iron metabolism 2013 - eth zacac1.ethz.ch/koppenol/iron_metabolism_2013_a.pdf · iron-sulfur...

IRON METABOLISM

November 2013Susanna [email protected]

OUTLINE

_ Introduction_General properties_Overview of iron-containing enzymes and their role

_Selected Fe-containing enzymes_Hemoglobin, myoglobin (O2-uptake and O2-transport)_Transferrin (Fe-transport), and receptor-mediated cellular iron uptake_Ferritin/hemosiderin (Fe-storage)

_ Iron metabolism_Duodenal iron uptake_Iron metabolism in cells and macrophages_Regulation of iron homeostasis

_Diseases related to the iron metabolism_Iron overload_Iron-deficiency anemia

2

OUTLINE

_Introduction_General properties_Overview of iron-containing enzymes and their role




3

INTRODUCTION (1)

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1) Iron is essential but toxic

2) Iron is not very bioavailable

1a) Iron is essential

_Only few bacteria can live without iron (e.g. the lyme disease pathogen Borrelia burgdorferi seems to use Mn instead of Fe)

_Involved in all essential metabolic processes_photosynthesis_respiration_electron transfer_elimination of noxious metabolites of O2

_DNA-synthesis_detoxification_fixation of N2 and H2

_hydratase_hydrolase_mineralization of theeth by invertebrates

INTRODUCTION (2)

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1b) Iron is toxic

_FeII/FeIII/FeIV: redox processes

_e−-donor/acceptor mostly O2 or one of its derivatives

_Reactions may lead to the formation of partially reduced

oxygen species („free radicals“, „reactive oxygen species“ or ROS“)

_Fenton reaction → cellular injury

_Nature has developed strategies to avoid the presence

of „free iron“

INTRODUCTION (3)

6

2) Iron is not bioavailable

_abundant element in the universe, on the earth crust1…

_but not in the ocean (as low as 50 pM!)

_Why?

→ Solubility of Fe3+, stability of Fe3+ compelxes

→ Bioavailabilty increased via reduction to Fe2+

(1) http://en.wikipedia.org/wiki/Abundance_of_the_chemical_elements

BASIC IRON CHEMISTRY

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„Free iron“ (aerobic environment): [Fe(H2O)6]3+

_pKa ~ 2

_neutral conditions: insoluble ferric hydroxides (rust)

Ksp(Fe(OH)3) = 10–38/10–39

at pH = 7: [Fe(H2O)6]3+ ~ 10–17/10–18 M

_Iron(III) concentration in the ocean: 50 pM (higher!)

→ chelators bind iron(III), e.g. citrate, EDTA, O-donor ligands

→ increase solubility

→ but chelated iron(III) is not available for uptake (rate of H2O

exchange, i.e. of ligand exchange is rather low)

STRATEGY TO MAKE IRON MORE BIOAVAILABLE

8

Reduce iron(III) to iron(II)

_rate of H2O exchange 3–4 orders of magnitude larger

_affinity of iron(II) to natural O-donor ligands 3–4 orders of

magnitude lower, e.g. EDTA complex 108 times lower

_ferrous hydroxide is more soluble

Ksp(Fe(OH)2) = 10–15

at pH = 7: [Fe(H2O)6]2+ ~ 0.08 M

9 Modified from: Henzte et al. Cell (2004) 117, 285–297.

SYSTEMIC IRON HOMEOSTASIS

Ferritin

Hemo-globin

Transferrin

Myoglobin (300 mg)∼100 enzymes (100 mg)

