ATP production in Mitochondria L8

Question 1

For ATP synthase the Fo domain is known as…

Answer 1

Fraction oligomycin

addition of oligomycin inhibits H+ permeability

Question 2

F1 domain

Answer 2

Separated head section has ATPase activity
No ATP synthesis activity
Faces mitochondrial matrix
In complex F1 region=ATP synthesis and NT hydrolysis
(can function in both directions)
Negative cooperativity b/w ATP binding sites
ATP release is very slow until ATP binds another subunit
PMF induces ATP release-lol u should know that moddafuckka

Question 3

Fo domain

Answer 3

High H+ permeability

No ATPase activity

Question 4

What are all the subunits in ATP synthase?

Answer 4

3alpha (NT binding sites do not participate in reaction/activity-redundant)
3beta (catalytic sites)
gamma/delta/epsilon (central region)
stalk region penetrates through catalytic region
OSCP (oligomycin sensitivity conferral protein)

a
b
oligomer of c subunits (8-15 copies)

Question 5

How much ATP is synthesised by these enzymes per hour?

Answer 5

50kg!!!

Question 6

What inhibiter inhibits the beta subunit?

Answer 6

Nbf-Cl
Binding at one beta subunit completely inhibits ATPase
Beta subunits cannot act independently
(3alpha 3 betas suggest there are 6 nucleotide binding sites)

Question 7

Where does the inhibitor DCCD bind to?

Answer 7

Inhibits at conserved asp/glu residues in c subunit. (carboxylic acids have ability to bind and release a proton)
Binding of just one c subunit completely blocks H+ transport
c subunits act as a whole unit, not independently

Question 8

alpha and beta subunits have sequence motif common in ATP-binding proteins. What are these?

Answer 8

Walker A (P-loop)/Walker B motif

Question 9

What is the Walker A motif

Answer 9

GXXXXGKT/S
P loop
Binding phosphate of ATP

Question 10

What is the Walker B motif

Answer 10

R/KXXXGXXL/VhhD
h=hydrophobic
Required for ATP hydrolysis/synthesis

Question 11

What is the binding change mechanism of ATP synthesis?

Answer 11

Tight–>open
Open–>loose
Loose–>tight
Loose ADP and Pi bind
Tight binds ATP and it can’t come off
The gamma subunit rotates and changes the structure of the 3 conformations (120degrees)
These changes are driven by conf changes transmitted to F1 though Fo (ATP translocation is indirectly driven by H+ translocation event)
The affinity for nucleotides of these sites changes in conjunction with the position of the gamma subunit
The gamma subunit always points to the OPEN subunit=defines open state of the beta subunit
Rotation of the gamma centurion subunit causes ATP synthesis because it forces off a tightly bound ATP

Question 12

c-ring

Answer 12

C ring rotates with gamma subunit
Driven by PMF
Involves glu or asp residue in c subunit
This is linked to 2 half channels in a-subunit
Conserved glu residue in c subunit forms salt bridge with arg residue in a-subunit
Protonation of glu through a channel to the P side side (IMS) neutralises this interaction
Neutralised c ring rotates so that the next non-protonated c subunit interacts with the arg residue
At some point/stage the protonated c subunit aligns with a second channel to the N side (matrix)
Proton is released to the N side, ready to start again.

1 full rotation requires proton transfer through each c subunit
Only one way in and one way out for protons (fail safe mechanism)

Question 13

How many ATPs are made per full c ring rotation?

Answer 13

3 ATPs (360 degrees and 3 catalytic sites)
The H+/ATP ratio depends on the number of c subunits
12 subunits = 4H+ per ATP

Question 14

How many subunits in the c ring can there be?

Answer 14

8-15

Question 15

Stator

Answer 15

holds the structure still
alpha3beta3 are kept fixed and still relative to a”” so that when c moves relative to “a” the catalytic sites don’t move as well.
Gamma subunit drives rotation

Question 16

How might the ATP synthase be reversed?

Answer 16

High concs of ATP reverses the process (ATP hydrolysis)
High ATP/ADP ratio-protons released back
Little/no PMF
Use ATP to create PMF (opposite to generate ATP)
Fermentative bacteria do this process (in reverse)

Question 17

What is the P/O ratio?

Answer 17

Number of ATP made per 1/2 O2 reduced

Question 18

If ATP synthase has 12 c subunits how many H+ are needed for each ATP synthesised in mitochondria?

Answer 18

5 per ATP
12/3=4
+1 H+ to transport ATP/ADP/Pi

Question 19

If an ATP synthase has 10 c subunits what is the theoretical P/O ratio for the oxidation of NADH?

Answer 19

2.31