Lecture 33 - Oxidative phosphorylation - finally making some ATP !! Flashcards
The mechanism of oxidative phosphorylation
Peter Mitchell 1961
Knew:
Inner mitochondrial membrane impermeable to protons
ETC in the inner mitochondrial membrane
Proposed:
ETC pumps protons out of the matrix
Generates a proton motive force
Proton motive force drives ATP synthesis
Proton motive force
The proton gradient across the inner mitochondrial membrane results in two energetic gradients …
1- a chemical gradient or pH gradient as a result of different H+ concentrations on either side of the membrane
2- an electrical gradient as a result of the charge difference across the membrane (positive in the inter membrane space, negative in the matrix) - ATP synthase uses the gradient
This electrochemical gradient is known as the proton motive force (pmf)
Mitchell proposed that the energy of the pmf approx 180 mV drives ATP synthesis
Proton gradient produced by…
ETC
Intermembrane space - increased H+, H3O+ (water picks up a hydrogen)
Matrix - Decreased H+, OH- (water can lose a hydrogen and become a hydroxide)
Chemiosmotic coupling hypothesis - experimental evidence
Isolate mitochondria from cells via centrifuge then …
Treat with strong detergent, soubise all the membranes which results in the ETC not working
Treat with mild detergent, only removes outer membrane, ETC still works but no ATP is made therefore shows that ATP is not synthesised in the ETC
Note - detergents disrupt membranes
Artificial liposome
Bacteriorhodopsin is a light inducible proton pump
In light will pump protons to make a proton gradient
ATP synthase - makes ATP when light switched on, does not make ATP when light is switched off
The proton gradient is sufficient for ATP synthesis
DNP
DNP is an uncoupler - shuttles H+ from the inter membrane space to the matrix therefore dissipating the proton gradient
In the present of DNP:
ETC functions
No ATP is made - therefore pmf is necessary for ATP synthesis, proton gradient is required to make ATP even though the ETC is still working
Therefore cells are going to sense that they are not making ATP and their response is to burn more fuel molecules such as fatty acids
Not good for you cells to not be able to make ATP when you need them. Only thing you are doing is making more and more heat which is how it kills
DNP is classified as a poison and is illegal to sell in NZ
What is the proton gradient used by?
Used by ATP synthase in order to make ATP
F1 of ATP synthase
Is in the matrix
F0 of ATP synthase
Is in the Inner mitochondrial membrane
ATP synthase
ATP synthase is an enzyme that creates the energy storage molecule adenosine triphosphate (ATP), forming it from adenosine diphosphate (ADP) and inorganic phosphate (Pi).
F1F0 - ATP synthase works as a molecular….
Motor
Actin filament moves and does the rotatory motion
How does the F1F0-ATP synthase work? (Refer to diagram whilst doing this card too)
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
Part of the stator in the membrane has proton channels going into the matrix (move down their concentration gradient into the matrix), channel for the protons going in is not connected to the channel of the protons going out into the matrix so protons go through part of the rotor and the movement of the protons down their concentration gradient is being used to turn the rotor (C subunit) and the gamma stalk is connected to the C subunits so this also begins to turn
3 pairs of Alpha and beta subunits and it is the alpha and beta subunits that do the catalysis and what happens is that the alpha and beta subunits are in different conformations and as this gamma stalk turns it changes the conformations in these sub units and it is this conformational change that drives ATP synthesis
Catalytic site and the three conformations/steps required for ATP synthesis. Linked proton flow to rotation.
Movement of the rotor causes conformational changes in the ….
Movement of the rotor causes conformational changes in the catalytic alpha and beta subunits of F1
O=open (leads to release/binding)
T= tight (leads to ATP formation)
L = loose (holds ADP and Pi in preparation for catalysis)
Changes for each dimer when rotor turns
Only the gamma stalk moves but the other parts change confirmations
OPEN TO LOOSE TO TIGHT
Energy accounting for the proton gradient
4 protons per 1 ATP (4 protons moving down their concentration gradient to release enough energy to phosphorylate one ADP to ATP)
NADH = 10 protons pumped in ETC - 2.5 ATP are made FADH2 = 6 protons pumped in ETC - 1.5 ATP are made