10 © Galenica Group 23.11.2013

IRON-CONTAINING PROTEINSNON-HEME-CONTAINING PROTEINS

Protein (class) Function %Fe body

Non-heme proteins

ribonucleotide reductase

Oxidative metabolism

DNA biosynthesis

Very little

Iron-sulfur proteins

aconitaseiron regulatory protein

Electron transfer

Citric acid cycleFe homeostasis

~1

Ferritin

Hemosiderin

Fe storage 20

variable

Transferrin Fe transport ~0.2

11 © Galenica Group 23.11.2013

IRON-CONTAINING PROTEINSNON-HEME-CONTAINING PROTEINS

_Non-heme containing proteins

Fe-S-proteinsRibonucleotide reductase

″rust″

Ferritin

IRON-CONTAINING PROTEINSHEME-CONTAINING PROTEINS

N

N N

N

Fe

Protein Function Localization %Fe body

Hemoglobin O2 uptake and transport Red blood cells 65

Myoglobin O2 transport and storage Heart, skeletal muscle 6

cyt c oxidase

cyt c

cyt P450

Oxidative production of cellular energy

Electron transfer

Oxidative degradation

Mitochondria

Endoplasmic reticulum

<1

Peroxidases Oxidation GranulocytesMacrophages

0.1

Catalase H2O2 breakdown e.g. liver macrophages 0.1

Cyt: cytochrome12 © Galenica Group 23.11.2013

IRON ENZYMES INVOLVED IN ENERGY METABOLISM

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_Respiratory chain in the mitochondria:

THE RESPIRATORY CHAIN IN THE MITOCHONDRIA

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Citric acidcycle

Glycolisis

NADHFADH2

IRON ENZYMES INVOLVED IN ENERGY METABOLISM

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_Respiratory chain in the mitochondria:

Cytochrome c oxidase reduction of oxygen to water

Cytochromes

Ferredoxins electron transfer

Rieske proteins

_Citric acid cycle:Aconitase _dehydratase/hydratase (isomerase)

_apo-aconitase (proetin without Fe-S-cluster)acts as iron regulatory protein

→ Possible effect of iron deficiency: reduced levels of these enzymes→ impaired physical performance→ fatigue

IRON ENZYMES INVOLVED IN IMMUNE DEFENCE

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_Nitric oxide synthase NO●-synthesis (iNOS)

_Myeloperoxidase

_Eosinophil peroxidase hypohalogenite production (HOCl, HOBr)(action against microorganisms)

_Lactoperoxidase

→ Possible effect of iron deficiency: reduced levels of these enzymes→ increased susceptibility to infectious diseases

IRON ENZYMES INVOLVED IN ANTIOXIDANT DEFENCE

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_Peroxidases reduction of lipid peroxides_Catalase disproportionation of hydrogen peroxide_Cytochromes reduction of antioxidants, e.g. vitamin C

→ Possible effect of iron deficiency: reduced levels of these enzymes→ increased susceptibility to oxidative stress

IRON ENZYMES INVOLVEDIN NEURONAL METABOLISM AND SIGNALLING

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_Nitric oxide synthase NO●-synthesis (nNOS)neuronal signalling function of NO●

_Hydroxylases involved in synthesis of neurotransmitters and hormones: dopamine, noradrenaline,

_Tyrosine peroxidase adrenaline, serotonine, epinephrine, norepinephrine

→ Effects of iron deficiency→ low brain iron levels→ decreased density of dopamine receptors→ decreased level of dopamine transporters

FURTHER ESSENTIAL IRON-CONTAINING ENZYMES

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_Ribonucleotide reductase first step of DNA synthesis

_Ferrochelatase heme synthesis

_Cytochrome P450 family detoxification reactions in the liver

_Cyclooxygenase (COX) prostaglandin, thromboxane,prostacyclin synthesis

_Lipoxygenase isoenzymes prostaglandin and leukotrienemetabolism

_Lipoate synthase lipoate synthesis(precursor of different enzymes)

OUTLINE





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HEMOGLOBIN, MYOGLOBINAND OTHER DIOXYGEN CARRIERS

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_Oxygen carriers needed because of low solubility of O2 in H2O

_Oxygen carriers are Fe- or Cu-containing proteins_Hemoglobin (Hb) heme_Hemerythrin (Hr) iron (non-heme)_Hemocyanin (Hc) copper

_Essential features of oxygen binding proteins_reversible binding_no redox reaction_no generation of partially reduced oxygen sepcies

Literature for Hb: any biochemistry or bioniorganic chemsitry text book!

