OxPhos - ATPSynthase

Question 1

What is the structure of the F1 fragment?

Answer 1

3α, 3β and γ subunits
- 3 of the α chains binding sites are inactive
Bind ATP but not catalytic
- nucleotide-protein interactions occur in the β subunits
- γ subunits rotates inside the subgroup

Question 2

Where are the nucleotide binding sites within F1?

Answer 2

interface of the α subunits β subunits

Question 3

What asymmetry is present within F1?

Answer 3

due to the asymmetric gamma subunit, this forces asymmetry from head groups in the β subunits

Question 4

What does the asymmetry in F1 mean in terms of ATP Synthase?

Answer 4

• ATP Synthase can run in reverse
• differing interactions between the C-terminal domain of the β subunit and the coiled coil of the γ subunit
• when gamma subunit rotates, it pushes beta subunits out differently so each one is different - inducing conformational changes

Question 5

What interactions do the β and γ subunit have?

Answer 5

A ring of Arg residues on the coiled-coil of the γ subunit electrostaticly interact with conserved carboxylate amino acids (DELSEED loop) in the β subunit
- electrostatic interactions are maintained as the gamma subunit rotates

Question 6

How can you achieve single-particle microscopy in real time?

Answer 6

• fluorescent label on actin filament attached to the γ subunit
• F1 is upside down and fixed onto glass side
• F1 γ subunit rotates when F1 catalyses the reverse reaction, hydrolysis of ATP
• Add ATP
• Gamma subunit rotates
• Observe a bright light that rotates every time ATP is added
• Can now calculate the force needed to hydrolyse the ATP

Question 7

What is the binding change mechanism?

Answer 7

2 of the active sites of β subunit are occupied with ATP and ADP, one is empty.

• head groups do not rotate
  1. Gamma subunit was in the Beta empty subunit
  2. Gamma subunit rotates into the ADP subunit
• intermediate
  3. After rotation of 120 degrees of the gamma subunit (fully positioned in the original ADP subunit)
• What was the empty active site is now the ATP active site, what was the ADP site is now empty and what was the ATP site is now the ADP site.

Question 8

How does ATP Synthase overcome the fact that ATP Hydrolysis is more thermodynamically favourable than ATP synthesis? And that ATP binds very tightly to the enzyme.

Answer 8

Hydrolysis & Synthesis both happen, reaction going backwards and forwards until the gamma subunit physically forces the ATP sub-unit out

Question 9

What is the Yeast F0 rotor formed from?

Answer 9

10 helical hairpins (c polypeptide)

- conserved acidic a.a on the outside helix

Question 10

What is the proposed two channel model of F0 rotor explaining?

Answer 10

• the reason as to why 2 acidic amino acids are present in the C polypeptide that sits in the membrane

Question 11

What is the proposed two channel model of F0 rotor ?

Answer 11

• protein formed 2 half channels (a subunit and c subunit)
• H+ enters from IMM (through A subunit)
• Goes halfway through membrane
• Meet the A.A’s
• Neutralise A.A
• Rotates around C ring
• Until it gets to next half channel
• Arginine wants negatively charged and so the proton is released
  Released into matrix space

Question 12

What is the overall structure of the F0F1ATPase?

Answer 12

Dimer

• contains an F0 that has a rotating subunit in the membrane
• contains a F1 that has a rotating gamma subunit

Question 13

How many ATP’s are produced from every half O2 used?

Answer 13

2.7