Nebraska Redox Biology Center Educational Portal


Iron (Fe) is the fourth most abundant element in the Earth's crust is an essential trace element required for survival by most organisms from bacteria to mammals. Iron functions as a component of wide range of proteins, including many enzymes and hemoglobin. Iron is necessary for normal growth, development, normal cellular functioning, and synthesis of several hormones. Iron can exist in oxidation states ranging from -2 to +6 [ 1, 2, 3, 4 ]. Fe2+ and Fe3+ are the major oxidation states of iron in biological systems. The redox cycling between these oxidation states defines o mechanism of Fe-mediated electron transfer and mechanism of reversible coordination of ligands. The major biological ligands for iron are oxygen, nitrogen, and sulfur atoms [ 1, 2, 3, 4, 5, 6 ].

There are four major classes of iron-containing proteins in the mammalian system: iron-containing heme proteins (hemoglobin, myoglobin, cytochromes), iron-sulfur enzymes (flavoproteins, hemeflavoproteins), proteins for iron storage and transport (transferrin, lactoferrin, ferritin, hemosiderin), and other iron-containing or activated enzymes (sulfur, nonheme enzymes) [ 1, 2, 3, 4 ]. The Recommended Dietary Allowance (RDA) for all age groups of men and postmenopausal women is 8 mg/day; the RDA for premenopausal women is 18 mg/day. The median dietary intake of iron is approximately 16 to 18 mg/day for men and 12 mg/day for women. The Tolerable Upper Intake Level (UL) for adults is 45 mg/day. Dietary iron has two main forms: heme and nonheme. Plants and iron-fortified foods contain nonheme iron only, whereas meat, seafood, and poultry contain both heme and nonheme iron. Heme iron, which is formed when iron combines with protoporphyrin IX, contributes about 10% to 15% of total iron intakes in western populations. Most of the elemental iron in humans is located in hemoglobin. Remaining iron is stored in the form of ferritin in the liver, spleen, and bone marrow or is located in myoglobin in muscle tissue. The major sources of heme iron in the human diet include meat and seafood. Dietary sources of nonheme iron include nuts, beans, vegetables, and fortified grain products [ 7, 8 ].

Disruptions in iron homeostasis resulting in iron deficiency or overload is associated with human diseases. Iron metabolism is depend on two distinct regulatory systems. One system is based on the hormone hepcidin and the iron exporter ferroportin, and another is based on iron-regulatory proteins that bind iron-responsive elements (IRE) in regulated messenger RNAs [ 1, 2, 3, 4 ]. Iron excess leads to the production of hydroxyl radical and resulting in oxidative stress in sensitive tissues. Iron mediated ROS production is recognized as important factor of pathological situations and cell death in a variety of organisms [ 9 ].

Fe2+ + H2O2 → Fe3+ + OH- + .HO;

In the United States, concentrations of Fe in soils range from less than 0.5% in the southern Gulf states to more than 5.5% in mountainous areas and many parts of the western United States (United States Geological Survey). In general, Fe in soils is tightly bound to chelating agents and is mostly unavailable for absorption by animals. Hovewer, exposure to an acidic environment may result in increased iron bioavailability [ 7, 8, 10 ].

Map of soil iron content in the U.S. (red = high iron areas). Courtesy of U.S. Department of the Interior, U.S. Geological Survey, Mineral Resources.

