ATP synthesis from proton gradient Flashcards
What are the two functional domains of mitochondrial ATP synthase/ATPase
- F1: Peripheral membrane protein, its function is to synthesize atp
- F0 Integral membrane protin (oligocycin binding), sits imedded in protien providing channel for protons
- F1 was first identified by Efin Racker in 60s
What is the F1 subunit composed of
- contains 9 subunits: α3 β3 γ δ ε
- contains the catalytic site for ATP synthesis
- three α subunits are identical molecules, three β subunits are identical
- Each β subunit contains a catalytical site for ATP synthesis
- THe γ subunit forms a stalk in the centre of the α3 β3 complex
- acting thorugh ε attached F1 to the membrane embedded “C” ring of Fo
*three catalytic sites can synthesise 3 ATP at once
What is the composition of Fo
ab2C10-12
- multiple C subunits, each a hairpin of two hydrophobic α helices, assemble to form a transmembrane C10 ring
- the number of c subunits vary among different kingdoms of life
- each C subunit contains a conserved aspartic acid residue (asp 61) in middle of one of its helices
- the two b subunits (acting through δ ) associate firmly with α3 β3
- the a subunit wraps partially around the C-10 raing and contains two half channels for movement of protons
*a is embedded in membrane providing two half channels
Who is Paul Boyer
paul boyer found the equilibrium constant for the reaction ADP + Pi -> ATP + H2O to be close to 1
- when the reaction occurs in the active site of the ATP synthase
- the formed ATP remains very tighty bound to the active site
*the enery required for the release of the formed ATP is provided by the proton gradient
*free energy change was about 0, energy levels of ATP and ADP are about same not what we expect, enzme is binding ATP very tightly (atp synthesized is not free atp is it bound to enzyme, this makes energy content low because if youre holding on to soemthing you restrict it and have less energy)
* energy required to remove ATP from enzyme not from synthesis itself
How does the proton gradient drive the release of ATP from the active site
- unlike typical enzyem catalyzed reaction, the energy barrier for ATP synthase is not reaching the transition state but the release of formed ATP from the enzyme
How does ATP synthase overcome the large energy barrier for release of ATP
- rotational Catalysis
- Boyer suggested that the ATP synthase active site cycles between a form that tightly binds ATP and form that releases ATP
*needs to change shape*
- the active site, in the cyclic fashion is formed, torn apart, then re formed to allow the continued synthesis of ATP
What is the structural basis for rotational catalysis?
Each β subunit of F1 can assume 3 different conformations
- the γ subunit interacts asymetrically with the α3 β3 complex causing the three β subunits to have different conformations each associated with differences in their ADP/ATP binding site
- one β subunit is able to cycle between these three forms
- the γ subunit rotates in centre of the α3 β3 complex
- with each 120 degree turn the γ subunit a differnt face comes into contact with each β subunit rotating them between the three different conformations
- A given β subunit starts in loose, binding ADP and Pi, then the tight form where ATP formed and finally open where ATP released
*at any given time each site in different conformation, as gamma moves around eahc beta changes shape , beta subunit is where catalytic site if found
What is held in place/what rotates
- the α, β, and δ subunits form the stator arm remaining fixed with resepct to the inner membrane
- the α3 β3 complex remains stationary helf in place by δ
- the sing of c subunits rotates with respect to the stator (a,b and δ) powered by the proton gradient
- the γ and ε subunits rotate along with the c-ring
- the net result is that the γ subunit turns within the core of the α3 β3 complex
Explain the rotary mechanism of ATP synthase
- the “a” subunit provides a passage for the movement of protons across the IMM by two half channels
- one half channel leads from the inter membrane space side of the membrane into the “a” subunit, the toher half channel leads from inside of the “a” subunit to the matrix with no direct routs from one half channel to other
- protons much jump from the “a” subunit onto adjacent “c” subunit, ride around the “c” subunit and move throough the second half of the channel
*goal is to get gamma to rotate*
How does passage of protons make the c ring rotate
*C 10 ring composed dorm 10 subunits each w. an asp
- C10 ring held in place by ionic interaction between Asp 61 of C and two conserved arginine residues of “a”
- a proton jumps from the intermembrane space half channel of “a” to the “c” and protonates Asp 61
- this breaks ionic interaction between asp and arg, setting the c10 ring free
- C10 ring rotates so that the protonated “c” moves away from “a” and into the hydrophobic milieu of the membrane
- Simultaneously, another “c” subunit (AspH) is forced into contact with the half channel of “a” which opens into the mitochondrial matrix
- Proonton carried by asp is released into the matrix
- the process will continue with another proton entering throught he IMS half channel to protonate Asp 61 of a second c subunit
- C10 ring will once again rotate, rbinging another protonated AspH into contact witht eh matrix half channel
- a proton will exit into the matrix through that half channel while another enters the c ring through the other
- the protonation/deprotonation event will continue to occur, moving rpotons fromt he IMS to the amtrix, adn rotating the C10 ring in the process
- after 10 such protonation deprotonation events, the C10 ring performed one full revolution
*rotation of C leads to the rotation the γ in the centre of α3 β3 . one full rotation of γ causes each β subunit to cycle thorugh all three possible conformations. leading to synthesis of thee ATP
What is the overall efficiency of oxidative phosphylation
x(ADP + pi) + NADH + H+ + 1/2O2 -> xATP + NAD+ + H2O
What is the P/O ratio
x
- if 10 c subunits, requires 1 full turn (10 pumped protons) to synthesise 3 ATP
- number of moles of ATP synthesized per mole of O reduced to water (or per 2e- passed along ETC: or per mol NADH,NAFH2 oxidized
P/O NADH: 2.5
P/O FADH2: 1.5
- would expect P/O ratio should be 3, but some energy is lost, some protons along gradient are used for other processes
* chemiosmotic coupling allows for non integral values for the P/O ratios. The P/O ratio depends on the mechanism by which th eproton gradient energy is used to power ATP synthesis
Each NADH oxidized produces ___ ATP
3
* to get 3 atp needs to turn 360 degrees, if only 8 subunits, only need 8 protons.
How many ATP come from the oxidation of a mole of palmitate (16:0) to CO2 and H2O
- need 7 rounds of B oxidation, producing 7 NADH, 7FADH2 and 8 Acyl CoA
- (7 x 2.5)= 17.5 ATP, (7x1.5)= 10.5 ATP
- in cirtic acid cycle produces 3 NADH and 1 FAD and 1 ATP: have 8 turns
3NADH per turn x 8 turns x 2.5 = 60
1 FADH2 per turn x 8 turns x 1.5 = 12
1 GTP/ATP x 8 turns = 8 ATP
TOTAL: 108
What steps produces NADH
(fatty acids)
enzymes:
B hydroxyacyl CoA dehydrogenase (B ox): if 16 carbon fat, 7 NADH
Isocitrate dehydrogenase (citric): if 16 carbon fat 8NADH
alpha ketoglutatrate dehydrogenase (citric): If 16 carbon fat 8 NADH
Malate dehydrogenase (citric): if 16 carbon fat 8 NADH
