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Iron (Fe 2+ /Fe 3+ ) Transport and Trafficking in Mammals Bertini et al Ch. 5 and 8

Iron (Fe 2+ /Fe 3+ ) Transport and Trafficking in Mammals Bertini et al Ch. 5 and 8. Prof . Arthur D. Tinoco University of Puerto Rico, Rio Piedras Campus. 1. Multiple Functions of Iron in Living Organisms. A. O 2 transport. Fe 2+. Hemoglobin. N. N. N. N. Hemerythrin

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Iron (Fe 2+ /Fe 3+ ) Transport and Trafficking in Mammals Bertini et al Ch. 5 and 8

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  1. Iron (Fe2+/Fe3+) Transport and Trafficking in Mammals Bertiniet al Ch. 5 and 8 Prof. Arthur D. Tinoco University of Puerto Rico, Rio Piedras Campus

  2. 1. Multiple Functions of Iron in Living Organisms A. O2 transport Fe2+ Hemoglobin N N N N Hemerythrin (Used by two phyla of marine invertebrates: sipunculids and brachiopods) Fe3+ Iron used in its Fe(II) and Fe(III) forms.

  3. 1. Multiple Functions of Iron in Living Organisms • B. Elimination of harmful O2 metabolites • Reactive oxygen species (ROS); although also responsible for the controlled production of ROS • C. Energy-transducing pathways of electron transport • The respiratory chain • D. Redox and oxygenation reactions • Relative ease of switching between Fe2+ and Fe3+ • E. Formation of deoxyribonucleotides by ribonucleotide reductase • DNA Synthesis • F. Fixation and activation of nitrogen and hydrogen

  4. Iron Containing Proteins Cytochromes RibonucleotideReductase Transferrin Ferritin Hemoglobin

  5. 2. How do we obtain soluble and bioavailable iron? Ingest iron in elemental form or as Heme (Fe2+) or nonheme Fe3+ www.Nineplanets.org pH ~ 3-4 (Normal) pH ~1-2 (Metabolism)

  6. 2A. Iron Digestion and Transport from the Intestine to Blood I. Production of Fe2+ in the stomach: A. Elemental iron is dissolved via acidification coupled with oxidation to Fe2+ 2 H+(aq) + Fe(s) H2(g) + Fe2+(aq) B. Dietary Fe(III) is reduced to Fe2+ Fe3+(aq) Fe2+(aq) II. Fe2+ is bound by gastroferrin and taken to the intestine www.Nineplanets.org Ferrireductase (cell surface) Or reductants in the diet (ascorbic acid)

  7. 2A. Iron Digestion and Transport from the Intestine to Blood • III. An Fe2+ uptake system then shuttles the iron from the intestinal lumen into the enterocytes lining the small intestine via the divalent metal transporter (DMT1) • A secondary active transport Fe2+-H+ symporter • Uses a H+ concentration gradient and the electrical potential of the membrane to drive Fe2+uptake • IV. Fe2+ is exported across the enterocyte membrane into the bloodstream by ferroportin/IREG1 • V. A critical oxidation event takes place before release into the bloodstream • Fe2+ (aq) Fe3+(aq) www.Nineplanets.org Hephaestin

  8. 2A. Iron Digestion and Transport from the Intestine to Blood www.Nineplanets.org

  9. But why oxidize Fe2+ when it is far more soluble than Fe3+ at pH 7.4???? www.Nineplanets.org

  10. 2A. Iron Digestion and Transport from the Intestine to Blood • Fe2+ can generate reactive oxygen species (ROS) via Fenton chemistry • If uncontrolled, these species cause great harm to the body • Harmful (potentially irreversible) cellular damage when ROS levels exceed the antioxidant defense and cells undergo oxidative stress • Fe3+ has to be regulated completely at the level of uptake • If not, Fe3+(aq) Fe(OH)3(s) www.Nineplanets.org X Low massmolecules (lmm) X Fe3+-lmm(aq) Fe2+(aq) Transferrin Reactive oxygen species Fe2-Tf

  11. 2B. Fe3+ Transport by Transferrin • Transferrin bound iron is dominant iron form in the blood. • Some nontransferrin bound iron (NTBI) but well-regulated • -High NTBI usually indicative of a diseased state

  12. 2BI. Receptor Mediated Endocytosis coupled with Redox and Transporter assisted transport Key Players: Apotransferrin (apoTf) Holotransferrin (holoTf) Transferrin Receptor (TfR1 and TfR2) Clathrin Fe3+Chelator Steap3 Divalent Metal Transporter 1 (DMT1)

  13. 2BI. Receptor Mediated Endocytosis coupled with Redox and Transporter assisted transport Steps involved: pH dependent process that modulates apoTf and holoTf affinity for TfR Endosome formation aided by clathrin-coating to protect against lysosomal degradation and to allow recycling of Tf and TfR ATP-dependent pump that lowers the endosome pH from 7.4 to 5.5 Iron release from Tf Iron escape from the endosome as Fe2+

