Formation of an Fe(III)-tyrosinate complex during biomineralization of H-subunit ferritin.

An iron(III)-tyrosinate complex was identified in ferritin by ultraviolet-visible and resonance Raman spectroscopies. Previously, a specific amino acid side chain coordinated to iron in ferritin was not known. Ferritin protein was overexpressed in Escherichia coli from complementary DNA sequences of bullfrog red cell ferritin. The purple iron(III)-tyrosinate intermediate that formed during the first stages of iron uptake was replaced by the amber multinuclear iron(III)-oxo complexes of fully mineralized ferritin. Only the H subunit formed detectable amounts of the iron(III)-tyrosinate complex, which may explain the faster rates of iron biomineralization in H- compared to L-type ferritin.

A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay?

The reaction of oxygen with protein diiron sites is important in bioorganic syntheses and biomineralization. An unusually short Fe-Fe distance of 2.53 angstroms was found in the diiron (mu-1,2 peroxodiferric) intermediate that forms in the early steps of ferritin biomineralization. This distance suggests the presence of a unique triply bridged structure. The Fe-Fe distances in the mu-1, 2 peroxodiferric complexes that were characterized previously are much longer (3.1 to 4.0 angstroms). The 2.53 angstrom Fe-Fe distance requires a small Fe-O-O angle (approximately 106 degrees to 107 degrees). This geometry should favor decay of the peroxodiferric complex by the release of H2O2 and mu-oxo or mu-hydroxo diferric biomineral precursors rather than by oxidation of the organic substrate. Geometrical differences may thus explain how diiron sites can function either as a substrate (in ferritin biomineralization) or as a cofactor (in O2 activation).

High resolution crystal structures of amphibian red-cell L ferritin: potential roles for structural plasticity and solvation in function.

Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated iron ions and protons to and from a mineralized core in the protein interior. We report here the high resolution structures of recombinant amphibian red-cell L ferritin and two mutants solved under two sets of conditions. In one mutant, Glu56, 57, 58 and 60 were replaced with Ala, producing a lag phase in the kinetics of iron uptake. In the second mutant, His25 was replaced with Tyr with, at most, subtle effects on function. A molecule of betaine, used in the purification, is bound in all structures at the 2-fold axis near the recently identified heme binding site of bacterioferritin and horse spleen L ferritin. Comparisons of the five amphibian structures identify two regions of the molecule in which conformational flexibility may be related to function. The positions and interactions of a set of 10 to 18 side-chains, most of which are on the inner surface of the protein, are sensitive both to solution conditions and to the Glu-->Ala mutation. A subset of these side-chains and a chain of ordered solvent molecules extends from the vicinity of Glu56 to 58 and Glu60 to the 3-fold channel in the wild type protein and may be involved in the transport of either iron or protons. The "spine of hydration" is disrupted in the Glu-->Ala mutant. In contrast, H25Y mutation shifts the positions of backbone atoms between the site of the mutation and the 4-fold axis and side-chain positions throughout the structure; the largest changes in the position of backbone atoms are in the DE loop and E helix, approximately 10 A from the mutation site. In combination, these results indicate that solvation, structural plasticity and cooperative structural changes may play a role in ferritin function. Analogies with the structure and function of ion channel proteins such as annexins are noted.

The influence of the base-paired flanking region on structure and function of the ferritin mRNA iron regulatory element.

Ferritin and transferrin receptors are co-ordinately regulated by the same RNA-protein interaction: the conserved iron regulatory element (IRE) in mRNA and the IRE-binding protein (IRE-BP/IRP/FRP/P-90). The 28 nucleotide IRE in ferritin mRNA is a single copy, with base-paired flanking regions (FL), located near the 5' cap. In the transferrin receptor mRNA, the IRE is located in the 3' untranslated region, as five variable copies and lacking predicted base-paired flanking regions; an alternate predicted structure without IREs has similar stability. When iron is scarce, ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated; when iron is abundant, ferritin mRNA forms polyribosomes and the transferrin receptor mRNA is degraded. To investigate structures which contribute to differences in the regulation of the two mRNAs, the effect of mutation of the ferritin FL was studied. Changes in structure (changes in reactivity with RNase V1 and RNase S1. Fe-bleomycin) and changes in function (translation in rabbit reticulocyte extracts) were compared for mutant and wild-type FL sequences in ferritin mRNA. The disruption of a triplet of base-pairs in the FL had diminished regulation; a second mutation to restore the triplet base-pairs conferred wild-type translational regulation. Conformation of the mutant RNA-IRE-BP complex was also different. We show that the triplet of base-pairs is conserved; the triplet is also the location of IRE-BP-dependent conformational changes in the FL structure previously observed. Increasing FL base-pairs had no effect on function. Structural changes associated with altered function included bleomycin sites in the IRE, suggesting an alternate conformation of the hairpin, and different base-stacking (V1 sensitivity) in the FL. The function of the FL, which is altered by mutation of phylogenetically conserved triplet base-pairs, may be enhancement of formation of a particular IRE stem-loop-protein interaction.

