Introduction to Bioinorganic Chemistry Nitrog.pdf · Laboratoire de Chimie Biomimétique des...

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1 Dominique Dominique Mandon Mandon Laboratoire de Chimie Biomimétique des Métaux de Transition Laboratoire de Chimie Biomimétique des Métaux de Transition INSTITUT DE CHIMIE INSTITUT DE CHIMIE UMR CNRS 7177 UMR CNRS 7177 Université de Strasbourg Université de Strasbourg 4 rue Blaise Pascal 4 rue Blaise Pascal 67070 Strasbourg cedex 67070 Strasbourg cedex FRANCE FRANCE Introduction to Introduction to Bioinorganic Chemistry Bioinorganic Chemistry Part 3: Iron/sulfur proteins, electron transfer, and nitrogenase

Transcript of Introduction to Bioinorganic Chemistry Nitrog.pdf · Laboratoire de Chimie Biomimétique des...

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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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The metal is easily removed

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Iron/sulfur proteins

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Iron/sulfur proteins as enzymes:

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Iron/sulfur proteins as electron transfer agents:

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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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One of the iron atoms is coordinated by water, not cystein!

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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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Electron transferAnd mitochondrial respiratory chain

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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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E° = -320 mV

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The role of organic electronic relays

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Initial reduction step

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The function of metal-containing proteins

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2H+ + 2e- H2 E1/2 = -420 mV (pH = 7 instead of pH = 0

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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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Different heme types

Heme a Heme b Heme c

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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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Cytochrome c, Fe(III) S = ½. Six-coordinated, His + Met. Narrow lines in1H NMR

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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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Homodimer200 kDa

13 subunits / monomerPresence of Fe, Cu, Mg, Na, and Zn

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Electron transfer site: CuA – heme a

Dioxygen reduction site: heme a3 - CuB

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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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Other hypotheses:Techniques: vibrational spectroscopy, Mössbauer

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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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The nitrogen cycle

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Nitrogen cycle: how biology is involved

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Nitrogen cycle: transition metals and biology

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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

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P cluster: [8Fe-8S] centre

All 8 iron atoms can be completely reduced

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« FeMoco or M cluster»[7Fe-9S-Mo] centre

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