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Metal Ion Transport and Storage

Metal Ion Transport and Storage. Tim Hubin March 3, 1998. References. J. J. R. Frausto da Silva and R. J. P. Williams The Biological Chemistry of the Elements , Clarendon Press, Oxford, 1991. J. A. Cowan Inorganic Biochemistry : An Introduction VCH Publishers, 1994.

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Metal Ion Transport and Storage

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  1. Metal Ion Transport and Storage Tim Hubin March 3, 1998

  2. References • J. J. R. Frausto da Silva and R. J. P. Williams The Biological Chemistry of the Elements, Clarendon Press, Oxford, 1991. • J. A. Cowan Inorganic Biochemistry: An Introduction VCH Publishers, 1994. • S. J. Lippard and J. M. Berg Principles of Bioinorganic Chemistry, University Science Books, 1994. • M. D. Yudkin and R. E. Offord A Guidebook to Biochemistry, Cambridge University Press, 1980. • CHM 986, Spring 1997, Prof. Grover Everett, University of Kansas.

  3. Outline • General Concepts • Abundance of Metal Ions in Biology • Challenges in Transport and Storage of Metal Ions • Membrane Transport • Specific Metal Ions • Sodium and Potassium • Calcium • Iron • Copper • Zinc

  4. Need for Metal Ions • Metal ions must be obtained for growth and development

  5. General Transport/Storage Problems • Capture of Trace Ions from the Environment • Homeostatic Control of Concentration is essential for life • Bulk ions present in high concentration • Trace ions must be actively accumulated • Trace ions are often insoluble • Selectivity of Ion Uptake is Essential • Toxic ions must be excluded • Beneficial ions must be accumulated • Specialized Moleculecules have evolved

  6. General Transport/Storage Problems • Charged Ions must pass through a Hydrophobic Membrane • Neutral gases (O2, CO2) and low charge density ions (anions) can move directly through the membrane • High charge density cations require help • Once inside the cell, metal ions must be transported to the location of their use, then released or stored for later • Release from ligand is often not trivial • Storage requires additional molecules

  7. Mechanisms for Membrane Transport • Ionophores: special carrier molecules that wrap around metal ions so they can pass through the membrane by diffusion • Ion Channels: large, membrane-spanning molecule that form a hydrophilic path for diffusion • Ion Pumps: molecules using energy to transport ions in one direction through a membrane

  8. Mechanisms for Membrane Transport • Passive Transport: moves ions down the concentration gradient, requiring no energy source • Ionophores and Ion Channels are Passive • Active Transport: moves ions against the concentration gradient, requiring energy from ATP hydrolysis • Ion Pumps are Active • Choice of Transport Mechanism • Charge • Size • Ligand Preference

  9. Sodium and Potassium • Function: • Simple Electrolytes to create potentials (along with Cl-) • Provide counter ions for DNA, membranes, etc... • Nerve action • Concentrations: [Na+] outside cells, [K+] inside cells • Inside Red Blood cells: [Na+] = 0.01 M [K+] = 0.09 M • Outside (Blood Plasma): [Na+] = 0.16 M [K+] = 0.01 M • Ion Pump is required to maintain concentration gradients

  10. Sodium and Potassium--Ion Pump • Na+/K+-ATPase • Membrane-Spanning Protein Ion Pump • a2b2 tetrameric 294,000 dalton protein • Conformational changes pump the ions: one conformation binds Na+ best, the other binds K+ best • Hydrolysis of ATP provides the energy for conformational changes (30% of a mammal’s ATP is used in this reaction) • Antiport transport: like charged ions are transported in opposite directions • Reversing the normal reaction can generate ATP • Reaction can occur 100 time per second 3Na+in + 2Kout+ + ATP4- + H2O 3Na+out + 2K+in + ADP3- + HPO42- + H+

  11. Sodium and Potassium--Ionophore • Nonactin: microbial Na+ and K+ ionophore • Makes Na+ and K+ membrane soluble when complexed • Oxygen Donors can be modeled by Crown Ethers Nonactin

  12. Sodium and Potassium--Ion Channel • Gramicidin: ion channel-forming molecule • Helical peptide dimer • Hydrophobic outer surface interacts with membrane • Carbonyls and Nitrogens on inner surface can interact with cations as they pass through • Potassium selective: pore size and ligands select for K+ • Channels can be Voltage-Gated or activated by the binding of a Chemical Effector which changes the conformation • 107-108 ion/second may pass (Emem = 100 mV)

  13. Calcium • Function: • Signal pathways (Ex: Muscle Contraction) • Skeletal Material • Concentration: • Outside of Cell [Ca2+] = 0.001 M • Inside Cell [Ca2+] = 10-7 M • Ca2+-ATPase in Cell Membrane controls concentration

  14. Calcium--Muscle Contraction • Muscle Cells • Sarcoplasmic Reticulum(SR): muscle cell organelle • Ca2+-ATPase pumps Ca2+ into SR to concentrations up to 0.03 M • Inside SR, Ca2+ is bound by Calsequestrin, a 40,000 dalton protein (50 Ca2+ per molecule) • Hormone induced stimulation of ion channels releases Ca2+ from the SR into the muscle cell causing contraction

  15. Calcium--Storage • CaCO3 in a protein matrix makes up egg shells and coral skeletons • Calcium Hydroxyapatite in a collagen framework is the stored form of Ca2+ in bones and teeth: Ca10(PO4)6(OH)2 • Collagen: triple helix fibrous protein • Hydroxyapatite crystallizes around the collagen • Replacement of OH- by F- prevents tooth decay because F- is a weaker base • When needed, Ca2+ can be released and reabsorbed

