Lecture 7 - ATP synthase

Question 1

How were mitochondrial particles used in early ATP syntase structure function studies

Answer 1

Early studies on ATP synthase involved sub-mitochondrial particles. These particles, formed by treating a membrane structure with ultrasonic vibrations, exhibited both electron transport and ATP synthesis capabilities. The F1 region, responsible for ATPase activity, was detached from the membrane using low ionic strength media, while the membrane vesicles remained H+ permeable. Ca+ was used to reattach the F1 region.

When substrates for the Electron Transport Chain (ETC) were added to these particles, they participated in synthesizing the proton gradient. In the presence of ADP and phosphate, ATP could be generated. Interestingly, blocking the ETC and adding ATP to the particles showed that the synthase could pump protons into vesicles, forming a proton gradient. This indicated the enzyme’s full reversibility.

Inhibitors of the synthase, such as oligomycin and DCCD, were identified. Oligomycin specifically inhibited proton translocation and led to the naming of the FO portion. DCCD, requiring only one molecule to inhibit proton translocation, guided the understanding of the mechanism for FO portion proton translocation. To summarize, ATPase is fully reversible, the F1 region is involved in ATP synthesis/hydrolysis, and the membrane-embedded FO portion forms the proton channel.

Question 2

Describe the Fo portion strucutre of E.coli - F-type ATPase

Answer 2

The structure is composed for 3 distinct subunits
* Subunit a has 6 TMS domains
* Subunit b has one TMS and has a stalk region which is important in binding the F1 portion
* Subunit c has an arginine 41 which is important in binding F1 and a glutamate/aspartate 61 which is crucial in H+ translocation and DCCD binding (Prevents H+ translocation)
Binding studies show that the binding of a single DCCD molecule to just one of the 10 C subunits completely inhibits H+ translocation.

Question 3

Describe the tertiary structure for F1 portion (Bovine heart)

Answer 3

The structure is composed of 5 distinct subunits: α, β, δ, γ, ε
* There are 3 α subunits and 3 β subunits that form an alternating ring around the γ subunit.
* The γ subunits is a 2 twisted α helices
* Each of the β subunits has a different state: Either loose, open or tight
* It was proposed that the γ subunit rotated coupling the opening and closing of the nucleotide binding sites on the β subunits

The rotation of the γ subunit was proven using a fluorescent actin engineered to the γ subunit. Histidine residues were engineered onto a subunits in order to attach it to an Ni coated plate. ATP was given and this resulted in ATP hydrolysis producing ADP and Pi. The actin filament rotated.

The forced rotation of the y subunit relative to a3b3 resulted in ATP synthesis. A magnetic bead attached to the y subunit was used to rotate the y subunit. It occurred in a medium containing ADP and pi as well as the luciferin-luciferase system which hydrolyses ATP and emits photons. A sensitive camera is used to measure photon emission. The greater the rotatory speed the greater the ATP synthesis.

Question 4

Describe rotational catalysis

Answer 4

• The translocation of H+ through FO generates the torque in the y subunit to rotate the a3b3 complex
  
  • The stator holds F1 head so that it is fixed relative to the rotation of the y subunit

Subunit c - Asp61 will be protonated when in the hydrophobic environment of the membrane
Subunit a - Two half channel structures provide aqueous pores that do not fully cross the membrane. One has an exit towards the N side the other towards teh P side.
The Asp61 residues in contact with the half channels are in a hydrophilic aqueous environment and tend not to be protonated
The Asp 61 residues in contact with the half channels are in a aqueous environment and tend not to be protonated in neutral environment.

Question 5

How does proton translocation through Fo drive rotation and generate the torque

Answer 5

1. H+ from the P side enters the half channel facing the P side
   
   2. The Asp 61 facing the P side protonates due to the acidic conditions
   3. The protonated Asp61 is rotated into the membrane
   4. The H+ dissociates from Asp61
   5. It diffuses to the N side according to the H+ gradient

Total = The movement of one proton across the membrane and the rotation of one c subunit clockwise
10 H+ are required to move the ring of c subunits a full 360 degrees

The binding change mechanism
1. OPEN state: ATP binding is poor and is therefore released
2. LOOSE state: ADP + Pi binding is weak
3. TIGHT state: Spontaneous formation of ATP from ADP and Pi. ATP tightly bound

The H+ electrochemical gradient is not directly needed for ATP synthesis - ATP synthesis is spontaneous
In the absence of a H+ gradient ATP is not released by the catalytic binding site
The role of the electrochemical gradient is not to form the ATP but to release it from the synthase enzyme.

It has been established that the rotation of the y subunit 120 degrees will synthesise 1 molecule of ATP
10 H+ are required for the full 360 and 3 ATP to be formed