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Dominique Dominique Mandon Mandon
Laboratoire de Chimie Biomimétique des Métaux de TransitionLaboratoire de Chimie Biomimétique des Métaux de Transition
INSTITUT DE CHIMIEINSTITUT DE CHIMIEUMR CNRS 7177 UMR CNRS 7177
Université de StrasbourgUniversité de Strasbourg4 rue Blaise Pascal4 rue Blaise Pascal
67070 Strasbourg cedex 67070 Strasbourg cedex FRANCEFRANCE
Introduction toIntroduction to Bioinorganic Chemistry Bioinorganic Chemistry
Part 3: Iron/sulfur proteins, electron transfer, and nitrogenase
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Preliminary Note:
All slides displayed in this presentation contain reproductions of drawings or schemes obtained from various sources.
The following presentation is provided on a non-commercial base and should be considered as a help-only tool for students.
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Iron/sulfur proteins
General aspects Rubredoxins, [2Fe-2S] centres, [4Fe-4S] centresDynamic considerations - Example of Aconitase
Part 5: Part 5: ironiron //sulfursulfur proteinsproteins , , electronelectron transfertransfer andand nitrogenasenitrogenase
Electron transfer
Organic electronic relays Metal-containing proteins
Nitrogenase
The Haber-Bosch industrial processNitrogen cycle Structure mechanistic hypotheses
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Ubiquitous proteins often involved in electron transfer at negative potentials.Fe/S centres are also important components within enzymes that catalyze
major reactions (hydrogenases, nitrogenases, sulfite reductase, oxidases…)
Iron/sulfur proteins
1% of iron in mamalians is found as iron/sulfur proteins
Sulfur is found in amino-acide residues (cystein for instance),but also as inorganic fragments. In that case its origin remains uncertain:
Inorganic sulfides, pyrites FeS (S22-) might have been involved in early life evolution mechanisms
by processes involving CO2 reduction.
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Two embedded tetrahedrons
The cysteins allow linkage to the proteinFe2+, 3+, Td => high spin
The [4Fe-4S] example
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Rubredoxins
Mononuclear proteins
Fe(II) almost colorlessFe(III) deep red (LMCT)
Electronic relays Stabilisation of ferric Fe(III) iron
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[2Fe-2S] centres
Ferredoxins
The two metal centres are inequivalent(different proteic environnement)i.e. Fe2+/Fe3+ and not Fe2.5+/Fe2.5+
(technique: Mössbauer)
2 S2-, 4RS-
Charge: Fe(III)/Fe(III) = 2-
Charge: Fe(II)/Fe(III) = 3-
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A particular case: the Rieske Centres [2Fe-2S]
Membrane proteins present in mitochondriae
Function: orientation of the electron flux in intramembrane transport pathways(high potential: along the membrane; low potential: throughout the membrane)
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[4Fe-4S] centres
The most commonly found
4 S2-, 4RS-
Charge: Fe(III)/Fe(III)/Fe(III)/Fe(III) = 0 unstable
Charge: Fe(II)/Fe(III)/Fe(III)/Fe(III) = 1-
Charge: Fe(II)/Fe(II)/Fe(III)/Fe(III) = 2-
Charge: Fe(II)/Fe(II)/Fe(II)/Fe(III) = 3-
Charge: Fe(II)/Fe(II)/Fe(II)/Fe(II) = 4- unstable
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[Fe3IIFeIII –4S] [Fe2
IIFe2III –4S] [FeIIFe3
III –4S]
# -400 mV # -50 mV
« regular » ferredoxins
2 FeII/FeIII equivalent pairs (Mössbauer)Diamagnetic ground state
Antiparallel coupling between the two pairs
- e- - e-
+ e- + e-
# -600 mV # +350 mV
« high potential » ferredoxins(HIPIP)
S = 0S = 1/2 S = 1/2
In HIPIP: hydrophobic amino acids:Access to water is hampered
=> Differences in potentials
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Dynamic aspects: from one cluster to anotherThe fourth iron atom is generally not bound to a cystein
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Reminder: transport and storage of iron: ferritin / transferrin regulation
The conversion [4Fe4S] => [3Fe4S] is beleived to induce conformationalmodifications and consequently affinity modifications for the IRE
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Simple example of a [3Fe-4S] centre enzyme: aconitase
Function: to catalyze this equilibrium involved in the Calvin cycle (photosynthesis)
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Iron/sulfur proteins
