Ferritin Mineral Research Articles

Peptides Selected for the Protein Nanocage Pores Change the Rate of Iron Recovery from the Ferritin Mineral

Pores regulate access between ferric-oxy biomineral inside and reductants/chelators outside the ferritin protein nanocage to control iron demineralization rates. The pore helix/loop/helix motifs that are contributed by three subunits unfold independently of the protein cage, as observed by crystallography, Fe removal rates, and CD spectroscopy. Pore unfolding is induced in wild type ferritin by increased temperature or urea (1-10 mM), a physiological urea range, 0.1 mM guanidine, or mutation of conserved pore amino acids. A peptide selected for ferritin pore binding from a combinatorial, heptapeptide library increased the rate of Fe demineralization 3-fold (p<0.001), similarly to a mutation that unfolded the pores. Conjugating the peptide to Desferal (desferrioxamine B mesylate), a chelator in therapeutic use, increased the rates to 8-fold (p<0.001). A second pore binding peptide had the opposite effect and decreased the rate of Fe demineralization 60% (p<0.001). The peptides could have pharmacological uses and may model regulators of ferritin demineralization rates in vivo or peptide regulators of gated pores in membranes. The results emphasize that small peptides can exploit the structural plasticity of protein pores to modulate function.

The Importance and Bioavailability of Phytoferritin-Bound Iron in Cereals and Legume Foods

Ferritin is present in several types of plants in low concentrations, but it is possible to enhance this content by plant breeding, or by inserting the gene for ferritin into staple foods. Since each ferritin molecule can bind thousands of iron atoms, this may be a sustainable means to increase the iron content of plants. Before launching such efforts it is important to determine whether ferritin-bound iron is bioavailable. We assessed this in vitro using Caco-2 cells and in vivo using radiolabeled ferritin and whole body counting in human subjects. In Caco-2 cells, we found that dietary factors affecting iron absorption, such as ascorbic acid, phytate, and calcium, had very limited effect on iron uptake from intact ferritin, suggesting that ferritin-bound iron is absorbed via a mechanism different from that of non-heme iron. Using in vitro digestion, we found that ferritin was relatively resistant against proteolytic enzymes. Binding of ferritin to Caco-2 cells was found to be saturable and the kinetics for binding characteristic for a receptor-mediated process. In human subjects, we found that iron absorption from animal ferritin was similar to that from ferrous sulfate, suggesting that iron is well absorbed from ferritin. We did not find any significant difference between iron absorption from ferritin reconstituted with high-phosphate (plant-type) and low-phosphate (animal-type) ferritin mineral, suggesting that plant ferritin-iron is bioavailable. In a subsequent human study we also found that iron from purified soybean ferritin given in a meal was as well absorbed as ferrous iron. In conclusion, iron is well absorbed from phytoferritin and may represent a means of biofortification of staple foods.

Iron absorption from soybean ferritin in nonanemic women

Dietary ferritin, a protein cage around an iron mineral, is an underestimated source of bioavailable iron. Plant ferritin, the most common dietary ferritin, has not been studied. Iron from animal ferritin is absorbed as well as is iron from FeSO4 in women. The objective was to examine iron absorption from purified soybean ferritin. Healthy, nonanemic women (n = 16) were fed a standardized meal (bagel, cream cheese, and apple juice) containing 1 microCi 59Fe/meal as FeSO4 or (extrinsically labeled) as iron-free soybean ferritin reconstituted with the high phosphate characteristic of plant ferritin (iron:phosphorus = 4:1). Iron-free, apo-soybean ferritin was prepared (with the use of thioglycolic acid and extensive dialysis) from purified ferritin. In a randomized crossover design, the other labeled meal, which contained FeSO4 or ferritin, was given after 4 wk. The subjects received 140 microg Fe as ferritin (2.5 mg) or as FeSO4. After 28 d, whole-body 59Fe and 59Fe in red blood cells were measured before and after dosing. There was no significant difference in whole-body iron absorption from soybean ferritin (29.9 +/- 19.8%) and that from FeSO4 (34.3 +/- 23.6%) or in iron absorption calculated from red blood cell incorporation (33.0 +/- 20.1% for soybean ferritin and 35.3 +/- 23.4% for FeSO4), which confirmed previous results with animal ferritin that was mineralized and labeled similarly. An inverse relation was observed between serum ferritin and iron absorption from both ferritin and FeSO4, which suggested that sensors regulating iron absorption respond similarly to iron provided as ferrous salts or as ferritin mineral. Iron from soybean ferritin is well absorbed and may provide a model for novel, utilizable, plant-based forms of iron for populations with a low iron status.

A protein carboxylate coordinated oxo-centered tri-nuclear iron complex with possible implications for ferritin mineralization

The crystal structure of an oxo-centered tri-nuclear iron complex formed on a protein surface is presented. The cluster forms when crystals of the class Ib ribonucleotide reductase R2 protein from Corynebacterium ammoniagenes are subjected to iron soaking. The tri-iron-oxo complex is coordinated by protein-derived carboxylate ligands arranged in a motif similar to the one found on the inner surface of ferritins and may mimic an early stage in the mineralization of iron in ferritins. In addition, the structure adds to the very limited data on protein–mineral interfaces.

