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.
• 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.
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
Need for Metal Ions
• Metal ions must be obtained for growth and development
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 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
• 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 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
• 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
• Nonactin: microbial Na+ and K+ ionophore
CH3 O
CH3
O
O
O
CH3
CH3
O
CH3
O
O
CH3
O
O
O
O
O
CH3
CH3
Nonactin
• Makes Na+ and K+ membrane soluble when complexed
• Oxygen Donors can be modeled by Crown Ethers
O
O
O
O
O
O
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
• 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+
Calcium
• Function:
– Signal pathways (Ex: Muscle Contraction)
– Skeletal Material
• Concentration:
– Outside of Cell
– Inside Cell
[Ca2+] = 0.001 M
[Ca2+] = 10-7 M
• Ca2+-ATPase in Cell Membrane controls concentration
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 Fis a weaker base
• When needed, Ca2+ can be released and reabsorbed
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
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
OH
OH
Catechol
O
OH
C N
R
R
Hydroxamic Acid
» Enterobactins: 3 catechols
» Ferrichromes: 3 hydroxamic acids, cyclic peptide
» Ferrioxamines: 3 hydroxamic acids, acyclic peptide
Iron-Enterobactin
• Structure: 3 catechol chelates bound to a 12-membered ring
• Kf = [Fe(ent)3-]/[Fe3+][ent6-] = 1049
OH
• Complex anion is soluble
OH
O C
• Spectroscopy:
– UV-Vis: like [Fe(cat)33-]
– D structure assigned by [Cr(ent)3-]
circular dichroism
HO
NH
O
O
O
O
O
HO
NH
C
HN
O
O
C
O
HO
• Crystal Structure:
[V(ent)2-]
HO
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
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
O
C
O
Tyr
O
O
O
Fe
O
Asp
O
N
NH
His
Tyr
• 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
Ferritin
The Gene Pool
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)
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
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
Type I
Type II
S
N
N
Type III
L
L
Cu
R
L
Cu
L
L
L
L
L
Cu
L
L L
O
R
R = S, N, O
L = N, O
Cu
L
L
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)
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+
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
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
– Zn2+-ATPase has been identified
• Storage: Metallothionein chemistry similar to Cu2+
Summary
• Transport and Storage of Metal ions:
– Necessary
– Diverse
– Evolved
– Largely Unknown