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Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1
Iron Chelation in BiologyIron Chelation in Biology
Alvin L. CrumblissDepartment of Chemistry
Duke UniversityBox 90346
Durham, NC 27708-0346
Telephone: (919) 660-1540Fax: (919) 660-1605
E-mail: [email protected]: http://www.chem.duke.edu/%7Ealc/labgroup/
Virtual Free Radical School
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 2
Iron Chelation in BiologyIron Chelation in Biology
Tutorial GuideIntroduction: Biological Iron Coordination Chemistry
Panels 3, 4 & 5
Common Iron Ligands in BiologyPanel 8
Chelate Stability DefinitionsPanel 9
Iron Chelation and TransportPanels 14, 15 &16
Chelation and Redox ControlPanels 10, 11 & 12
Influence of pH on Chelate StabilityPanel 17
Influence of Chelate Stability on E0
Panel 18 Influence of Chelation on Kinetics
Panel 19
Chelation and SolubilityPanel 6
Chelation and Redox PotentialPanel 7
Oxidation State Influence on Chelate StabilityPanel 13
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 3
Introduction: Introduction: Biological Iron Coordination ChemistryBiological Iron Coordination Chemistry
Iron is the second most abundant metal on the earth’s surface, falling closely behind aluminum and in near equivalent concentration to calcium and sodium. It is an essential element for virtually every living cell.
The biochemistry of iron is controlled to a large extent by its coordination chemistry; i.e. the immediate chemical environment in the first coordination shell. This first coordination shell controls iron’s biological activity in small molecule storage (e.g.O2), electron transport, and catalysis.
Fe
1st coordination shell;immediate chemical environment
Common oxidation states: +2, +3
Common coordination numbers: 4, 5, 6
3 References [1,2]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 4
Introduction: Introduction: Biological Iron Coordination ChemistryBiological Iron Coordination Chemistry
Examples of the extensive use of iron in biological systems, all of which are controlled or mediated by chelation, are as follows:
redox chemistry involved in simple electron-transfer reactions;
redox chemistry involved in reactions with O2, ranging from O2 transport and storage to O2 reduction by cytochrome oxidase, and O atom insertion catalyzed by cytochrome P450; and
substrate activation by the electrophilic behavior of iron; for example, hydrolase enzymes such as purple acid phosphatase.
4 References [1,2]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 5
The first coordination shell Prevents hydrolysis/precipitation Influences molecular recognition Controls redox potential Controls mobility
Introduction: Introduction: Biological Iron Coordination ChemistryBiological Iron Coordination Chemistry
Fe
5
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 6
Fe insoluble due to hydrolysis
Iron Chelation and SolubilityIron Chelation and Solubility
FeH2O OH2
OH2H2O
OH2
OH2
FeH2O OH
OH2H2O
OH2
OH2
3+ 2+
FeH2O O
OH2OOH2
OH2
FeOH2
OH2
OH2
OH2H
H 4+
H+
H+
Higher insoluble polymers
Fe
L:
[Feaq3+]tot = 10-10 M @ pH 7
Strong chelators prevent hydrolysis and precipitation
6
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 7
-0.4
0.0
0.4
0.8
1.2
Fe(OH2)6
Iron(II) stabilized
Iron(III) stabilized
Fe(terpy)2
Fe(phen)3
Fe(bipy)3
Fe(salicylate)
Fe(CN)6-
Fe(EDTA)
Fe(oxinate)3
HEMEDERIVATIVES
hemoglobin
myoglobin
+
+
+
-
Easy to
reduce
Eo
volts
Fe(oxalate)3
hydroxamate siderophores
FeL L
LLL
L
n+
Iron Chelation and Redox PotentialIron Chelation and Redox Potential
Fe(III/II) redox potential varies significantly with ligands in 1st coordination shell
7
e.g. Desferal
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 8
Common Iron Ligands in BiologyCommon Iron Ligands in Biology
Iron(III) is a hard Lewis acid and prefers ligation to hard Lewis base donors (e.g. O, amine N) and iron(II) is a borderline soft Lewis acid and prefers ligation to soft Lewis base donors (e.g. S, pyrrole N).
N
N
N
N
Fe
Fe
O
OFe
O
O
R1
N
R2
Fe
O
O
O
Fe
O
O
Fe
OFe
H2N
Fe
N
NH
Fe
S
Common iron ligand donor groups in biology include amino acid side chains, such as amine (I), carboxylate (II), imidazole (III), phenol (IV), and thiol (V). Other ligating groups include -hydroxy carboxylate (VI), catecholate (VII), hydroxamate (VIII) and porphyrin (IX).
