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Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University Box 90346 Durham, NC 27708-0346 Telephone: (919) 660-1540 Fax: (919) 660-1605 E-mail: [email protected] Website: http://www.chem.duke.edu/%7Ealc/labgroup/ Virtual Free Radical School

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Page 1: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 2: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 3: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 4: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 5: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 6: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 7: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 8: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 9: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 10: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 11: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 12: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 13: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 14: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 15: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 16: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 17: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 18: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 19: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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]

Page 20: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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

Page 21: Iron in Biology Society For Free Radical Biology and Medicine Crumbliss 1 Iron Chelation in Biology Alvin L. Crumbliss Department of Chemistry Duke University

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.