3+) ions are not readily reduced under biological conditions, the ion cannot be transported from endosomes by the same mechanism as iron that utilized divalent metal ion transporters. Cr(3+) has been hypothesized to potentially be transported as small ligand complexes with a free carboxylate functionality by monocarboxylate transporters (MCT), in a similar fashion to that proposed for Al(3+). Consequently, mouse C2C12 muscle cells were utilized to determine if Cr(3+) is potentially transported by MCT by examining the effects of MCT inhibitors on Cr and Fe transport and subcellular distribution when the metals are added as their transferrin complexes. The results suggest that Cr is not primarily transported by MCT from the endosomes to the cytosol, and that another mechanism for this transport needs to be identified.     

The interaction of resealed reticulocyte ghosts with Fe3+-transferrin-CO3(2-)

The present studies were designed to investigate the interaction of Fe3+transferrin-CO3(2-) with the transferrin receptors of the resealed reticulocyte ghost and to assess the degree to which the iron release reaction can be reconstituted in resealed ghosts supplemented with entrapped cytoplasmic components. Reticulocyte, but not erythrocyte, ghosts displayed an intact Fe3+transferrin-CO3(2-) binding capability. When ATP, NADH and ferritin were included during the resealing process, some iron release to the ghosts was observed.     

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.     

New perspectives on the structure and function of transferrins.

Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.     

Binding of [Cr(phen)3]3+ to transferrin at extracellular and endosomal pHs: Potential application in photodynamic therapy

BackgroundTransferrin is an iron-binding blood plasma glycoprotein that controls the level of free iron in biological fluids. This protein has been deeply studied in the past few years because of its potential use as a strategy of drug targeting to tumor tissues. Chromium complex, [Cr(phen)₃]³⁺ (phen = 1,10-phenanthroline), has been proposed as photosensitizers for photodynamic therapy (PDT). Thus, we analyzed the binding of chromium complex, [Cr(phen)₃]³⁺, to transferrin for a potential delivery of this diimine complex to tumor cells for PDT.MethodsThe interaction between [Cr(phen)₃]³⁺ and holotransferrin (holoTf) was studied by fluorescence quenching technique, circular dichroism (CD) and ultraviolet (UV)–visible spectroscopy.Results[Cr(phen)₃]³⁺ binds strongly to holoTf with a binding constant around 10⁵ M⁻¹, that depends on the pH. The thermodynamic parameters indicated that hydrophobic interactions played a major role in the binding processes. The CD studies showed that there are no conformational changes in the secondary and tertiary structures of the protein.ConclusionsThese results suggest that the binding process would occur in a site different from the specific iron binding sites of the protein and would be the same in both protein states. As secondary and tertiary structures of transferrin do not show remarkable changes, we propose that the TfR could recognize the holoTf despite having a chromium complex associated.General significanceUnderstanding the interaction between [Cr(phen)₃]³⁺ with transferrin is relevant because this protein could be a delivery agent of Cr(III) complex to tumor cells. This can allow us to understand further the role of Cr(III) complex as sensitizer in PDT.     

Fe3+ coordination and redox properties of a bacterial transferrin

The Fe(3+) binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe(3+) binding constant in the presence of 350 microm phosphate at pH 6.5 and 25 degrees C was determined as 2.4 x 10(18) m(-1). EPR spectra for the recombinant Fe(3+)nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe(3+) in a strong ligand field of low (orthorhombic) symmetry. (31)P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe(3+) in apo-nFbp. Finally, an uncorrected Fe(3+/2+) redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 degrees C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe(3+) binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.     

Multivalent metal-induced iron acquisition from transferrin and lactoferrin by myeloid cells

We previously described a unique, high-capacity, ATP-independent mechanism through which myeloid cells acquire Fe from low-m.w. chelates. The rate of this Fe acquisition is markedly increased by cellular exposure to multivalent metal cations. Because most Fe in vivo is bound to transferrin or lactoferrin, we examined whether this mechanism also contributes to myeloid cell acquisition of Fe from transferrin and/or lactoferrin. Using HL-60 cells as a model system, we show cellular acquisition of (59)Fe from both lactoferrin and transferrin that was unaffected by conditions that depleted the cells of ATP or disrupted their cytoskeleton. Fe acquisition was dramatically increased by cell exposure to various metals including Ga(3+), Gd(3+), Al(3+), Fe(3+), La(3+), Zr(4+), Sn(4+), Cu(2+), and Zn(2+) by a process that was reversible. Exposure to these same metals also increased binding of both transferrin and lactoferrin to the cell surface by a process that does not appear to involve the well-described plasma membrane receptor for transferrin. Approximately 60% of the Fe acquired by the cells from transferrin and lactoferrin remained cell associated 18 h later. HL-60 cells possess a high-capacity multivalent metal-inducible mechanism for Fe acquisition from transferrin and lactoferrin that bears many similarities to the process previously described that allows these and other cell types to acquire Fe from low-m.w. Fe chelates. The biologic importance of this mechanism may relate to its high Fe acquisition capacity and the speed with which it is able to rapidly adapt to the level of extracellular Fe.     

Interference of aluminium on iron metabolism in erythroleukaemia K562 cells

It has been suggested that aluminium (Al) has a deleterious effect on erythropoiesis. However, there is still uncertainty as to its action mechanism. The present work was designed to determine how Al could affect the iron (Fe) metabolism in the human erythroleukaemia cell line K562. These cells, that express surface transferrin receptors (TfRs), were induced to erythroid differentiation by either haemin or hydroxyurea in 72 h cultures in media containing apotransferrin (apoTf). In the presence of aluminium citrate, the number of benzidine-positive cells decreased 18% when the cultures were induced by haemin, and 30% when hydroxyurea was the inducer. Cell viability was always unaffected. From competition assays, surface binding of 125I-Tf-Fe2 was found to be inversely related (p < 0.05) to Tf-Al2 concentration (from 2.5 to 10 nM). The dissociation constants (Kd) of the binding reaction between TfRs and the ligands Tf-Fe2 and Tf-Al2 were calculated. Kd values of the same order of magnitude demonstrated that TfR has a similar affinity for Tf-Fe2 (Kd = 1.75 x 10(-9) M) and Tf-Al2 (Kd = 1.37 x 10(-9) M). The number of surface TfRs, measured by kinetic 125I-Tf-Fe2 binding assays, was higher in induced cells cultured in the presence of Al. Nevertheless, in spite of the inhibition of cell haemoglobinization observed, 59Fe incorporation values were not different from those measured in control cultures for 72 h. As a consequence, it can be suggested that cellular Fe utilisation, and not Fe uptake, might be the main metabolic pathway impaired by Al.     

The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy

A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+-transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000-fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.