(I)
(II)
(III)(IV) (V) (VI)
(VII)(VIII)
(IX)8
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 9
Compilations of metal-ligand complex stabilities, such as that edited by Martell and Smith, use pH independent equilibrium constants,
FeLH, as defined below for the reaction between Fe(III) and a hexadentate triprotic ligand, LH3, in aqueous solution.
Fe(OH2)63+ + L3- FeL
110 =
However, in an in vivo or in vitro situation protons compete for the Fe(III) binding sites and the degree of complexation of the metal will be influenced by the ligand pKa values and the pH of the medium. Fe(OH2)6
3+ + H3L FeL + 3 H+ K = Since stability constants and K are determined as concentration quotients, their units differ on changing the denticity of the ligand. Consequently, 110 for a hexandentate ligand and
130 for a bidentate ligand cannot be directly compared. A pFe scale circumvents this problem and the problem of H+ competition due to different ligand pKa values. The pFe value for a particular ligand is the negative log of the free Fe(III) concentration at a fixed set of conditions: [total ligand] = 10 M, [total Fe(III)] = 1 M, and pH = 7.4. A high pFe value denotes a stable chelate complex. Panel 17 illustrates the influence of pH on Fe(III)-siderophore complex stability, using pFe values to express the stability of the complex.
[FeL] [Fe(OH2)6
3+][L3-]
[FeL] [H+]
[Fe(OH2)63+][H3L]
Iron Chelate Stability DefinitionsIron Chelate Stability Definitions
9 References [3,4,5,6]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 10
Iron Chelation and RedoxIron Chelation and Redox Control Control
A mechanism for preventing iron from participating in a catalytic cycle to produce toxic hydroxyl radicals and/or reactive oxygen species (ROS) (e.g. via the Fenton reaction or Haber Weiss cycle) is to control its redox potential by selective chelation. Through chelation, the redox potential for iron may be removed from the region where it can undergo redox cycling and produce hydroxyl radicals and ROS. This is illustrated in Panel 12. From the following thermochemical cycle, Equation (1) can be derived which relates the redox potential of an Fe complex to the chelator’s ability to discriminate between Fe(III) and Fe(II), as expressed by βIII and βII. This relationship illustrates that the selectivity of a chelator for Fe(III) over Fe(II) increases with decreasing redox potential. Fe(H2O)6
3+ + L Fe3+L
Fe(H2O)62+ + L Fe2+L
βIII
βII
E0aq
E0complex E0
aq – E0complex = 59 log(βIII/βII) [1]
Why is it important?Why is it important?
10 Reference [6]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 11
From Equation (1) it is evident that the redox potential and stability of an iron complex are inter-related. These inter-relationships are important in characterizing the biological chemistry of iron because controlling the oxidation state of iron is a method of controlling both the thermodynamic and kinetic stability of a coordination compound. This is illustrated in Panel 13. As a result, the redox potential of a complex may be viewed as a measure of the sensitivity of a molecular level switch for changing the chemical environment of the iron (1st coordination shell). Data in Panel 13 show that for high spin complexes, changing the oxidation state of iron from +2 to +3 changes both the kinetic lability and thermodynamic stability of an iron chelate complex.
Iron Chelation and RedoxIron Chelation and Redox ControlControl
Why is it important?Why is it important?
11 Reference [6]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 12
Prevent redox cycling & ROS productionFe(III) selectivity
Control stability
Control ligand exchange kineticsControl "switch" sensitivity
Iron Chelation and RedoxIron Chelation and Redox ControlControl
Why control EWhy control E00??
12Reference [6]
-480
-320E
(m
V v
s N
HE
)NAD(P)+/NAD(P)H
-160 O2/O2. -
+940 O2. -/H2O2
+460 H2O2/HO., HO-
Fe3+
Fe2+ O2
O2. -
H2O2
HO.
HO-
ROSRH
Haber-Weiss Cycle
Eoaq - Eo
complex = 59 log(II)
Fe(H2O)63+/Fe(H2O)6
2++770
1020
1010
100
-500
Ferrioxamine B
Transferrin
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 13
FeIII
L L
LLL
LL L
LLL
L
L
n+(n-1)+
+ e-
- e-FeII
StableInert
Less stableLabile
Fe(III)transferrin log K @ pH 7.4 = 20 Fe(II)transferrin log K @ pH 7.4 = 3 Fe(III)ferrioxamine B log 110 = 30.6 Fe(II)ferrioxamine B log 110 = 10.3
ThermodynamicsThermodynamicsIllustration of the loss of several orders of magnitude of stability on reduction of high spin Fe(III) complex to Fe(II).
