Electron transport chain and oxidative phosphorylation

Question 1

Describe the path of the electrons from NADH in the ETC

Answer 1

Complex I, complex Q, complex III, cytochrome C, complex IV, oxygen

Question 2

Why use several complexes in the electron transport chain ?

Answer 2

It slowly uses up the reducing power of the NADH / FADH2, maximising the effeciency.

Question 3

What types of structures carry the electrons in the ETC ?

Answer 3

Metals:
Fe, or Cu

Lipids with stabalised rings
riboflavin ring
quinone ring (ubiquinone)

Question 4

Where in the membrane are each of the ETC components ?

Answer 4

Complexes I, II, III, and IV are integral membrane proteins
Conezyme Q is lipid and inside the membrane
Cytochrome C is peripheral

Question 5

How does complex III and ubiquinone transport electrons in the ETC

Answer 5

When reduced, ubiquinol has 2 hydrogens, which are transferred to complex III when it is oxidised back to ubiquinone.
2 more are pumped by complex III

Question 6

Stoichemistry of ATP

Answer 6

1 NADH = 10 H+ = 2.5 ATP

1 FADH2 = 6 H+ = 1.5 ATP

Question 7

What are poisons that inhibit the ETC

Answer 7

Complex I - rotenone or amytal

Complex III - antimycin

Complex IV - cyanide

Question 8

END OF LECTURE Q: Typically, how much energy does it take to make one ATP under normal operating conditions with high cellular ATP levels?

Answer 8

Typically it will take 4 H+ to make one ATP

NADH will give 10 H+,
FADH2 will give 6 H+

Question 9

END OF LECTURE Q: Why does FADH2 generated in β-oxidation only give 1.5 ATPs/ FADH2 whereas NADH + H+ gives 2.5 ATPs/NADH?

Answer 9

FADH2 is fed into complex II, which does not pump any protons

NADH is fed into Complex I, which pumps 4 protons.

It takes approx 4 protons to make one ATP.

Question 10

END OF LECTURE Q:
Q3. Why does the H+ gradient not dissipate by leaking out from the intermembranal space to the cytosol through the outer mitochondrial membrane (porin)?

Answer 10

The porin is fairly leaky, but is specific enough to keep protons in.

H+ is positive and small, whereas many things that can pass (eg fatty acids) are negative

Question 11

What is the consequence on respiration if the ETC is inhibited ?

Answer 11

Oxygen consumption is halted when the ETC is inhibited

Question 12

What is the effect of oligomycin ?

Answer 12

It inhibits the Fo sub unit in ATP synthase (which is what the Fo subunit is named for)

Question 13

What is the coupling factor between ATP synthase and the electron transport chain ?

Answer 13

Proton gradient

Question 14

What is the mitchell hypothesis and what is the evidence to support it ?

Answer 14

The Mitchell hypothesis suggests that the ETC complexes pump a proton gradient that drives ATP synthesis (proton motive force)

Evidence:

• pH difference across mitochondrial membrane (approx 1pH unit difference)
• cleaving the F1 of ATP synthase will allow H+ to pass but does not make ATP
• Damage to the inner mitochondrial matrix disrupts H+ gradient and stops ATP production
• Uncouplers (get rid of the H+ gradient) stop the production of ATP
• Racker and Stoeckenius experiment: Bacteriorhodopsin creates a proton gradient under light, ATP synthase spins when gradient is formed
• ATP synthase will function when artificial gradient is introduced
• Evolutionary similarities

Question 15

What part of ATP synthase spins ?

Answer 15

The rotor (Fo) and the γ unit spin,
And the stator (F1) is stationary

The stator has 3α3β subunits and actually makes the ATP

Question 16

What are the 3 states of the stator and what do they do ?

Answer 16

The beta units have 3 conformations, loose, tight, open

The L state binds ADP and Pi, the T state hydrolyses them int ATP, the o state releases the ATP

Question 17

What is the Kinosita experiment ?

Answer 17

The stator of ATP synthase was His tagged and bound to a Ni complex,
Actin was bound to the rotor with avidin an biotin.
In the presence of ATP, the actin spun around in 120 degree segments.

Question 18

END OF LECTURE QUESTION

What are the differences between an inhibitor of ETC or Ox-Phos and an uncoupler? How could you distinguish between the two agents?

Answer 18

An inhibitor of the ETC will prevent the function of one or more of the ETC complexes (transferring electrons and pumping H+)
Likewise, an OxPhos inhibitor will inhibit ATP synthase.
An uncoupler simply destroys the proton gradient without directly inhibiting any of these complexes.

If ATP synthase is inhibited, it will also inhibit the ETC, because the proton gradient will build up to a point where the ETC will not be able to work against the gradient
Likewise, if part of the ETC is inhibited, the ETC will not be functional.
In both these scenarios, oxygen will not be consumed, as the ETC is nonfunctional

An uncoupler removes the proton gradient without disturbing the function of the ETC, and so oxygen will still be consumed.

Question 19

END OF LECTURE QUESTION

Why is the proton channel of the ATP-synthase called Fo?

Answer 19

It was named for its inhibition by oligomycin

Question 20

END OF LECTURE QUESTION

The pH of the intermembranal space and matrix differ by 1 pH unit. Which way round is this, which is acid and which is alkaline? Why is the intermembranal space sometimes called the P-side & matrix N-side?

Answer 20

The intermembranal space has a large amount of protons (positive, p-side) and is therefore acidic (low pH)

The matrix is by comparison negative (n-side) and basic (high pH)