Oxidative Phosphorylation 2 Flashcards
How can the transfer of 2e- from NADH to O2 be written as
- NADH + H+ + ½ O2 NAD+ + H2O
How would you calculate gibbs energy for the transfer of 2e-
- DGo’ = –nFDEo’ = –nF[Eo’(acceptor) – Eo’(donor)]
How do we determine DG available from H+ gradient across mitochondrial membrane
- DG = RT ln (c2/c1) + ZFDy
- (c2/c1) = concentration ratio for the ion that moves
- Z = absolute value of its charge
- F = Faraday (96.5 kJ mol-1 V-1)
- Dy = electrical potential difference across membrane (V)
In actively respiring mitochondria what is DG from H+ gradient per NADH
- DpH is about 0.75 pH units
- Dy is about –0.15 V
- DG = –0.74 kJ mol-1 + –14.48 kJ mol-1 = –15.21 kJ mol-1
- 10 H+ are available from each NADH, so DG = –152.1 kJ mol-1 per NADH
Where does most of the free energy available from the H+ gradient come from
- Most of the free energy available from the H+ gradient (the proton-motive force) in mitochondria is derived from Dy (–14.48 kJ mol-1) rather than DpH (–0.74 kJ mol-1)
What is the efficiency of energy conversion by electron transport system based on energy difference
- Efficiency of energy conversion by electron transport system is about 70%:
- 152 kJ mol-1 (generated from proton gradient) / 220 kJ mol-1 (energy required to transfer electrons?)≃ 0.7
- Energy difference of about 70 kJ mol-1 is lost as heat and contributes to thermogenesis
- DG (as opposed to DGo’) of NADH is likely to be more negative than –220 kJ mol-1
What is the efficiency of energy conversion by electron transport system based on energy difference based on number of ATP molecules made
- estimated DG for ATP synthesis in mitochondrial matrix is –40 kJ mol-1
- Hence theoretical yield per NADH oxidized is 152 kJ mol-1 / 40 kJ mol-1 ≃ 3.8 ATP
- Actual yield per NADH oxidised is about 2.5 ATP, so efficiency is 2.5 / 3.8 ≃ 66%
Describe the main components of ATP synthase
- F1
- F0
- Stalk
- Associated polypeptides
Describe the subunit composition of F1 and its function
- a3b3yδe
- Beta- contains the ATP synthase site
- δ- forms the gate coupling the F0 proton channel with F1
Describe the subunit composition of F0 and its function
- 4-5 types of subunit including 6-10 copies of DCCD-binding proteolipid
- DCCD-binding proteolipid oligomer forms the proton channel
Describe the subunit composition of the stalk and its function
- One copy each of OSCP and F6
2. Required to bind F0 to F1
Describe the subunit composition of the associated polypeptides and its function
- IF1 and F(B)
2. IF1 inhibits ATP- hydrolyses and binds to the F1 B subunit
Describe the difference between the bacterial and yeast F1 component
- The bacterial ATP synthase complex contains a subunit called ε that associates with the F1 component. – Sits in cytoplasm as does not have mitochondria
- The δ subunit of the yeast ATP synthase complex is homologous to the bacterial ε subunit.
- Equivalent subunits with different names e.g. yeast δ = bacterial ε
What does the OSCP subunit stand for
- The OSCP subunit in the yeast ATP synthase complex is the oligomycin-sensitivity-conferring protein.
- C sub unit is part that rotates
- Rotary motor- One direction – synthesises ATP, Other direction- hydrolyses ATP
What is the main role of F1 and F0
- F1= catalytic activity
2. F0= H+ channel
What are the general structures of the F0 and F1 subunit
- F0 is water insoluble transmembrane protein composed of 10-12 subunits
- F1 is water soluble peripheral membrane protein composed of 5 types of subunits
What can oligomycin do
- Inhibits ATP synthase by binding to a subunit of F0 (not OSCP)
- This means it interferes with H+ transport through F0
What are the 3 functional units of ATP synthase
- Stator:
2) Rotor:
3) Headpiece:
Describe the stator
- a subunit with half-channels for H+ to enter and exit FO, plus stabilizing arm (b, d, h and OSCP
- The stator holds the headpiece in place so that it does not turn with the rotor.
Describe the rotor
- c + g + d + e rotate as H+ enter and exit c-ring
2. the rotor is responsible for translating proton-motive force into protein conformational changes in the headpiece.
Describe the headpiece
- hexameric a3b3 unit responsible for ATP synthesis
- a3b3 hexamer contains the 3 catalytic sites, on outer edge of each b subunit
- g subunit extends inside a3b3 hexamer and is attached to the c ring
What ae the 3 phases of the binding change mechanism
- Translocation of protons carried out by F0
- Catalysis of formation of the phosphoanhydride bond of ATP carried out by F1
- Coupling of the dissipation of the proton gradient with ATP synthesis which requires interaction of F1 and F0
What are the different ATP binding affinities of the 3 beta subunits
- Tight: ATP bound
- Loose: ADP and Pi bound
- Open: ATP is released and accept ADP and phosphate
Why are there 3 different beta conformations
- g directly contacts all three b subunits, but each interaction is distinct giving rise to 3 different b conformations
What drives the conformational change that transitions the subunits to different sites
- The number of H+ required for each 120° turn of the γ subunit depends on the number of subunits in the c ring; the yeast mitochondrial ATP synthase complex appears to require ∼3 H+ for each ATP synthesized
What drives the conformational change that transitions the subunits to different sites
- Driven by free energy supplied by proton flow
- The impermeability of the inner mitochondrial membrane allows an electrochemical gradient to be established across the membrane during the H+ translocation associated with electron transport
- The only way for the H+ to reenter the matrix is through the F0 portion of the proton-translocating ATP synthase
- The electrochemical gradient builds up until the free energy required to transport H+ balances the free energy of electron transport
- Electron transport then must cease
- ATP synthesis by dissipating the electrochemical gradient allows the electron transport to continue.
How many H+ are required for each 120 degrees turn of Y subunit
- The number of H+ required depends on the number of subunits in the c ring;
- the yeast mitochondrial ATP synthase complex appears to require ∼3 H+ for each ATP synthesized
Describe the first experiment that shows that ATP synthase rotates
- Took catalytic part of ATP -gamma sub-unit and attached long fluorescent actin filament to the gamma sub-unit
- Attached to glass slide
- Fed it ATP
- Rotary motion can be seen from fluorescence
- The g subunit rotates in 120º steps.
- The fluorescence micrographs show the position of the fluorescent actin polymer at 133 ms intervals.
Describe the second experiment that shows that ATP synthase rotates
- F1 component as a nanomotor driving ATP synthesis in absence of electrochemical proton gradient
- Attach a magnetic bead to the gamma subunit
- Made a chamber with electromagnets
- Fed atp
- Use electric field to drive synthesis or hydrolysis – can reverse the field to change direction of rotation
- ATP synthesis or ATP hydrolysis can be catalysed by a3b3 headpiece simply as function of rotational direction of g subunit imposed by electromagnets
- Proof that rotational motion of g subunit drives ATP synthesis, independent of electrochemical H+ gradient
How does H+ movement cause rotation of g subunit?
- Two-channel model
- H+ neutralizes D59 allowing c subunit to rotate 36° into hydrophobic membrane
- Proton binds to aspartate side chain – neutralises positive charge- can move into hydrophobic membrane
- This rotation allows D59 in a different c subunit to access the second half-channel in the a subunit and exit the channel because of the low H+ concentration on matrix side
- Carousel analogy: each H+ must ride once around the c ring carousel to exit into matrix
