ATP synthase

Question 1

2 components of the proton motive force

Answer 1

chemical potential energy: due to difference in concentration in H+
electrical potential energy: due to separation of charges

Question 2

Chemiosmotic theory

Answer 2

• explains how the concentration of protons is transformed to ATP
• free energy used by ETC to pump proteins moving H+ to IMS
• protons flow back into the mitochondrial matrix down its concentration gradient
• energy of the electrochemical gradient is released and used for generation of ATP

Question 3

obligatory coupling of electron transfer and ATP synthesis

Answer 3

1. an inhibitor of electron transfer will inhibit both oxygen consumption and ATP synthesis
   - since the energy for ATP synthesis is derived from the oxidation process
   - succinate and ATP are required to synthesize ATP
2. Inhibition of ATP synthase blocks the ETC
   - when ATP synthase is blocked the H+ remain in the IMS and concentration builds up
   - energy to pump protons against gradient will eventually exceed energy available from NADH

Question 4

uncoupling oxidative phosphorylation

Answer 4

• if the IMM is disrupted, the proton gradient is eliminated
• electron transport continues but ATP synthesis stops
• certain chemicals can act as uncouplers without damaging the mitochondrial membrane (ex. DNP)

Question 5

action of 2,4-DNP

Answer 5

• DNP- picks up a proton from DNPH and transports it across the IMM
• releases H+ into the matrix
• DNP- -ve charge is delocalized over the ring making it hydrophobic
• the proton gradient is collapsed and ATP synthesis stops

Question 6

2 functional domains of ATP synthase

Answer 6

F1: peripheral membrane proteins
- each beta-subunit contains a catalytic site for ATP synthesis
F0: integral membrane proteins
- multiple c-subunits contain an Asp
- a-subunit contains 2 half channels for the movement of protons

Question 7

what provides the energy required for the release of formed ATP

Answer 7

the proton gradient
- the energy barrier (hump) is not the transition state, but the release of formed ATP from the enzyme

Question 8

how does ATP synthase overcome the energy barrier for release of ATP

Answer 8

• Rotational catalyst
• the active site of ATP synthase cycles between a form that tightly binds ATP and one that releases ATP
• the active site is formed, broken then reformed in a cyclic fashion

Question 9

Beta and gamma-subunit in the rotational catalyst

Answer 9

• beta-subunit has 3 confirmations: lose, tight and open
• gamma-subunit rotates in the centre and rotating the beta-subunits between the 3 confirmations
• a beta-subunit starts as loose, binding ADP and Pi, then moving to tight where ATP is formed, and open where ATP is released

Question 10

a-subunit half channels

Answer 10

• one half channel leads from the IMM and the other leads to the maxtrix
• there is no direct route from one channel to the other
• protons must jump from a to c, then ride around c and jump to the second half of a

Question 11

how does the passage of protons make the c ring rotate

Answer 11

• c10 ring is held in place by ionic interaction of Asp of c and Arg of a
• when a proton jumps from a to c it breaks the ionic bond which makes c10 rind rotate
• simultaneously another c subunit Asp is forced to make contact with a
• the proton carried by that Asp is released into the matrix
• the process continues, after 10 protonation events the c10 ring has completed one full revolution

Question 12

how does ATP synthase release ATP

Answer 12

• when the beta-subunit cycles through all 3 confirmations, it leads to the synthesis and release of 3 ATPs

Question 13

P/O ratio

Answer 13

• number of moles of ATP synthesized per moles of O reduced to eater
  P/O NADH ~ 2.5
  P/O FADH2 ~ 1.5