Iron circulates in plasma in complex with glycoprotein transferrin, which has two high-affinity binding sites for Fe3+. Binding to transferrin maintains iron in a soluble form, serves as a major vehicle for iron delivery into cells via the transferrin receptor, TfR1, and limits the generation of hydroxyl radicals. In humans, plasma transferrin is normally about 30% saturated with iron. A transferrin saturation <16% indicates iron deficiency, whereas >45% saturation is a sign of iron overload. When the saturation exceeds 60%, non-transferrin-bound iron is accumulating in the circulation and damaging parenchymal cells [ 4, 11, 12, 13, 14 ]. The iron homeostatic system is maintaining transferrin saturation at physiological levels, responding to signals from pathways that consume iron and sending signals to the cells that supply iron to the blood stream. Iron is released into the circulation from duodenal enterocytes, which absorb 1-2 mg of dietary iron per day, and from macrophages, which internally recycle 20-25 mg of iron from senescent erythrocytes. Hepatocytes are the major site of iron storage and they secrete the regulatory hormone hepcidin (Hamp, LEAP1) [ 4, 14 ]. Hepcidin regulates systemic iron fluxes and controls plasma iron levels by binding to the iron exporter ferroportin (SLC40A1) on the surface of iron-releasing cells, triggering its lysosomal degradation and hence reducing iron transfer to transferrin. Hepcidin is a defensin family member 25 amino acid peptide is generated from an 84 amino acid prepropeptide by furin mediated cleavage. Hepcidin is secreted from hepatocytes and circulates in plasma bound to α 2-macroglobulin. Disorders that perturb hepcidin production consequently cause iron deficiency (high hepcidin levels) or iron overload (hepcidin deficiency) [ 4, 13, 14 ].

Regulation of Systemic Iron Homeostasis. Divalent metal transporter 1 (DMT1) at the apical membrane of enterocytes takes up iron from the lumen of the duodenum after DCYTB reduces Fe3+ to Fe2+. Ferroportin at the basolateral membrane cooperates with hephaestin that oxidizes Fe2+ to Fe3+. Iron-loaded (diferric) transferrin (Tf-Fe2), indicated by red dots, supplies iron to all cells by binding to the transferrin receptor 1 (TfR1) and subsequent endocytosis. TfR1 is highly expressed on hemoglobin-synthesizing erythroblasts. Hepatocytes sense transferrin saturation/iron stores and release hepcidin accordingly. Red cell iron is recycled by macrophages via ferroportin and the ferroxidase ceruloplasmin. In iron overload (left), high hepcidin levels inhibit ferroportin-mediated iron export by triggering internalization and degradation of the complex to reduce transferrin saturation. Hepcidin expression is high. In iron deficiency (right), iron is released by ferroportin into the circulation. Hemoglobin-derived heme is catabolized in macrophages by hemoxygenase-1 (HOX1). Hepcidin expression is low [ 4 ].

Iron Absorption. Inorganic dietary iron is absorbed by mammalian duodenal enterocytes using divalent metal transporter 1 (DMT1/SLC11A2). The iron adopts the oxidized state and must first be reduced by the membrane-associated ferrireductase DcytB (Cybrd1). Heme iron is absorbed independently by uncertain mechanisms. Hemoxygenase 1 (HOX1) is involved in intracellular heme iron release. Cytosolic iron can then be exported into the circulation by the basolateral iron exporter ferroportin. Enterocytic iron export through ferroportin requires hephaestin, a multicopper oxidase homologous to ceruloplasmin, which oxidases Fe2+ to Fe3+ for transferrin binding [ 4, 15, 16, 17, 18, 19 ].

Majority dietary iron (approximately 25 mg/day) is dedicated to hemoglobin synthesis. TfR1 mediates erythroid iron acquisition. An additional possible way of erythroblast iron acquisition is a ferritin released from macrophages. Erythroblasts not only acquire but also handle large amounts of iron. Potentially, iron may be directly transported from endosomes into mitochondria through a direct contact between both organelles. Iron is imported into mitochondria by the inner membrane protein mitoferrin 1 (Mfrn1/ SLC25A37). This process is facilitated by the ABCB10 (ATP-binding cassette, subfamily B, member 10) protein, which is thought to stabilize Mfrn1. Although erythroblasts consume large amounts of iron, they have to maintain safety mechanisms to avoid iron and/or heme excess: iron can be stored in ferritin or exported by ferroportin [ 4, 20, 21, 22, 23, 24 ].