  16. Iron • Iron is the most abundant transition metal ion in biological systems--almost all organisms use it • Availability: • Most abundant transition metal on the Earth’s crust • Nuclear Binding Energy is maximized at 56Fe • Versatility: • Fe2+/Fe3+ • High Spin/Low Spin • Hard/Soft • Labile/Inert • Coordination Number: 4,5,6

  17. Iron--Evolution • When life began: • Reducing Atmosphere: H2, H2S, CH4, NH3---> Fe2+ used • Ksp(Fe(OH)2) = 4.9 x 10-17 [Fe2+] = 5.0 x 10-3 • After Photosynthesis: • Oxidizing Atmosphere: O2---> Fe3+ used • Ksp(Fe(OH)3) = 2.6 x 10-39 [Fe3+] = 2.6 x 10-18 • Specialized Molecules were developed to solubilize Fe3+ and protect Fe2+ from oxidation • Functions:O2 transport, electron transfer, metabolism

  18. Iron--Siderphores • Siderophores: class of bacterial ionophores specific to Fe3+ • Small molecules released into the environment • Complexation of Fe3+ solubilizes it for uptake • Ligands are Catechol and Hydroxamic Acid chelates • Enterobactins: 3 catechols • Ferrichromes: 3 hydroxamic acids, cyclic peptide • Ferrioxamines: 3 hydroxamic acids, acyclic peptide Catechol Hydroxamic Acid

  19. Iron-Enterobactin • Structure: 3 catechol chelates bound to a 12-membered ring • Kf = [Fe(ent)3-]/[Fe3+][ent6-] = 1049 • Complex anion is soluble • Spectroscopy: • UV-Vis: like [Fe(cat)33-] • D structure assigned by [Cr(ent)3-] circular dichroism • Crystal Structure: [V(ent)2-]

  20. Iron-Enterobactin • Getting Fe3+ into the cell • [Fe(ent)3-] binds to an uncharacterized receptor on cell surface • Active transport process takes the complex inside • Mechanism of iron release is still unknown • Hydrolysis of ligand • Reduction to Fe2+ would labilize ion • Ered = -750 mV vs NHE at pH = 7 • Lowering pH would facilitate reduction • Intracellular ligand

  21. Iron-Transferrin • Transferrin: Mammalian transport ab dimer protein • 80,000 dalton protein carries 2 Fe3+ ions in serum • Iron captured as Fe2+ and oxidized to Fe3+ • CO32- must bind at same time: Synergism • Taking Iron into the cell--Endocytosis

  22. Iron--Ferritin • Family of protein found in animals, plants, and bacteria • Structure: • symmetric, spherical protein coat of 24 subunits • Subunits are 175 amino acids, 18,500 daltons each • Channels on 3-fold axes are hydrophilic: iron entry • Inside surface is also hydrophilic • Inner cavity • 75 Å inner diameter holds 4500 iron atoms • Iron stored as Ferrihydrate Phosphate [(Fe(O)OH)8(FeOPO3H2) . nH2PO4] • Iron-protein interface: binding of core to protein is believed to be through oxy- or hydroxy- bridges

  23. Iron-Ferritin • Iron thought to enter as soluble Fe2+, then undergo oxidation by O2 in channels or inside the cavity • Biomineralization: synthesis of minerals by organisms • Ferritin is synthesized as needed • Normal iron load is 3-5 grams in a human • Ferritin is stored in cells in the bone marrow, liver, and spleen • Siderosis: iron overload (60 g can be accumulated) • doposits in liver, kidneys, and heart • treated by Chelation Therapy (desferrioxamine)

  24. Copper • Function • O2 transport (hemocyanin in crustacean and mollusks) • O2 activation (Cu oxidases) • electron transfer (plastocyanin) • Availability • Third most abundant transition metal ion in organisms • 300 mg in a human body • Ksp(Cu(OH)2) = 2.6 x 10-19 [Cu2+] = 2.6 x 10-5 • Solubility means less specialized transport and storage

  25. Copper--Transport • Ceruloplasmin • 132,000 dalton glycoprotien (7% carbohydrate) • Binds 95% of the Cu2+ in human plasma • 6 Cu2+ sites: 1 Type I, 1 Type II, 4 Type III

  26. Copper--Transport • Ceruloplasmin • Biological role not fully understood • transport • oxygen metabolism • Wilson’s Disease • genetic disorder of low ceruloplasmin levels • Cu2+ accumulates in the brain and liver • treated by chelation therapy (EDTA)

  27. Copper--Storage • Metallothioneins • Small (6000 dalton) metal storage protein family • 20 cysteine residues select for soft metals: • Cu+, Zn2+, Cd2+, Hg2+, Pb2+ • X-Ray structure of Cd2+/Zn2+ complex shows tetrahedrally coordinated metal clusters • Up to 20 Cu+ can bind • Mechanism of Cu+ and Zn2+ homeostasis • Detoxification by removal of soft ions: Cd2+, Hg2+, Pb2+

  28. Zinc • Function: • Lewis Acid catalyst • Structural control • Substrate binding • 200 Zn2+ proteins known • Availability: • abundant in biosphere, highly soluble • all forms of life require it (2 g in a human) • Versatile: labile, varied geometries (no LFSE), hard/soft • No redox chemistry

  29. Zinc • Transport: Serum Albumin • Constitutes more than half of all serum protein • plays a role in Cu2+ transport as well • 600 amino acid protein • poorly described • Zn2+ pumps? • high concentrations in some vesicles suggest pumps • [Zn2+]cytoplasm = 10-9 M [Zn2+]vesicle = 10-3 M • Storage: Metallothionein chemistry similar to Cu2+

  30. Summary • Transport and Storage of Metal ions: • Necessary • Diverse • Evolved • Largely Unknown

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