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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

Metal Ion Transport and Storage

Tim Hubin

March 3, 1998

  • 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.
  • 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
need for metal ions
Need for Metal Ions
  • Metal ions must be obtained for growth and development
general transport storage problems
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 Molecules have evolved
general transport storage problems8
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
mechanisms for membrane transport
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 molecules that form a hydrophilic path for diffusion
  • Ion Pumps: molecules using energy to transport ions in one direction through a membrane
mechanisms for membrane transport12
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
sodium and potassium
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
sodium and potassium ionophore
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


sodium and potassium ion channel
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)

Active Form

Inactive Form

sodium and potassium ion pump
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+

  • 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
calcium muscle contraction
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
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
  • 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
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
iron siderophores
  • 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


Hydroxamic Acid

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-]
iron enterobactin35
  • 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
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
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


The Gene Pool

iron ferritin44
  • 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)
  • 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
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
copper transport47
  • 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)
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+
  • 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
  • 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
    • Zn2+-ATPase has been identified
  • Storage: Metallothionein chemistry similar to Cu2+
  • Transport and Storage of Metal ions:
    • Necessary
    • Diverse
    • Evolved
    • Largely Unknown