Oxidation State Influence on Chelate StabilityOxidation State Influence on Chelate Stability
KineticsKineticsIllustration of an increase in 1st coordination shell lability on reduction of Fe(III) to Fe(II).
Fe(OH2)63+ + *OH2 Fe(OH2)5(*OH2)
3+ + OH2
Fe(OH2)6
2+ + *OH2 Fe(OH2)5(*OH2)2+ + OH2
-e- +e -
t1/2 = 4 ms
t1/2 = 0.2 s
13 References [7,8,9,10,11]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 14
Iron Chelation and TransportIron Chelation and TransportIn humans, the host protein transferrin (Tf) is produced in excess of circulating free iron and sequesters extracellular iron at extremely high affinity (Kd ~10-20 M). This chelation of iron prevents it from precipitation and also has a bacteriostatic effect by keeping iron as an essential nutrient from being available to bacterial pathogens.
FeIII
O
Otyrosine
OtyrosineO
Nhistidine
O
aspartate
C
O
Human Transferrin Fe(III) Binding Site
Fe3+ + apo-Tf + CO32-
Kb ~ 1020 M-1
FeIIITf(CO32-)
Reference [7]14
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 15
Iron Chelation and TransportIron Chelation and Transport
OOOO
O
O
Ion Recognition
microbial cell
O
O
OO
OO
Fe2O3.6H2O or Fe(OH)3
Fe3+
Molecular Recognition
Ksp ~ 10-39
environmental iron
siderophore
Fe Release
Complexation
AlZnCr CuMn Ni
KCa Pb
Fe
(i)
(ii)
(iii)
(iii)
(iv)
(iv)(v)
Microbes solubilize environmental iron by a chelation process, whereby the microbe secretes chelators called siderophores which have a high and specific affinity for Fe(III). Siderophore mediated iron acquisition by microbes is illustrated here where the cell synthesizes and releases a polydentate siderophore (i) which solubilizes insoluble iron deposits by chelation (ii). The Fe(III) chelate diffuses back to the cell (iii) where it is recognized by a cell receptor (iv) and the iron is released into the metabolic processes within the cell (v).
15 References [4,5,6]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 16
Iron Chelation and TransportIron Chelation and TransportSiderophores, microbially synthesized Fe(III) specific chelators, are low molecular weight molecules that usually incorporate bidentate catechol, hydroxamic acid, and/or -hydroxy carboxylic acid donor groups. These chelators exhibit high Fe(III) complex stabilities (high and pFe) to enhance delivery of iron to the cell, and large negative redox potentials (Panels 7, 12 and 18) for Fe(III) complexing specificity and to prevent redox cycling leading to the production of toxic hydroxyl radicals and ROS (Panels 10, 11 and 12). Shown below are the structures of two hexadentate siderophores; enterobactin, a tris catecholate (in red), and ferrioxamine B, a tris hydroxamate (in blue).
enterobactin
Fe(III)-enterobactin complex110 = 1049 ; pFe = 35.5
HO
O
OH HO
O
NHN
O
N
HN
O
N
NH3+
O
NH
O
O
O
O
HN
O O
NH
O
O
OH
OH
O
OH
HO
OH
HO
O
O
O
O
OO
NHN
O
N
HN
O
N
NH3+
Fe
desferrioxamine B
ferrioxamine B complex110 = 1030.2 ; pFe = 26.6
16
O
O
O
O
O O
Fe
O
O
OO
O OHN
O
OHN
O
NH
References [4,5,6]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 17pH
0 2 4 6 8 10 12
pF
e
0
10
20
30
40
Exochelin MN (I)Ferrioxamine B (II)
Influence of pH on Fe(III)-Chelate StabilityInfluence of pH on Fe(III)-Chelate Stability
Plot of Fe(III) complex stability, expressed as pFe (Panel 9), as a function of pH for two siderophores, exochelin MN (I) and ferrioxamine B (II). Although they have approximately the same stability at pH 6.0, above this pH exochelin MN has a higher affinity for Fe(III) and below this value ferrioxamine B exhibits a higher affinity. This is due to different levels of competition from H+ for the Fe(III) binding sites, due to different pKa values for the donor groups (shown in blue) in these two siderophore chelators.
NNH
O
NHN
NH
O
NH
NH2
O
N
O
OHOH
NHO
NH
OOHNH2
(I)
(II)
HOO
OH HOO
NHN
O
NHN
O
N
NH3+
O
17 Reference [9,12]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 18
pFe
5 10 15 20 25 30
-E1/
2 (m
V)
vs N
HE
200
250
300
350
400
450
500
8
6
53
12
7
4
Influence of Fe(III)-Chelate Stability on EInfluence of Fe(III)-Chelate Stability on E00
Plot of the reversible Fe(III/II) redox potential (-E1/2) as a function of the stability of the complex, as expressed by pFe values (Panel 9). Data are for hexadentate (1,2,4), tetradentate (3,5) and bidentate (6,7,8) hydroxamic acid siderophores and siderophore mimics. Note that:
as the stability of the Fe(III)-complex increases, the complex becomes more difficult to reduce; and
the stability of the complex decreases with decreasing denticity.