Photochemical Reactivity of Ferritin for Cr(VI) Reduction

The iron storage protein ferritin was used to catalyze the photoreduction of aqueous Cr(VI) species to Cr(III). Ferritin is a 24 subunit protein of roughly spherical shape with outer and inner diameters of approximately 12 and 8 nm, respectively. The native mineral core of ferritin is the ferric oxyhydroxide ferrihydrite (Fe(O)OH). Fe(O)OH particles that were used in these experiments ranged from 5 to 7.5 nm in diameter. The ferritin protein without the Fe(O)OH core (i.e., apoferritin) was inactive toward Cr(VI) reduction under our experimental conditions, suggesting that the Fe(O)OH provided the active catalytic sites in the redox chemistry. Experiments using photon band-pass filters suggested that the reaction occurred out of a photoinduced electron−hole pair and the optical band gap for the Fe(O)OH semiconductor was determined to be in the range 2.5−3.5 eV. Comparison of ferritin and protein-free Fe(O)OH mineral nanoparticles indicated that ferritin provided a photocatalyst with significantly more stab...

Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex, the first detectable intermediate in ferritin mineralization.

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by Mössbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench Mössbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

Forming the phosphate layer in reconstituted horse spleen ferritin and the role of phosphate in promoting core surface redox reactions.

Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe3+/HoSF) by depositing Fe(OH)3 within the hollow HoSF interior by air oxidation of Fe2+. Fe2+ and phosphate (Pi) were then added anaerobically at a 1:4 ratio, and both Fe2+ and Pi were incorporated into the HoSF cores. The resulting Pi layer consisted of Fe2+ and Pi at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe2+. The total amount of Fe2+ and Pi bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the Pi layer decreases with increasing core size. Mössbauer spectroscopic measurements of the Pi-reconstituted HoSF cores using 57Fe2+ show that 57Fe3+ is the major species present under anaerobic conditions. This result suggests that the incoming 57Fe2+ undergoes an internal redox reaction to form 57Fe3+ during the formation of the Pi layer. Addition of bipyridine removes the 57Fe3+ bound in the Pi layer as [57Fe(bipy)3]2+, showing that the bound 57Fe2+ has not undergone irreversible oxidation. This result is related to previous studies showing that 57Fe2+ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the Pi layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the Pi layer "floats" on the developing iron mineral core.

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis.

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (vmax = 24.4 pmol/(mg min), K(m) = 64.4 microM) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.

Correlations between Bone Histopathology and Serum Biochemistry in Uremic Patients on Chronic Hemodialysis

To define which noninvasive investigations are of value in predicting bone histology, we analyzed transiliac bone specimens (66 biopsies, 14 autopsies) from 80 uremic patients on chronic dialysis. Results were compared with values of different measurements of parathyroid hormone (PTH), alkaline phosphatase (APH), osteocalcin, calcitonin, baseline and post-deferroxamine (DFO) aluminium (Al), - β2 microglobulin, ferritin and bone mineral density Among histomorphometric parameters, woven osteoid, active osteoblastic surface and resorption surface showed the best correlations with dynamic and biochemical marks of active bone metabolism. Among biochemical parameters, intact PTH and APH were better related to histomorphometric and dynamic bone parameters than other PTH measurements as well as osteocalcin, while calcitonin was related to no parameters. Stainable Al alone, and not total bone Al content was related to bone histology. Baseline Al was related to lamellar osteoid, while post-DFO Al was related to stainable Al. β2 microglobulin was positively related to active osteoid surface and ferritin was inversely related to the mineral apposition rate, while bone mineral density was related only to total bone volume. We conclude that, though definite diagnosis requires the use of histological methods, few simple biochemical parameters may offer insight to the bone metabolic status, useful to the physician in day to day clinical practice.

Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization.

Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Ferritin mineralization: Ferroxidation and beyond

Ferritin mineralization: Ferroxidation and beyond

Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization.

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Formation of the ferritin iron mineral occurs in plastids.

Ferritin in plants is a nuclear-encoded, multisubunit protein found in plastids; an N-terminal transit peptide targets the protein to the plastid, but the site for formation of the ferritin Fe mineral is unknown. In biology, ferritin is required to concentrate Fe to levels needed by cells (approximately 10(-7) M), far above the solubility of the free ion (10(-18) M); the protein directs the reversible phase transition of the hydrated metal ion in solution to hydrated Fe-oxo mineral. Low phosphate characterizes the solid-phase Fe mineral in the center of ferritin of the cytosolic animal ferritin, but high phosphate is the hallmark of Fe mineral in prokaryotic ferritin and plant (pea [Pisum sativum L.] seed) ferritin. Earlier studies using x-ray absorption spectroscopy showed that high concentrations of phosphate present during ferritin mineralization in vivo altered the local structure of Fe in the ferritin mineral so that it mimicked the prokaryotic type, whether the protein was from animals or bacteria. The use of x-ray absorption spectroscopy to analyze the Fe environment in pea-seed ferritin now shows that the natural ferritin mineral in plants has an Fe-P interaction at 3.26A, similar to that of bacterial ferritin; phosphate also prevented formation of the longer Fe-Fe interactions at 3.5A found in animal ferritins or in pea-seed ferritin reconstituted without phosphate. Such results indicate that ferritin mineralization occurs in the plastid, where the phosphate content is higher; a corollary is the existence of a plastid Fe uptake system to allow the concentration of Fe in the ferritin mineral.