Cell Biology of Iron Metabolism Most cells acquire plasma iron via transferrin receptor 1 (TfR1)-mediated endocytosis of transferrin-bound iron. In endosomes, iron is freed from transferrin and reduced to Fe2+ by STEAP metalloreductases prior its release into cytosol via divalent metal transporter 1 (DMT1); transferrin and TfR1 return to the plasma membrane to be used for further cycles. DMT1 also functions in the apical absorption of dietary iron after reduction by DCYTB and possibly other ferrireductases. Other iron acquisition pathways are symbolized (e.g., acquisition of heme iron from red blood cells by macrophages). Iron uptake systems feed the so-called labile iron pool (LIP). The LIP is utilized for direct incorporation into iron proteins or iron transport to mitochondria via mitoferrin (Mfrn), where the metal is inserted into heme and Fe/S cluster prosthetic groups. Proteins promoting heme transport into and out of cells have been identified. The fraction of the LIP that is not utilized for metalation reactions can be exported via ferroportin, which works together with ferroxidases for iron loading onto transferrin, or stored in a nontoxic form in ferritin shells. Ferritin can be released into the extracellular milieu by unknown mechanisms and interact with specific receptors on the cell surface. Some cells also express a mitochondrial form of ferritin to protect the organelle against iron-induced toxicity. The size of the LIP is determined by the rate of iron uptake, utilization, storage, and export; these processes must be coordinately regulated to avoid detrimental iron deficiency and prevent iron excess [ 4 ].

Cellular iron homeostasis. The regulation of iron homeostasis by cells is very similar to similar control at the systemic level an involve regulation of iron uptake, utilization and storage to maintain physiological level avoid toxicity. However, the machinery and the mechanisms are entirely different. In contrast to systemic iron metabolism, cellular iron traffic also involves regulated iron excretion. Transferrin (Tf-Fe2) is a major iron source for mammalian cells, which they import using the high-affinity transferrin receptor TfR1. The Tf-Fe2/TfR1 complex is internalized by clathrin-dependent endocytosis. Subsequent acidification triggers conformational changes in both transferrin and its receptor that promote the release of iron. The iron is then reduced to Fe2+ by STEAP familly of metalloreductases for transport into the cytosol via DMT1 transporter. The specialized cells are able to acquire iron in the form of heme. The nature of the enterocytic heme importer remains unclear [ 4, 25, 26 ]. Iron export occurs from all cells, hovewer, it is particularly important in cells that maintain plasma iron levels. Such cells include macrophages and duodenal enterocytes, and in fetal develoment iron export is mediated by cells of the extraembryonic visceral endoderm (ExVE) and placental syncythiotrophoblasts. These cells express relatively high levels of ferroportin (SLC40A1) involved in iron release. Thus, ferroportin transports Fe2+ and acts with ferroxidases or ceruloplasmin that determine iron extraction from the ferroportin channel and subsequent loading onto plasma transferrin. The cells are able to export iron in the form of heme and FLVCR1 is suggested to promote heme efflux [ 4, 27, 28, 29 ]. Intracellular iron is stored in redox-inactive form within the nanocavity of ferritin heteropolymers made of 24 subunits of heavy (FtH1) and light (FtL) chains [ 30 ].

Intracellular iron homeostasis is mainly regulated post-transcriptionally by the IRE/IRP regulatory system. The iron regulatory proteins (IRP1 and IRP2) can recognize a regulatory mRNA motif named iron responsive element (IRE), a conserved RNA element located in the 3' and 5' untranslated regions (UTR) of mRNAs that encode proteins involved in iron metabolism [ 4, 31 ].

Human and mouse sequences of iron-responsive elements [ 32 ].

A canonical iron responsive element structure is composed of a 6-nucleotide apical loop (5'-CAGWGH-3'; whereby W stands for A or U and H for A, C or U) on a stem of five paired nucleotides, a small asymmetrical bulge with an unpaired cytosine on the 5' strand of the stem, and an additional lower stem of variable length. Iron regulatory proteins inhibits translation initiation when bound to the 5' UTR IREs. IRP binding to the IRE motifs within the 3' UTR prevents mRNA endonucleolytic cleavage and subsequent degradation. IRP/IRE interactions regulate the expression of the mRNAs encoding proteins for iron acquisition (transferrin receptor 1, Tfrc; divalent metal transporter 1, Dmt1), iron storage (ferritin H, Fth1; ferritin L, Ftl), iron utilization (erythroid 5-aminolevulinic acid synthase, e-Alas; mitochondrial aconitase, Aco2; Drosophila succinate dehydrogenase, Sdh), iron export (ferroportin, Fpn), oxygen availability (hypoxia-inducible factor 2 alpha, Hif2aplha) and cell cycle (CDC14A). The mRNAs of Fth1, Ftl, e-Alas, Aco2, Sdh, Fpn and Hif2alpha contain one single IRE in their 5'UTRs. The mRNA of Slc11a2 and CDC14A contain also one single IRE in its 3'UTR and Tfrc mRNA is so far the only known mRNAs with five IREs, all of them located in its 3'UTR Thus, the IRPs act as key regulators of cellular iron homoeostasis via expression control of iron metabolism-related genes [ 31, 33, 34, 35 36, 37 ].