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies.

BACKGROUND: The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms. RESULTS: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern. CONCLUSIONS: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.

Targeting mRNA to regulate iron and oxygen metabolism.

A family of non-coding sequences in the mRNA (iso-IREs [iron-responsive elements]) regulate synthesis of key proteins in animal iron and oxidative metabolism such as ferritin and mitochondrial aconitase. Differential recognition between iso-IREs and iso-IRPs (iron regulatory proteins) regulates the translation or degradation of the IRE-containing mRNAs. IREs are hairpin loop structures with an internal loop/bulge or bulge that influence the binding of the iso-IRPs. The iso-IRPs have sequence homology to the aconitases and at least one IRP can be converted to an aconitase. Signals that target the iso-IRE/iso-IRP interactions in mRNA include environmental iron, O2, nitric oxide, H2O2, ascorbate, growth factors, and protein kinase C-dependent IRP phosphorylation. Iso-IRE structural specificity suggests a means of pharmacologically targeting mRNA function with chemicals such as Fe-bleomycin and other transition metal complexes that could be extended to other mRNAs with specific structures. With the iso-IRE/iso-IRP system, nature has evolved coordinated combinatorial control of iron and oxygen metabolism that may exemplify control of mRNAs in other metabolic pathways, viral reproduction, and oncogenesis.

Structural variations in soluble iron complexes of models for ferritin: an x-ray absorption and Mössbauer spectroscopy comparison of horse spleen ferritin to Blutal (iron-chondroitin sulfate) and Imferon (iron-dextran).

Variations in the turnover of storage iron have been attributed to differences in apoferritin and in the cytoplasm but rarely to differences in the structure of the iron core (except size). To explore the idea that the iron environment in soluble iron complexes could vary, we compared horse spleen ferritin to pharmaceutically important model complexes of hydrous ferric oxide formed from FeCl3 and dextran (Imferon) or chondroitin sulfate (Blutal), using x-ray absorption (EXAFS) and Mössbauer spectroscopy. The results show that the iron in the chondroitin sulfate complex was more ordered than in either horse spleen ferritin or the dextran complex (EXAFS), with two magnetic environments (Mössbauer), one (80%-85%) like Fe2O3 X nH2O (ferritinlike) and one (15%-20%) like Fe2O3 (hematite); since sulfate promotes the formation of inorganic hematite, the sulfate in the chondroitin sulfate most likely nucleated Fe2O3 and hydroxyl/carboxyls, which are ligands common to chondroitin sulfate, ferritin and dextran most likely nucleated Fe2O3 X nH2O. Differences in the structure of the iron complexed with chondroitin sulfate or dextran coincide with altered rates of iron release in vivo and in vitro and provide the first example relating function to local iron structure. Differences might also occur among ferritins in vivo, depending on the apoferritin (variations in anion-binding sites) or the cytoplasm (anion concentration).

A comparison of an undecairon(III) complex with the ferritin iron core.

The iron core of ferritin is comprised of up to 4,500 Fe(III) atoms as Fe2O3.nH2O, which is maintained in solution by a surrounding, spherical coat of protein. Organisms as diverse as bacteria and man use the ferritin iron-protein complex as a reservoir of stored iron for other essential proteins. To extend studies of the steps in polynuclear iron core formation, a recently characterized undecairon(III) oxo-hydroxo aggregate [Fe11 complex] (Gorun et al., J. Am. Chem. Soc. 109, 3337 [1987]) was examined by x-ray absorption spectroscopy as a model for an intermediate. The results, which are comparable to the previous x-ray diffraction studies, show near neighbors (Fe-O) at 1.90 A that are distinct from those in ferritin and a longer distance of 2.02 A. However, contributions from neighbors (Fe-C) known to exist at ca. 2.7 A were obscured by a highly ordered Fe-Fe interaction and were not detectable in the Fe11 complex in contrast to a previously characterized Fe(III) cluster bound to the protein coat. Of the two Fe-Fe interactions detectable in the Fe11 complex, the shortest, at 3.0 A is particularly interesting, occurring at the same distance as a full shell (CN = 6) in ferritin, but having fewer Fe neighbors (CN = 2-3) characteristic of an intermediate in core formation. The incomplete Fe-Fe shell is much more ordered than in ferritin, suggesting that the disorder in ferritin cores may be associated with the later steps of the core growth. Differences between the Fe11 complex and the full core of ferritin indicate the possibility of intermediates in ferritin iron formation that might be like Fe11.

A sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control.

OBJECTIVES: To stimulate novel sustainable solutions to the problem of the nutritional iron deficiency, we asked: How does Nature insure proper iron nutrition of embryos and neonatal animals? Estimates of iron deficiency world-wide are 30% of the population, with women and children at the greatest risk. Recent studies linking iron deficiency with impeded cognitive development emphasizes the enormity of the impact of iron deficiency. Sustainable solutions to the problem of dietary iron deficiency have been elusive. RESULTS: Data for storage iron was examined in seeds, developing plants, embryos and developing animals. In all cases, the common source of stored iron for development was ferritin. The protein component of ferritin concentrates iron billions of times above the solubility of the free metal ion. High conservation of ferritin sequences in bacteria, plants and animals and the specificity of ferritin bioavailability either added extrinsically or intrinsically enriched in a selected soybean cultivar, showed high efficacy in curing dietary iron deficiency in the rat model. Older data on ferritin were reevaluated in light of contemporary knowledge. CONCLUSIONS: Enhancement of natural seed ferritin content by biotechnology and breeding has the potential for a sustainable solution to the problem of global dietary iron deficiency.

Structure and function of IREs, the noncoding mRNA sequences regulating synthesis of ferritin, transferrin receptor and (erythroid) 5-aminolevulinate synthase.

The synthesis of least three proteins involved in iron metabolism is coordinately regulated in animals through noncoding sequences in mRNA, the IREs; the transcription of the genes encoding the proteins are also regulated. Cellular iron is the best known effector of changes in regulation of mRNA with IREs. A hairpin loop is the secondary structure of IRES which conserve the hairpin loop sequence, CAGUGU/C. However, variable stem sequences, apparently related to mRNA-specific function, create a family of IRE regulatory sequences. At least three types of proteins recognize IRE regions: (1) Nucleases which degrade mRNAs with 3' noncoding IRES; the IRE/IRE-BP stabilizes mRNAs with 3' noncoding IRES (transferrin receptor mRNA). (2) Initiation factors/ribosomes; the IRE/IRE-BP blocks ribosome binding of mRNAs with 5' noncoding IREs (ferritin, eALAS mRNAs). (3) Initiation factors to enhance translation (ferritin mRNA) when the IRE-BP does not bind; the ferritin IRE is thus both a negative and positive control element depending on which type of protein is bound. The IRE in ferritin mRNA is the most studied IRE to date. Site-directed mutagenesis shows that sites throughout the IRE alter negative control and IRE-BP binding reflecting the fact that the footprint of the IRE-BP is over the entire IRE. Base paired flanking regions (FL) which are ferritin IRE specific, enhance the effects of IRE-BP binding on negative control. Positive control is altered by modifying the single sites in stem/internal loop but not in the hairpin loop.(ABSTRACT TRUNCATED AT 250 WORDS)

The IRE (iron regulatory element) family: structures which regulate mRNA translation or stability.

Iron regulatory elements (IREs) are a family of 28 nucleotide, non-coding elements which regulate the translation of ferritin mRNA (iron storage), erythroid delta-aminolevulinic acid synthase mRNA (heme synthesis) and the stability of the transferrin receptor (TfR) mRNA (iron uptake). IREs in the 5' end control translation (ribosome binding) and IREs in the 3' end control turnover (degradation). The specific regulator protein, the IRE-BP, is a member of the aconitase family but binds RNA only in the apo form without the Fe-S cluster. Cellular iron alters the IRE/IRE-BP interaction leading to translation of ferritin and eALAS mRNAs but degradation of the TfR mRNA. IRE function requires proximity to the 5' cap, achieved either by a short leader (eALAS) or a long, base-pairing flanking region (FL) (ferritin); a conserved triplet of FL base pairs enhances repression of ferritin mRNA. TfR mRNA has five AU-rich IREs which can also form an alternate structure with inter-IRE base pairs, in the absence of the IRE-BP. Ferritin IREs regulate both translation repression (negative control-IRE-BP dependent) and enhancement (positive control-initiation factor dependent); IRE-BP binding induces conformational changes in the FL. IREs use CAGUGU/C to form a hairpin loop with specific variations in the stem such as internal or bulge loops. A current structural model obtained using metallonucleases (1,10-phenanthroline-Cu, Fe-EDTA, Fe-bleomycin) and a preliminary analysis of the NMR spectrum, is a distorted helix with folds. The effect of cellular iron, Fe-S clusters and heme on the IRE-BP/RNA is not completely understood.(ABSTRACT TRUNCATED AT 250 WORDS)

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen.