  14. Transferrin Receptor (TfR) Transports Fe2TF into Cells dimer axes • TfR is a homodimeric protein found at the cell membrane • Binds two molecules of Fe2TF • At pH 7.4, Fe2TF preferentially binds to the TfR (Kd is nM affinity) • At low pH (~5.5) apo hTF preferentially binds to the TFR (nM affinity). Ectodomain (AA 89 - 760) Lawrence et al. Science1999, 286, 779-782. Cytoplasmic region (AA1 - 67)

  15. 2BII. One proposed mechanism for the TfRMediated Endocytosis: Chelator driven 1. pH decrease from 7.4 to 5.5 does not trigger Fe3+ release from transferrin even with the higher affinity of apoTf for the TfR 2. Fe3+ chelation (possibly by ATP or citrate) coupled with metal binding site residue protonation results in Fe3+dissociation 3. Dissociated Fe3+ is reduced to Fe2+ by Steap3 4. Fe2+ is transported to the cytosol via DMT1

  16. 2BII. One proposed mechanism for the TfR Mediated Endocytosis: Chelator driven • Labile Fe2+ Pool • Chelator • Fe3+ • Steere et al. BiochemBiophysActa2012, 1820, 326-333.

  17. 2BII. Another proposed mechanism for the TfR Mediated Endocytosis: Lowered Metal Affinity driven 1. The redox potential of Fe3+ bound to transferrin is modulated by holoTf bound to TfR E (V) (Fe3+)2-Tf (Fe2+)2-Tf -0.56 [(Fe3+)2-Tf]2-TfR [(Fe2+)2-Tf]2-TfR -0.30 2. Fe2+has a much lower affinity for Tf and easily dissociates log β (Fe3+)2-Tf 43.5 (Fe2+)2-Tf 13 3. Dissociated Fe2+ is transported to the cytosol via DMT1 TOO NEGATIVE! Physiologically Feasible; Reduced by Steap3

  18. 2BII. Another proposed mechanism for the TfR Mediated Endocytosis: Lowered Metal Affinity driven log β = 43.5 Steap3 • Labile • Fe2+ Pool E1/2 Fe2+ Fe3+Fe2+ log β = 13 (Fe3+ )2Tf: -526 mV [(Fe3+)2Tf]2-TfR: -300 mV Kraiter, D.C.; Zak, O.; Aisen, P.; Crumbliss, A.L. Inorg. Chem., 1998, 37, 964-968. Dhungana, S.; Taboy, C.H.; Zak, O.; Larvie, M.; Crumbliss, A.L.; Aisen, P. Biochemistry, 2004, 43, 205-209. Bou-Abdallah, F. Bioenergetics, 2012, 3.

  19. 2C. Iron Trafficking in the Cell In reticulocytes (immature red blood cells), the transferrin endocytotic cycle is complete within 2 mins; longer times in other cells. The labile Fe2+ pool generated is distributed for storage and for function. Labile iron pool Iron Storage by Ferritin Utilization of Iron

  20. 2CI. Acquisition of Iron in Mitochondria for Heme, Iron-Sulfur Cluster Synthesis • Some of the Fe2+ shuttled to the mitochondrion is acquired by ferrochelatase and inserted into protoporphyrin-IX to form heme • Heme transported out of the mitocondria and into the cytoplasm becomes part of hemoglobin • 75% of iron in the body is in hemoglobin • Fe-S clusters (of several different forms) are synthesized and integrated into electron transport proteins and redox-regulated enzymes, amongst others

  21. 2CII. Ferritin: Iron Storage Protein • a. Huge protein found in plants, animals, and bacteria • 24 polypeptide subunits (~20 kDa each); • 480 kDa • - Assemble into a hollow sphere • - Feature symmetry around • 2-, 3-, and 4-fold axes • - Three subunits create 8 pores • that serve as ion channels • b. Very soluble protein that raises cellular • iron levels to “mM” amounts • (iron is present in cells at μM) • c. Deletion of the ferritin gene is deadly

  22. 2CII. Ferritin: Iron Storage Protein • d. Nanoreactor core • Site of iron oxide mineralization • The majority of the subunits are ferroxidase (Fox) sites ~36-2,200 Fe2+ (aq) + ~18-1,100 O2 ~18 -1,100 [Fe3+-O-O-Fe3+ ] (aq) ~36 - 2,200 Fe3+ (aq) + ~18 - 1,100 H2O2 [Fe 2 O3· (H2O)x]~18-1,100 + ~54 - 3,300 H+ (aq) ms timescale ms timescale Travel from Fox site to the nanocavity takes minutes to hours Slow metal hydrolysis rate believed to prevent a large pH drop that would hydrolyze and destroy the protein

  23. 2CIII. Maintaining Iron Homeostasis When the iron level in our body falls below the optimal level, we mobilize ferritin to access our stored iron.