General aspects Rubredoxins, [2Fe-2S] centres, [4Fe-4S] centresDynamic considerations - Example of Aconitase
Part 5: Part 5: ironiron //sulfursulfur proteinsproteins , , electronelectron transfertransfer andand nitrogenasenitrogenase
Electron transfer
Organic electronic relays Metal-containing proteins
Nitrogenase
The Haber-Bosch industrial processNitrogen cycleStructure mechanistic hypotheses
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Energy released: reduction of O2 in H2O
NADH + ½ O2 + H+ H2O + NAD+ ∆G°= -52.6 kcal/mol
This energy is further recovered in the process of ATP generation from ADP
ADP + H3PO4 ATP + H2O ∆G°= +7.3 kca/mol
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Complex I:NADH reductaseMM # 850 kDa
Complex III:Cytochrome c reductase
Complex IV:Cytochrome c oxidase
Complex II:Succinate dehydrogenase
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Fe/S proteins: [4Fe-4S], [2Fe-2S], Rieske centres…Vide supra
Cytochromes a, (a3…), cytochromes b (b566, b562…), cytochromes c (c, c3…)
Heme-containing proteins
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Cytochrome c
Cytochrome c (cyt.c) acts as a shuttle between complex III (cyt. c reductase) and complex IV (cyt. c oxidase)
E° = 260 mV
Small heme-containing protein, around 100 amino acids, MM # 12kDaExternal side of the membrane
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A major question poorly understood:
How are electron transferred at the active site of cytochrome c oxidase?
Electronic relays via amino acid residues ? Directional dependency ?
Tunnel effect ?
Estimated distance of around 20 Å
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Cytochrome c oxidase
The last step of the respiratory chain4 e- reduction of molecular oxygen into water
2 O2 + 4 ferrocytochromes c + 4H+ 2 H2O + 4 ferricytochromes c
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How does it work ?one hypothesis among others:
Dioxygen binding
Reduction to peroxide (O22-)
Reduction of CuB
Protonation of the peroxide: => (HO2-)
Reduction of heme by CuB
Protonation and homolysis of the O-O bond
Reduction of heme a, then back to resting state
Protonation (H2O release) and reduction of the two other metal centres
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In resting state, the spin state of the enzyme is S = 2Techniques: EPR, Mössbauer …
dxz, dyz
dxy
dz2
dx2-y2
(eg)
dxz, dyz
dxy
dz2
dx2-y2
St = 3
St = 2
J < 0 # -200 cm-1
S1 = 5/2 S2 = 1/2
Strong antiparallel interaction between Fe(III) high spin and Cu(II)
St = S1 - S2 (ground state)
Fe(III)
OH
CuBH(II)
HO
Fe(III)
CuB(II)
O
Fe(III)
OH
CuB(II)
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Protons are transferred through specific channels
( E° = + 0.815 mV/NHE)
Cyt. C. Ox also acts as a proton pump!
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Iron/sulfur proteins
General aspects Rubredoxins, [2Fe-2S] centres, [4Fe-4S] centresDynamic considerations - Example of Aconitase
Part 5: Part 5: ironiron //sulfursulfur proteinsproteins , , electronelectron transfertransfer andand nitrogenasenitrogenase
Electron transfer
Organic electronic relays Metal-containing proteins
Nitrogenase
The Haber-Bosch industrial processNitrogen cycleStructure mechanistic hypotheses
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Industrial reduction of dinitrogen by theHaber – Bosch process
1909: discovery of the process1910: submission of the patent1918: Fritz Haber wins Nobel prize in chemistry1931: Karl Bosch wins Nobel prize in chemistry (with Friedrich Bergius)
The first industrial process using high pressure !
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Concomitant release of dihydrogen and ammonia formation
A postulated mechanism: preliminary hydrogenation
Dihydrogen in excess inhibits the reaction
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Structure of nitrogenase: a complex molecular machinery
Fe/Mo nitrogenase: two proteins: (α2β2)(γ2)
Dinitrogenase reductase: (γ2)60 kDa
Dinitrogenase or FeMo protein (α2β2)220 kDa
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Dinitrogenase reductase: (γ2)60 kDa
[4Fe-4S] centre
E°red = -0.35V
Two Mg2+/ATPreceptors
When ATP/ADPbound, E°red = -0.45V
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Dinitrogenase or FeMo protein (α2β2)220 kDa
[8Fe-8S] centre«P cluster»
[7Fe-9S-Mo] centre« FeMoco or M cluster»
20 Å