HEMOGLOBIN AND MYOGLOBIN → BLOOD

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_Blood: ca. 7–8% of body weight, ca. 5 L, pH 7.35−7.45_Plasma

H2O, Na+Cl− (ca. 0.9% w/w), glucose, proteins, O2,CO2/HCO3

− , …_White blood cells (immune system)_Red blood cells

_Red blood cells (RBC)_optimized form (large surface, shape)_half of the volume of human blood (2.5 ×1013)_most weight of RBC is hemoglobin

(5 mM, ca. 800 g in men and 550 g in women)_life span: 80−120 days

→ Fe recycled, porphyrin metabolized (CO!)_production: 1010 RBC/h

MYOGLOBIN (Mb)

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_ca. 10% of the amount of hemoglobin

_Transports O2 from Hb to mitochondria of muscle cells (cyt c oxidase)

_Correlation in concentration: Mb and cyt c oxidase

MYOGLOBIN (Mb) STRUCTURE

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_Monomer

_First X-ray of a protein(John Kendrew andMax Perutz,1958)

_ca.150 amino acids,mostly α-helices (globin), heme, highly conserveddistal and proximal histidine

http://en.wikipedia.org/wiki/Myoglobin

MYOGLOBIN (Mb) O2-BINDING SITE

25 http://www.chm.bris.ac.uk/webprojects2001/hoyle/page2.htm

Distal His

Proximal HisDistal His

Proximal His

MYOGLOBIN (Mb) OXYGEN BINDING CURVE

26http://www.aw-bc.com/mathews/ch07/c07obm.htm

_P50 ≈ 2 mm Hg

_Muscle ≈ 35−40 mm Hg

_Lung ≈ 80−100 mm Hg

Binding fraction

HEMOGLOBIN (Hb) STRUCTURE

27 http://en.wikipedia.org/wiki/Hemoglobin

_Tetramer (α2β2)

_Each chain:ca.150 amino acids,mostly α-helices (globin), heme, highly conserveddistal and proximalhistidine

_Cooperative oxygen binding(sigmoidal oxygen bindingcurve)

HEMOGLOBIN (Hb) OXYGEN BINDING CURVE


_Lower affinity for O2 thanmyoglobin

_P50 ≈ 27 mm Hg

_Sigmoidal oxygen bindingcurve

OXYGEN BINDING CURVES (Hb, Mb)

29 http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindhemoglobin.html

HEMOGLOBIN (Hb) OXYGEN BINDING CURVE


_Different (higher) affinity foreach subsequent O2-moleculebinding to the protein

_Allosteric ligands (effectors)regulate the binding of O2

to Hb: H+, CO2, andbisphosphoglycerate(see later)

MOLECULAR BASES FOR COOPERATIVE O2-BINIDING

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_Two main conformational states:_deoxy (or T, tense) → low O2-affinity_oxy (or R, relaxed) → high O2-affinity

http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindhemoglobin.html

COOPERATIVE O2-BINIDING AT AN ATOMIC LEVEL (1)

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_deoxyHb (T-state): Fe(II), high spin, 0.78 Å, d6, paramagnetic

_oxyHb (R-state): diamagnetic

_two possibilities: Fe(III)−O2●−, low spin, 0.55 Å, d5; S = ½;

antiferromagnetic coupling

Fe(II)−1O2, low spin, 0.61 Å, d6; S = 0;∆E(1O2/ 3O2) = ~22 kcal/mol (94 kJ/mol)

COOPERATIVE O2-BINIDING AT AN ATOMIC LEVEL (2)

33 http://www.ul.ie/~childsp/CinA/Issue65/TOC28_Haemoglobin.htm

Transition from T(deoxyHb)- to R(oxyHb)-state:

_conformational change of the porphyrin/heme

_cleavage of 8 salt bridges (that stabilize T-state)

CLEAVED SALT BRIDGES

34

6 of the salt bridges are between different subunits, with 4 of those involving the C- or N-terminus

http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindhemoglobin.html

35

_H-bonds between Tyr140 (α-chain) or Tyr145 (β-chain) and the

carbonyl O of Val93 (α-chain) or Val98 (β-chain) are cleaved

_When Fe binds O2, Fe is pulled into the plane of the heme ring

(shift of about 0.2 nm)

