Electron Transport and ATP synthesis

Question 1

Give an overview of the electron transport chain?

Answer 1

The electron transport chain (ETC) is located in the mitochondrial inner membrane
The electron transfer components are arranged in order from most negative to most positive standard reduction potential (εo’)

The energy released by redox reactions within the ETC is used to move protons from the matrix to the intermembrane space
The proton gradient is used to drive ATP synthesis

Question 2

What are redlx active molecules in the electron transport chain?

Answer 2

These components include Flavins, Iron-sulphur (FeS) centres, Quinones, Cytochromes, Haems, Copper centres
These are associated with the protein complexes of the ETC
Some are hydrogen carrier (H+ and e-) whereas others carry electrons only

Question 3

Describe flavins?

Answer 3

Reduced flavins act as hydrogen (proton and electron) carriers
They can exist in the semiquinone intermediate

Question 4

Describe iron-sulphur clusters?

Answer 4

The iron atom can undergo oxidation and reduction Fe3+ +e- → Fe2+ therefore carry electrons
The Cys are cysteine residues in the polypeptide to which the FeS cluster is bound

Question 5

Describe ubiquinone?

Answer 5

Both complex I and II reduce ubiquinone
Note the similarity to flavins - as both carry electrons and protons (i.e. hydrogen atoms)

Ubiquinol is the reduced form, ubiquinone in the oxidised form
Because of its lipophilic nature Q can acts as a mobile carrier between protein complexes

Question 6

Describe cytochromes?

Answer 6

They differ in the nature of the side chains on the porphyrin ring, the linkage to the protein and the protein environment
They can bind covalently as seen here for c type cytochromes or non covalent as in cyt a and b
They can only have one electron at one time
All these factors influence the redox potential

Question 7

Describe complex I - NADH dehydrogenase?

Answer 7

AKA NADH-Q oxidoreductase
1MDa in size
14 central and ~30 peripheral subunits
Some subunits are mitochondrially encoded others nuclear
Eukaryote - found in inner membrane, Prokaryote - similar structure found in the cytoplasm

Ubiquinone redox chemistry induces conformational changes within the complex driving proton pumping 4H+ translocated per pair of electrons

It contains 1 favin mononucleotide (FMN) - a redox active prosthetic group
8 iron-sulfur clusters (mammals)

Question 8

Describe the mechanism of the electron transport chain using NADH - complex I?

Answer 8

1. NADH transfers 2 electrons to complex I NADH dehydrogenase, accompanied by a H+
   ○ NADH is an electron donor as its redox potential is more negative
2. 2 electrons and 2 protons pass through complex I
3. The 2 electrons are donated to ubiquinone, the 2 protons accompany the e- and react to form QH2
   ○ This leaves regenerate NAD+, which can go back to the TCA cycle in the matrix
4. Energy released by the movement of electrons through complex I is used to pump 4 protons from the matrix into the intermembrane space

Question 9

What is the overall equation at complex I?

Answer 9

NADH + H+ + Q -> NAD+ + QH2

For each time of this reaction 4H+ from the matrix move across the inner membrane

Question 10

Describe complex II - succinate dehydrogenase?

Answer 10

It contains a linear chain of redox cofactors
Complex II is a mushroom-shaped homotrimer
It’s protomers each consist of two hydrophilic subunits, a flavoprotein (Fp) and an iron–sulfur subunit (Ip) and two hydrophobic membrane-anchor subunits, CybL and CybS
Fp binds the substrate and the FAD prosthetic group
Ip binds via the 3 Fe-S clusters and passes its electrons on to ubiquinone

No protons are pumped by complex II - due to insufficient energy produced from the redox reaction
Succinate + FAD+ -> fumarate + FADH2

Question 11

Describe the mechanism of the electron transport chain using FAHD2- complex II?

Answer 11

This enzyme is also used in the TCA cycle

1. FADH2 transfers 2 electrons directly to ubiquinone, accompanied by 2 protons producing QH2
   Complex II doesn’t pump protons as there isn’t enough energy produced by the transfer of these electrons to facilitate this movement

FADH2 + Q -> FAD + QH2
The following electron transport chain is the same as in NADH

Question 12

Describe complex III - Q-cytochrome c oxidoreductase?

