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 Moleculecules 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 molecule 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--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 mammals 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+

Sodium and Potassium--Ionophore

Nonactin: microbial Na+ and K+ ionophore
CH3 O O O CH3 O O CH3 O CH3 O CH3 O O CH3 O CH3 O O CH3

Nonactin

Makes Na+ and K+ membrane soluble when complexed Oxygen Donors can be modeled by Crown Ethers

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)

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 Earths 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--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
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 Complex anion is soluble
O C OH OH

Spectroscopy: UV-Vis: like [Fe(cat)33-] D structure assigned by [Cr(ent)3-] circular dichroism HO

Crystal Structure: [V(ent)2-]

NH O O O HO NH C O HO HO HN O C O O O

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 Fe O O O N NH His Asp

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 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

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 S N N L Cu R L Type II L Cu L L L L L Cu L L O R R = S, N, O L = N, O Type III L Cu L L

Copper--Transport
Ceruloplasmin Biological role not fully understood transport oxygen metabolism Wilsons 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

Storage: Metallothionein chemistry similar to Cu2+

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