ATP production in Mitochondria L8 Flashcards
For ATP synthase the Fo domain is known as…
Fraction oligomycin
addition of oligomycin inhibits H+ permeability
F1 domain
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
Fo domain
High H+ permeability
No ATPase activity
What are all the subunits in ATP synthase?
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)
How much ATP is synthesised by these enzymes per hour?
50kg!!!
What inhibiter inhibits the beta subunit?
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)
Where does the inhibitor DCCD bind to?
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
alpha and beta subunits have sequence motif common in ATP-binding proteins. What are these?
Walker A (P-loop)/Walker B motif
What is the Walker A motif
GXXXXGKT/S
P loop
Binding phosphate of ATP
What is the Walker B motif
R/KXXXGXXL/VhhD
h=hydrophobic
Required for ATP hydrolysis/synthesis
What is the binding change mechanism of ATP synthesis?
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
c-ring
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)
How many ATPs are made per full c ring rotation?
3 ATPs (360 degrees and 3 catalytic sites) The H+/ATP ratio depends on the number of c subunits 12 subunits = 4H+ per ATP
How many subunits in the c ring can there be?
8-15
Stator
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
How might the ATP synthase be reversed?
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)
What is the P/O ratio?
Number of ATP made per 1/2 O2 reduced
If ATP synthase has 12 c subunits how many H+ are needed for each ATP synthesised in mitochondria?
5 per ATP
12/3=4
+1 H+ to transport ATP/ADP/Pi
If an ATP synthase has 10 c subunits what is the theoretical P/O ratio for the oxidation of NADH?
2.31