1. Fe(desferrioxamine B)+ 2. Fe(Desferrioxamine E) 3. Fe2(alcaligin)3 4. Fe(saccharide-trihydroxamate) 5. Fe2(rhodotorulic acid)3 6. Fe(N-methylacetohydroxamate)3 7. Fe(acetohydroxamate)3 8. Fe(L-lysinehydroxamate)3
18 References [6,10,13,14]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 19
Influence of Fe(III)-Chelation on KineticsInfluence of Fe(III)-Chelation on Kinetics
Fe
OH2
O
OO
O OH2
H3C
N
CH3
Fe
OH2
OH2
OH2H2O
H2O OH2
Fe
OH2
O
OH2O
H2O OH2
H3C
N
CH3
N
H3C
H3C
O
HO
H3C
N
CH3
O
OHN
H3C
H3C
H+
H+
3+ 2+
1+
k1 = 1.8 M-1s-1
k2 = 8.1 x 102 M-1s-1
Iron chelate formation places a strong electron donor in the first coordination shell, which labilizes the remaining aquated coordination sites. This is illustrated here for the reaction of hexa(aquo)iron(III) with N-methylacetohydroxamic acid, a siderophore mimic. Incorporation of the bidentate hydroxamate group in the first coordination shell labilizes the remaining aquo ligands by a factor of ~500 (k2/k1).
19 Reference [5,15]
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 20
1. Crichton, R. (2001) Inorganic Biochemistry of Iron Metabolism, John Wiley & Sons, Ltd, New York.
2. Harris, W. R. (2002) in Molecular and Cellular Iron Transport (Templeton, D. M., Ed.) pp 1-40, Marcel Dekker, Inc., New York.
3. Martell, A. E. and Smith, R. M., Eds. (1974, 1975, 1976, 1977, 1982, 1989) Critical Stability Constants, Plenum Press, New York.
4. Raymond, K. N. and Stintzi, A. (2002) in Molecular and Cellular Iron Transport (Templeton, D. M., Ed.) pp. 273-320, Marcel Dekker, New York.
5. Albrecht-Gary, A.-M., and Crumbliss, A. L. (1998) in Metal Ions in Biological Systems Vol. 35, Iron Transport and Storage in Microoganisms, Plants and Animals (Sigel, A. and Sigel, H., Ed.) pp. 239-327, Marcel Dekker,New York.
6. Crumbliss, A. L. and Boukhalfa, H. (2002) BioMetals 15, 325-339. 7. Aisen, P. (1998) in Metal Ions in Biological Systems Vol. 35, Iron
Transport and Storage in Microoganisms, Plants and Animals (Sigel, A. and Sigel, H., Ed.) pp. 585-632, Marcel Dekker,New York.
8. Harris, W. R. (1986) J. Inorg. Biochem. 27, 41-52. 9. Schwarzenbach, G., and Schwarzenbach, K. (1963) Helv. Chem. Acta
46, 1390-1400.
ReferencesReferences
20
Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 21
10. Spasojevic, I., Armstrong, S. K., Brickman, T. J., and Crumbliss, A. L. (1999) Inorg. Chem. 38, 449-454.
11. Helm, L. and Merbach, A. E. (1999) Coord. Chem. Rev. 187, 151-181. 12. Dhungana, S., Miller, M.J., Dong, L., Ratledge, C. and Crumbliss, A. L.
(2002) manuscript in preparation. 13. Wirgau, J. I., Spasojevic, I., Boukhalfa, H., Batinic-Haberle, I., and
Crumbliss, A. L. (2002) Inorg. Chem. 41, 1464-1473. 14. Dhungana, S., Heggemann, S., H., Gebhardt, P. Möllmann, U. and
Crumbliss, A.L. (2002) Inorg. Chem. 41, submitted for publication. 15. Caudle, M. T., and Crumbliss, A. L. (1994) Inorg. Chem. 33, 4077-4085.
ReferencesReferences
21
AcknowledgementsAcknowledgementsI thank my co-workers, some of whose names appear in the References, for their hard work, questions, ideas, and intellectual stimulation. Our work in this area is supported by the National Science Foundation, the National Instutites of Health, and the American Chemical Society Petroleum Research Fund.