_This small shift leads to larger conformational changes since the

subunits are packed so tightly that compensatory changes in their

arrangement must occur

_The proximal His is pulled toward the heme, which causes the F

helix to shift, causing a change in the FG corner (the sequence

separating the F and G helices) at the alpha-beta interface

(→ cleavage of salt bridges)

SUMMARY: CHANGES T-STATE → R-STATE

MOLECULAR BASES FOR COOPERATIVE O2-BINIDING

36 http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindhemoglobin.html

37

_Lower pH → lower O2 affinity stabilization of T-state, moresalt bridges because Hisprotonated

_Not so relevant when PO2 high

_But in tissues PO2 low,

pH is lower

→ O2 easily dissociated

→ Hemoglobin even more effective

ALLOSTERIC EFFECTORS, BOHR EFFECT

http://www.aw-bc.com/mathews/ch07/c07be.htm

38

_CO2 is one of the end products of respiration

_Slightly better soluble than O2 in the blood:_8% dissolved as CO2_25% bound to Hb*_67% as HCO3

− (carbonic anhydrase, Zn-containing protein)

_*Binds α-amino group (terminal): R−NH2 + CO2 → R−NH−COO− + H+

_ Proton dissociation, lower pH → reduces affinity for O2

_Sequence of events in O2-consuming tissues:_CO2 produced binds to Hb → O2 dissociates

→ H+ taken up by Hb

→ helps carbonic anhydrase

CO2 + H2O → HCO3− + H+

→ H+ taken up by Hb….

ALLOSTERIC EFFECTORS, CO2

O2 AND CO2 METABOLISM

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CO2

CO2

NH2

O2

H+

NH–COO–

Carbonic anhydraseCO2 + H2O

H+

O2

Carbonic anhydrase

NH2

CO2 + H2O

CO2

CO2NH–COO–

HCO3–

Cl–

HCO3–

Cl–

Tissues Plasma

Red blood cell

HbFeO2 HbFeII

HCO3– + H+

Lungs Plasma

Red blood cell

HbFeO2 HbFeII

HCO3– + H+

40

_Lower pH → lower O2 affinity stabilization of T-state, moresalt bridges because Hisprotonated

_Not so relevant when PO2 high

_But in tissues PO2 low,

pH is lower

→ O2 easily dissociated

→ Hemoglobin even more effective

ALLOSTERIC EFFECTORS, BOHR EFFECT

http://www.aw-bc.com/mathews/ch07/c07be.htm

Binding of CO2 to Hb also shifts the curve towards right, i.e. lowers thebinding affinity of Hb for O2

41

_2,3,-bisphosphateglycerate (BPG) binds in the opening between theβ-chains of deoxyHb

_BPG concentration in red blood cells: ~5 mM

ALLOSTERIC EFFECTORS, bis-PHOSPHOGLYCERATE (1)

http://www.aw-bc.com/mathews/ch07/c07bh.htm

42

_The opening is much smaller in oxyHb: BPG cannot bind to theoxyHb form


http://www.aw-bc.com/mathews/ch07/c07bh.htm

43

_The higher the BPG content in red blood cells, the more stable thedeoxyHb form

→ decrease in O2-affinity → O2-release

_At high altitudes (low PO2) the BPG levels increase within a few days

Example:_At 4500 m, PO2 in lungs drops from ~100 to ~50 mm Hg (12 to 7 kPa)

→ within 24 h the BPG concentration raises from 5 to 8 mM→ lowers O2-affinity → enables tissues to obtain O2 in spite of its diminished availability

_On returning to low altitude the concentration of BPG (half-life 6 h)returns rapidly to normal levels



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_In the lungdecreased affinity makes littledifference to the amount of O2 bound

_In the tissuesdecreased affinity allows muchmore O2 to be released

_At sea level, with normal BPG levels,occupancy goes from 0.97 in lungs to0.50 in tissues