Regulation of Cellular Iron Metabolism In iron-deficient cells (right), iron regulatory protein 1 (IRP1) or IRP2 bind to cis-regulatory hairpin structures called iron-responsive elements (IREs), present in the untranslated regions (UTRs) of mRNAs encoding proteins involved in iron transport and storage ( Muckenthaler et al., 2008). The binding of IRPs to single IREs in the 5' UTRs of target mRNAs inhibits their translation, whereas IRP interaction with multiple 3' UTR IREs in the transferrin receptor 1 (TfR1) transcript increases its stability. As a consequence, TfR1-mediated iron uptake increases whereas iron storage in ferritin and export via ferroportin decrease, thereby increasing the LIP. In iron-replete cells (left), the FBXL5 iron-sensing F-box protein interacts with IRP1 and IRP2 and recruits the SKP1-CUL1 E3 ligase complex that promotes IRP ubiquitination and degradation by the proteasome; IRP1 is primarily subject to regulation via the assembly of a cubane Fe/S cluster that triggers a conformational switch precluding IRE-binding and conferring aconitase activity to the holoprotein. IRPs also modulate the translation of the mRNAs encoding the erythroid-specific ALAS2 heme synthesis enzyme, the mitochondrial aconitase (ACO2), and the HIF2α hypoxia-inducible transcription factor. Single 3' UTR IRE motifs are present in the DMT1 and CDC14A mRNAs, but their role and mechanism of function are not yet fully defined [ 4 ].

Iron-sulfur clusters are [Fe-S] ubiquitous and evolutionary ancient prosthetic groups that represents one of the major biological function of iron and are required for life processes. [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. More then 120 distinct types of proteins are known to bind [Fe-S] clusters [ 38, 39, 40 ]. The ability to delocalize electron density over both Fe and S atoms makes [Fe-S] clusters ideal for their primary role in mediating biological electron transport. [Fe-S] clusters are major components in the photosynthetic and respiratory electron transport chains, they define the electron transport pathways in numerous redox enzymes, and constitute the redox-active centers in ferredoxins [ 39, 41, 42 ].

Functions of some biological [Fe-S] clusters. SAM is S-adenosylmethionine; acetyl-CoA is acetyl coenzyme A; FNR is fumarate and nitrate reduction; IRP is iron-regulatory protein; IscR is iron-sulfur cluster assembly regulatory protein; PRPP is phosphoribosylpyrophosphate [ 39 ].

For example, the SoxR protein senses oxidative stress via oxidation of the [2Fe-2S]2+ cluster and stimulates transcriptional expression of SoxS gene, which is responsible for activating the transcription of numerous enzymes in the oxidative stress response [ 43, 44 ]. The fumarate and nitrate reduction protein is an oxygen sensing transcriptional regulator that functions by oxygen-induced conversion of the DNA-binding dimeric [4Fe-4S]2+ form to a monomeric [2Fe-2S]2+ form to control the expression of genes involved in the aerobic and anaerobic respiratory pathways of Escherichia coli [ 43 ].

Structures and core oxidation states of crystallographically defined [Fe-S] clusters. Iron is shown in red, and sulfur is shown in yellow. The [3Fe-4S]- cluster has only been observed as a fragment in heterometallic [M-3Fe-4S]+ clusters (not shown) in which M is a divalent transition metal ion [ 39 ].

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