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

• Na+/K+-ATPase

– Membrane-Spanning Protein Ion Pump

– 22 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

Sodium and Potassium--Ion Pump

3Na+in + 2Kout

+ + ATP4- + H2O 3Na+out + 2K+

in + ADP3- + HPO42- + H+

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

OOO

O

O O O

O

CH3

O

CH3CH3

O CH3CH3

O

CH3CH3

OCH3

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 [Ca2+] = 0.001 M

– Inside Cell [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 F- is 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--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

OH

OH

C NR

O

R

OH

Catechol 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-]

– structure assigned by [Cr(ent)3-]

circular dichroism

• Crystal Structure: [V(ent)2-]

OHNC

OH

OH

C

NH

O

O

O O

O

NH

CHO

HO

O

O

O

HO

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

• Transferrin: Mammalian transport 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-Transferrin

FeO N

OO

O

O

Asp

NHHis

Tyr

Tyr

O

C

O

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

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

Cu

SN

N R

Cu

LLL

O LL

L LL

Cu

R

Cu

LLL

L L

R = S, N, O L = N, O

Type I Type II Type III

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

• Storage: Metallothionein chemistry similar to Cu2+

Summary

• Transport and Storage of Metal ions:

– Necessary

– Diverse

– Evolved

– Largely Unknown