Lecture 30: Oxidative phosphoryltion Flashcards
Mitchell proposed the chemiosmotic coupling hypothesis. What is this?
“Chemiosmotic Coupling hypothesis”
Knew:
- ETC in the inner mitochondrial membrane
- membranes impermeable to protons
Proposed:
- ETC pumps protons out of the matrix
- Generates a proton-motive force
- Proton-motive force drives ATP synthesis
What is the proton motive force (PMF) and what role does it play in ATP generation?
The proton gradient across the inner mitochondrial membrane results in two energetic gradients:
- A chemical gradient or pH gradient due to different H+
concentrations on either side of the membrane - An electrical gradient due to the charge difference across the membrane (positive in the intermembrane space, negative in the matrix)
This electrochemical gradient is known as the proton-motive force (pmf).
Mitchell proposed that the energy of the pmf drives ATP
synthesis
What are the two components of proton-motive force (PMF)?
Chemical AND electric gradient across the inner membrane created by the ETC which can then power ATP synthase
Name the three experiments that provide evidence to support chemiosmotic coupling hypothesis for ATP synthesis:
Detergent test: Removes outer membrane
The Light test: Light induced proton pump
The H+ shuttler: DNP uncoupler
Describe the detergent test and how it provides evidence to support the chemiosmotic coupling hypothesis for ATP synthesis:
Steps:
Isolate mitochondria, treat with mild detergent to remove outer membrane.
Results:
ETC still works, but no ATP is made.
Conclusion:
The outer membrane is required to maintain the proton gradient for ATP synthesis.
Describe the Light test and how it provides evidence to support the chemiosmotic coupling hypothesis for ATP synthesis:
Setup:
Artificial liposome with bacteriorhodopsin (light-induced proton pump) and ATP synthase.
Results:
- In the presence of light, protons are pumped, creating a proton gradient, and ATP is made. (No light = no protons pumped and no ATP being made)
- ETC is not present.
Conclusion: The proton gradient alone is sufficient for ATP synthesis.
Describe the DNP (2,4-Dinitrophenol) uncoupler test and how it provides evidence to support the chemiosmotic coupling hypothesis for ATP synthesis:
Setup:
Add DNP (an uncoupler that shuttles H⁺ from the intermembrane space to the matrix).
Results:
ETC functions, but no ATP is made.
Conclusion:
A proton gradient is essential for ATP synthesis, as DNP dissipates the gradient by pumping H+ back into the matrix
What is an “uncoupler” in oxidative phosphorylation?
an uncoupler “uncouples” the ETC from ATP production by eliminating the proton gradient required for ATP synthesis.
How does ATP-synthase act like a turbine to generate ATP?
The “rotor subunits” turn
The “stator subunits” do not turn
Proton flow drives rotor movement
Rotor movement causes conformational changes in
the stator that drives ATP synthesis
More detail of above:
3 alpha-beta pairs
As gamma stalk turns it changes shape/conformation of alpha-beta pairs
This change in shape leads to ATP synthesis
Describe the conformational changes of the alpha-beta subunits of F1: Describe Open, Loose, Tight
O = open (ATP release/ADP and Pi binding)
L = loose (holds ADP and Pi in preparation for catalysis)
T = tight (catalysis - ATP formation)
Changes for each dimer when rotor turns
- each dimer eg. (O > L > T > O > L etc)
How many protons are required to generate 1 ATP?
4 protons per 1 ATP
One NADH entering the ETC leads to 10 protons being pumped. How many ATP will this produce?
NADH = 10 protons pumped in ETC
2.5 ATP made
One FADH2 entering the ETC leads to 6 protons being pumped. How many ATP will this produce?
FADH2 = 6 protons pumped in ETC
1.5 ATP made