The life of aerobes is dependent on iron and oxygen for efficient bioenergetics. Due to potential risks associated with iron/oxygen chemistry, iron acquisition, concentration, storage, utilization, and efflux are tightly regulated in the cell. A central role in regulating iron/oxygen chemistry in animals is played by mRNA translation or turnover via the iron responsive element (IRE)/iron regulatory protein (IRP) system. The IRE family is composed of three-dimensional RNA structures located in 3' or 5' untranslated regions of mRNA. To date, there are 11 different IRE mRNAs in the family, regulated through translation initiation or mRNA stability. Iron or oxidant stimuli induce a set of graded responses related to mRNA-specific IRE substructures, indicated by differential responses to iron in vivo and binding IRPs in vitro. Molecular effects of phosphorylation, iron and oxygen remain to be added to the structural information of the IRE-RNA and IRP repressor in the regulatory complex.

Cellular regulation and molecular interactions of the ferritins.

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature's unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies.

Bioiron - central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen - depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plant, and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers: heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Regulation of ferritin and transferrin receptor mRNAs.

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.

The abundance of ferritin in yolk-sac derived red blood cells of the embryonic mouse.

Circulating red blood cells formed early in development have several distinctive properties which include retention of the nucleus (mammals), large size and characteristic haemoglobin type (mammals, birds, amphibia). The primitive or embryonic red cells of early development are replaced by the definitive red cells which contain fetal or adult haemoglobin; a second developmental change occurs in the haemoglobin of some mammals (man, cattle, sheep) but does not involve a cell replacement. Circulating yolk-sac derived red cells from embryonic mice are siderocytes; elevated ferritin levels are associated with the circulating red cells of bullfrog tadpoles, but not with those of the adult frog, again indicating that red cell iron metabolism can change during development. In order to extend the observations made on an amphibian to a mammal, the ferritin content of circulating red cells from embryonic mice was determined and found to be 0.65 mg/100 mg of soluble protein; no ferritin (less than or equal to 0.007 mg/100 mg of soluble protein) was detected in adult mouse red cells. Elevated ferritin levels appeared to be specifically associated with the yolk-sac derived population of red cells since a decline in red-cell ferritin content coincided with the replacement of yolk-sac derived red cells by definitive red cells derived from the liver. Fractionation of mixtures of yolk-sac derived and liver derived red cells showed that fractions rich in the definitive red cells contained less ferritin than the mixture. The results suggest that elevated ferritin levels may be a general characteristic of the circulating, haemoglobinized red cells formed early in development.

The ferritin family of iron storage proteins.

The ferritins are a family of proteins produced in a variety of amounts and types depending on the state of development of an animal, or the state of differentiation of a particular cell type, or the environment. Iron storage is the main function of the ferritins when iron is needed for intracellular use (housekeeping) for iron proteins such as ribonucleotide reductase, cytochromes, oxidases, nitrogenases, or photosynthetic reaction centers or for extracellular use by other cells (specialized). Under abnormal conditions, such as the breach of transferrin-receptor-controlled incorporation of iron, ferritin can also serve to detoxify excess intracellular iron. The structure of ferritin is very complex, consisting of a protein coat of 24 polypeptide subunits, approximately 20 kDa, which surrounds an inorganic phase of hydrous ferric oxide. The polypeptide subunits, bundles of four alpha helices, display remarkable conservation of sequence among plants and animals, which is probably related to the necessity of forming the hollow sphere pierced by 14 channels through which iron may pass. In spite of the conserved regions of sequence, there are multiple genes for ferritin polypeptide subunits within an organism; at the moment three distinct subunit types, H H'(or M), and L, have been identified which are expressed in a cell-specific fashion. How many different subunit types exist, the influence on function, and the number of genes required to encode them are currently being actively investigated. Not only does the protein coat of ferritin display variations, the inorganic phase of ferritin can vary as well. For instance, differences can occur in the number of Fe atoms (up to 4500), as well as in the phosphorus content and in the degree of hydration and order. Such observations have depended on the use of a variety of physical techniques such as X-ray diffraction, EXAFS, and Mössbauer spectroscopy. The same approaches, as well as EPR spectroscopy, have been used to monitor the path taken by Fe as it passes from mononuclear Fe(II) outside the protein coat to polynuclear Fe(III) inside the protein coat. Both mononuclear Fe(II) and Fe(III) have been observed, as well as dimeric Fe(II)-O-Fe(III), and Fe(III)-oxo bridged clusters attached to the protein. A possible protein site for the Fe(III) cluster is a groove on the inner surface of the dimeric interface, suggested by the structure and from the affect of natural cross-links between subunit pairs.(ABSTRACT TRUNCATED AT 400 WORDS)