Answer 12

4 subunits - Rieske iron-sulphur center, heme c1, heme bL, heme bH
In Rieske one of the Fe atoms is coordinated by two His residues - this stabilises the reduced form thereby raising its reduction potential

Overall reaction - QH2 + 2 cyt cox + 2H+matrix → Q + 2 Cyt cred + 4H+cytosol

Question 13

Describe the proton motive Q-cycle in complex III?

Answer 13

2 electrons from QH2 bind at the Qo site
The electrons take different routes - as cytochrome can only have 1e- at one time
One is transferred via the Rieske FeS centre and cytochrome c1 to cytochrome c, the other via the two b cytochromes to a second oxidised Q molecule at site Qi
The 2 protons from QH2 are released to the cytosol

The Q at Qo is replaced by a second QH2 molecule and the cycle repeated
The semiquinone intermediate at Qi is reduced to QH2 by the transfer of a second electron from the Qo site and the uptake of 2 protons from the matrix

Question 14

Describe complex IV - cytochrome c oxidase?

Answer 14

410 kDa homodimer
The component protomers are composed of 13 subunits
The core has 3 large hydrophobic subunits
It has 4 redox centers: cytochrome a & a3 and copper atom (CuB) & a pair of copper atoms (CuA center)

Overall reaction - 4 Cyt c(red) + 4H+ matrix +O2 →4 Cyt c(ox) + 2H20
These 4 protons come from the matrix

Question 15

Describe the mechanism of the electron transport chain - complex IV?

Answer 15

1. Electron transfer from cytochrome C through: CuA, heme a, heme a3 to CuB
2. The second e- is transfered to the Fe in heme a3 = both CuB and Fe in heme a3 are reduced
3. O2 binds between haem a3 and CuB as a peroxide bridge
4. An additional e- from cyt c reduces the peroxide bridge = cleavage of O-O bond and a H+ is taken up from the matrix
5. The ferryl group is reduced as a further e- from cyt c is transfered and another H+ is taken up
6. Finally 2 more H+ result in release of H2O

Question 16

Describe proton pumping by complex IV - cytochrome oxidase?

Answer 16

As well as 4 ‘chemical’ protons removed from the matrix as part of the reaction
4 additional protons are pumped by the complex per 4e- transferred and per O2 reduced

Question 17

Give a recap of ATP synthesis?

Answer 17

Electron transport and ATP synthesis are coupled by the proton gradient
If we inhibit electron transport ATP synthesis stops
If we inhibit ATP synthesis electron transport stops
If we provide an alternative route for protons back through the membrane via an uncoupler the electron transport can continue but no ATP is formed because the proton gradient is dissipated by the uncoupler

Question 18

Describe the structure of ATP synthase?

Answer 18

It is made up of two parts
The first sits in the membrane, called F0 - essentially a proton channel
F0 comprises a transmembrane ring of hydrophobic proteins that act as a H+ channel
As protons flow through the F0 channel it rotates
This in turn drives rotation of the gamma subunit which drives conformational changes in a and b subunits

The second sits in the matrix, called F1 - acts as an enzyme to catalyse the synthesis of ATP
F1 is composed of 3 alpha and 3 beta subunits
b is the catalytic subunit, a is regulatory
gamma connects F1 to F0

Question 19

What is the mechanism of ATP synthesis?

Answer 19

Within the beta subunit there are different conformations that are more preferable for ATP synthesis
There is a T, O and L conformation (in a clockwise circle order) - tight, open, loose
1. ADP + Pi bind in the L (loose) binding site. there is a conformational change to T state
2. The T conformation has such a high affinity for ATP that bound ADP +Pi are converted to ATP (forming the phosphoanhydride bond)
3. This ATP is released after the conformational change driven by the rotation of the gamma subunit to the O conformation (driven 120° anticlockwise)
4. ADP and Pi bind to the vacant L site after further rotation converting O to L and L to T resulting in the synthesis of a second molecule of ATP

Question 20

What are P:O ratios?

Answer 20

The P:O ratio describes how many molecules of ATP can be made per oxygen atom reduced to water
Reduction of 1 oxygen atom (O) requires 2 electrons coming from substrates and transferred along the ETC

The P:O ratio is 2.7 for NADH linked substrates and 1.6 for FADH linked substrates
Other ATP synthases such as bacterial, yeast or chloroplast have a larger number (10-15) of c subunits in the Fo part of the ATPase therefore the P:O ratio is lower