  24. Mobilization in Metabolism www.Nineplanets.org

  25. Metabolism of biopolymers Polysaccharides Triacylglycerols Nucleic Acids Proteins DNAse/RNAse Lipase Protease Amylase Monosaccharides Fatty Acids Nucleotides Amino Acids • Catabolism of nucleotides yields an insignificant amount of metabolic energy. Citric Acid Cycle in Mitochondria Produces 90% of the energy used by aerobic human cells.

  26. Excess Monomers Stored in Polymeric Form • Some tissues are specialized for the long-term storage of nutrients that are not catabolized for energy. • Fatty acids are stored as triacylglycerols in adipose tissue. • Monosaccharides are stored as glycogen in liver and muscle. • Branch structure allows for efficient addition and removal of sugar units.

  27. Excess Monomers Converted as Source of other Biomolecules • A. While amino acids (AA) can be used to generate more proteins, proteins are not a storage pool for amino acids. • Excess AA can be converted to carbohydrates or to fats. • B. AA and glucose are required for synthesis of nucleotides.

  28. Mobilization of Stored Polymers to Extract Monomers • AA, monosaccharides, and fatty acids are known as metabolic fuels. When these fuel supplies are exhausted, the body mobilizes its stored resources by convertings its biopolymers into their monomeric units. • Most of the body’s tissues uses glucose as primary metabolic fuel. • Liver mobilized to break down glycogen via phosphorolysis.

  29. Mobilization of Stored Polymers to Extract Monomers • When glycogen stores are depleted, amino acids are mobilized to generate energy. • Regardless of needs for amino acids, cellular proteins are continuously degraded via two major mechanisms. • Lysosome: An organelle containing proteases and other hydrolytic enzymes. • Proteasome:A barrel-shaped multiprotein complex. • - A protein has to be covalently tagged at a Lys residue with a small protein called ubiquitin (C- terminus) in order to be hydrolyzed by the proteasome.

  30. Mobilization of Stored Polymers to Extract Monomers • - A chain of at least 4 ubiquitins is required to mark a protein for destruction by the proteasome.

  31. Mobilization of Stored Polymers to Extract Monomers • C. Only when the supply of glucose and amino acid runs low does adipose tissue mobilize its fat stores. • A lipase yields fatty acids, which are released into the bloodstream. • The released fatty acids bind to circulating proteins (i.e. albumin) due to their poor aqueous solubility. • Even if a diet includes almost no fat, it is very hard to mobilize stored fat.

  32. 2CIIIa. Two Proposed Routes for Ferritin Mobilization • i. Digestion of ferritin by the lysosome • The protein is “torn apart” by acid hydrolysis • Acidification helps to solubilize the metal • - A very uncontrolled method for metal release • ii. Regulated unfolding/opening of the ferritin pores • A signal is sent indicating that iron resources are being depleted • The gates of the ferritin pores open or unfold • Reducing agents such as NADH (reduced nicotinamide adenine dinucleotide) and FMN (flavin mononucleotide) enter to reduce Fe3+to Fe2+ • Chelators bind the iron and solubilize it

  33. 2CIIIb. Ferritin Regulation on DNA and mRNA level • DNA as a target for regulating transcription • - Ferritin genes are regulated by iron and dioxygen levels in addition to hormones, growth factors, etc. • mRNA as a target for regulating translation • The iron-responsive element (IRE) is a ferritin mRNA regulatory sequence, which has an iron-binding pocket • As iron concentrations increase, IRE senses this increase via iron binding and this triggers the expression of more ferritin to uptake and store this iron

  34. 2CIIIc. Depleting excess iron There are intracellular mechanisms for dealing with reasonable excess of metals.

  35. 2CIIIc. Depleting excess iron • Intracellular chelation • Extrusion • A transporter is activated to expel excess metal from the cell • A metal is shuttled to a storage compartment to make it inert • Exclusion • A metal’s route of entry is removed • No specific physiological route for clearing iron from the body except from skin shedding.

  36. 2CIIId. Iron Overload Diseases • Toxic levels of iron can be achieved in a number of ways: • Excessive intake in food • Multiple blood transfusions • Mutation of proteins responsible for iron uptake and transport • Hemochromatosis is the result of a mutation of the HFE protein • HFE is associated with the TfR and acts to depress iron uptake when the iron limit is reached • Treatment of iron overload diseases can consist of bleeding patients or chelation therapy.

  37. Iron Related Diseases • WHO considers iron deficiency to be the #1 nutritional disorder world-wide with ~80% of diets iron deficient • Iron related diseases - Sickle cell anemia - Thalassemia • Iron overload - Hemochromatosis (1 in 250 persons of Northern European descent) - Acquired iron overload (from multiple transfusions) • Iron implicated in a wide variety of other diseases, i.e., heart and liver disease, diabetes, neurodegenerative diseases, cancer and restless leg syndrome

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