_At 4500 m, with 8 mM BPG, occupancygoes from 0.75 in lungs to 0.35 in tissues

http://vohweb.chem.ucla.edu/voh/classes%5Csummer08%5C153A-2ID19%5CHbMb.pdf

OTHER DIATOMIC LIGANDS THAT BIND TO THE HEME IRON

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1) CO: degradation of RBC, heme oxygenase (heme oxidase, see later)

2) NO● (endothelium-derived relaxing factor, EDRF): produced byeNOS, responsible for relaxation of smooth muscles surroundingthe blood vessels, resulting in vasodilation and thus increasedblood flow

1) CO: competitive binding vs. O2

Heme oxygenase: 1 CO/porphyrin

Normal conditions: ca. 1% HbCO (smokers up to 20%)

Hemoglobin structure reduces affinity for CO:

Fe-Porphyrin KCO/KO2 = 25’000

Hb(Mb) KCO/KO2 = 200

Why?

http://web.virginia.edu/Heidi/chapter15/chp15.htm

CO BINDING TO Hb

46

Why reduced affinity for CO?Old (wrong!) explanation (still found in many textbooks and internetpages)

http://web.virginia.edu/Heidi/chapter15/chp15.htm

HisE7 forces the CO molecule to tilt away from a preferred perpendicular alignment with the plane of the heme (“confirmed” by first X-ray structures)

First X-ray structures of Hb(Mb)CO: 135−150°Recent, more accurate, X-ray structures of Hb(Mb)CO: 167°

CORRECT EXPLANATION FOR REDUCED AFFINITY FOR CO

47

ElectronIc, not steric effect!

O2 binding: electrostatic stabilzation

CO binding: little electrostatic stabilization

Olson et al. JBIC (1997) 2, 544–552.

48 Olson et al. JBIC (1997) 2, 544–552.

NO● BINDING TO Hb(Mb)

49

NO● binding: partial electrostatic stabilization

Olson et al. JBIC (1997) 2, 544–552.

COMPARISON OF CO AND NO● BINDING TO Hb(Mb)

http://www.inchem.org/documents/ehc/ehc/ehc213.htm#1.950

CO is significantly more toxic than NO●

_WHO, levels of CO for which HbCO maximally 2.5%(even during light or moderate exercise):

→ 87 ppm for 15 min or 9 ppm for 8 h

_NO● is used in critical care to promote capillary and pulmonarydilation to treat primary pulmonary hypertension in neonatal patients

→ up to doses of 80 ppm/h

Why?_CO binding to Hb leads to stabilization of R-state

→ high O2-affinity, O2 cannot dissociate

_ NO● binding to Hb leads to stabilization of T-state→ low O2-affinity, O2 dissociates

WHO: world health organization

SUMMARY OF REACTIONS WITH HEMOGLOBIN

51blood HbFeO2

OUTLINE


_Selected Fe-containing enzymes_Hemoglobin, myoglobin (O2-uptake and O2-transport)_Transferrin (Fe-transport), and receptor-mediated cellular iron uptake_Ferritin/hemosiderin (Fe-storage)_Iron regulatory protein/aconitase (regulation of iron metabolism/citric acid cycle)



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HEMOCYANIN

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Dioxygen carrier in most molluscs, and some arthropods (tarantula, 24-mer), including crabs.

OTHER DIOXYGEN CARRIERS(1)

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_Invertebrates_Hemerythrin (Hr) iron (non-heme)_Hemocyanin (Hc) copper

_Hemerythrin: large variety, several X-ray structures, from monomericto octameric (cooperativity)Active site: deoxyHr: 2 FeII; oxyHr: 2 FeIII/peroxo; metHr: 2 FeIII/oxo

http://en.wikipedia.org/wiki/Hemerythrin

OTHER DIOXYGEN CARRIERS (2)

55

_Hemocyanin_Large proteins, mostly 6–10 domains, each domain 2 Cu_Active site:

_deoxyHc: 2 CuI

_oxyHc: 2 CuII/peroxo

http://www.ml.duke.edu/projects/Magnus/images/hemocyanin_color.gif

Intense blue (ligand tometal charge transfer)

iron metabolism 2013 - eth zacac1.ethz.ch/koppenol/iron_metabolism_2013_a.pdf · iron-sulfur...

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