ATP synthesis Flashcards
What are the different energy transducing membranes?
The proton pumps within these membranes use energy by exergonic electron transport reactions in order to build up a proton gradient
Membranes of different organisms:
The mitochondrial inner membrane - the H+ ions are pumped into intermembrane space from the matrix
Chloroplast thylakoid membrane -the H+ ions are pumped from the stroma into thylakoid lumen
Bacterial plasma membrane - H+ pumped from cytosol to exterior
The proton gradient = imbalance of charge and pH = proton motive force (pmf) - this powers ATP synthesis
What is membrane potential used for?
ATP synthesis Active transport Flagellar rotation NADHPH synthesis Generate heat and electron potential
What is the chemiosmotic theory?
The free energy of electron transport is conserved by pumping H+ from the mitochondrial matrix to the intermembrane space to create an electrochemical H+ gradient across the inner mitochondrial membrane
The electrochemical potential of this gradient is harnessed to synthesize ATP
How is ATP synthesised in the mitochondria?
As H+ has just been transported into the intermembrane space by the complexes of the electron transport chain, there is a higher concentration of H+ ions in the intermembrane space
- Protons travel down their concentration gradient back into the matrix through the ATPase
- For every three protons passing through the ATPase, one molecule of ATP is formed
ADP and Pi are needed as substrates by the ATPase, therefore they enter the matrix via transporters
How do the substrates for ATP move into the matrix of the mitochondria?
Adenine nucleotide translocase, moves ADP into the matrix, is driven by the charge different across the inner membrane - as ADP has a 3- charge but ATP has a 4- charge
Phosphate translocase moves Pi across the inner membrane into the matrix
How many H+ are required for ATP synthesis?
ATPase mechanism: 3
Transport of Pi and ADP into matrix: 1
Total: 4
How much ATP is produced, when looking at the coupled reaction of O2 reduction?
Electrons from NADH
10 protons pumped out of the matrix
4 protons must travel into the matrix to synthesise one ATP molecule
10/4 = 2.5 molecules of ATP formed
Electrons from FADH2
6 protons pumped out of the matrix
4 protons must travel into the matrix to synthesise one ATP molecule
6/4 = 1.5 molecules of ATP formed
What is the structure of ATPase?
It is made up of two parts
The first sits in the membrane, called F0 - essentially a proton channel
F0 comprises a transmembrane ring of hydrophobic proteins that act as a H+ channel
As protons flow through the F0 channel it rotates
This in turn drives rotation of the gamma subunit which drives conformational changes in a and b subunits
The second sits in the matrix, called F1 - acts as an enzyme to catalyse the synthesis of ATP
F1 is composed of 3 alpha and 3 beta subunits
b is the catalytic subunit, a is regulatory
gamma connects F1 to F0
How was the structure and function of ATPase confirmed?
You can wash the F1 part from the membrane as it isn’t actually integrated into the membrane, and only remains attached due to the gamma subunit interaction with F0
F1 alone is able to hydrolyse ATP by the beta subunit - which can then drive rotation of the gamma subunit (proved by attaching a fluorescent actin filament to the gamma subunit)
What is the mechanism of ATP synthesis?
Within the beta subunit there are different conformations that are more preferable for ATP synthesis
There is a T, O and L conformation (in a clockwise circle order) - tight, open, loose
1. ADP + Pi bind in the L (loose) binding site. there is a conformational change to T state
2. The T conformation has such a high affinity for ATP that bound ADP +Pi are converted to ATP (forming the phosphoanhydride bond)
3. This ATP is released after the conformational change driven by the rotation of the gamma subunit to the O conformation (driven 120° anticlockwise)
4. ADP and Pi bind to the vacant L site after further rotation converting O to L and L to T resulting in the synthesis of a second molecule of ATP
What gives evidence to support the chemiosmotic theory?
Electron transport chains pump protons
An artificially produced proton gradient can drive ATP synthesis
The effects of uncouplers and inhibitors can be predicted
What experiment can prove the chemiosmotic theroy?
Place an isolated mitochondrial ATPase and bacteriorhodopsin in a synthetic vesicle
(Bacteriorhodopsin is a light driven proton pump)
When light is provided the bacteriorhodopsin creates a proton gradient, and if provided with ADP and Pi the ATPase can use this to form ATP
This confirms the need for a proton gradient
Give an overview of the events of the electron transport chain?
- NADH and FADH2 are oxidised by transferring electrons to Complexes I and II respectively
- These electrons travel through the electron transport chain components
- Oxygen is reduced to water when it accepts the electrons from the last component of the electron transport chain, cytochrome oxidase
- Protons are pumped out of the mitochondrial matrix at Complexes I, III and IV as the electrons travel through them
Synthesis of ATP
- Protons are pumped out of the matrix, their concentration in the inter-membrane space is greater than that in the mitochondrial matrix
- Protons move back into the matrix via the ATPase, driving it to synthesize ATP
What are some inhibitors of the electron transport chain?
Complex I - blocked by rotenone and amytal
QH2 - blocked by antimycin A
Complex IV - blocked by CN-, N3- and CO
What is the effect on the electron transport chain by inhibiting complex I?
Inhibition at Complex I prevents NADH donating electrons to the electron transport chain
However, FADH2 can still donate electrons to Complex II
Therefore oxygen is still reduced to water, protons can still be pumped out of the matrix and ATP can still be made
What is the effect on the electron transport chain by inhibiting complex II?
Inhibition at Complex II prevents FADH2 donating electrons to the electron transport chain
NADH can still donate electrons to Complex I, which can pass via ubiquinone to Complex III
Therefore, oxygen is still reduced to water, protons can still be pumped out of the matrix and ATP can still be made
What is the effect on the electron transport chain by inhibiting complex III?
Electrons donated to the electron transport chain components cannot be passed through Complex III - meaning electron flow is inhibited
Therefore oxygen is not reduced to water, protons cannot be pumped out of the matrix and ATP cannot be made
What is the effect on the electron transport chain by inhibiting complex IV?
Electrons donated to the electron transport chain components cannot be passed through Complex IV - meaning electron flow is inhibited
Therefore oxygen is not reduced to water, protons cannot be pumped out of the matrix and ATP cannot be made
What is the effect on the electron transport chain by inhibiting ATPase?
This is not a direct inhibition of the electron transport chain
Protons cannot enter the matrix from the inter-membrane space
ATP cannot be made
Due to a high concentration of H+ ions that have been pumped into the intermembrane space, without the H+ ions being moved back in the concentration gradient doesn’t allow complexes to keep pumping H+ ions out
This prevents the electron transport and oxygen is therefore not reduced to water
What do uncouplers do?
It provides a mechanism for protons to come back into the matrix without going through ATPase
An uncoupler dissolves in the inner mitochondrial membrane and carries protons across the membrane down their concentration gradient
This is more efficient to move protons into the matrix but ATP synthesis stops
How do uncouplers effect the electron transport chain?
The proton gradient across the membrane is reduced as more protons can travel across
Protons are pumped out of the matrix at a faster rate by the complexes, therefore electrons travel at a faster rate
Oxygen is reduced to water at a faster rate
What is an example of an uncoupler?
Dinitrophenol acts as an uncoupler as it can cross the membrane in both protonated and deprotonated form - allowing it to diffuse back and forth
How are different metabolites transported in/out of the mitochondria?
Transport proteins are needed for: ATP - ATP/ADP translocase Phosphate - Dicarboxylate carrier or phosphate carrier Malate - tricarboxylate carrier Pyruvate - pyruvate carrier
What metabolites don’t require transporter proteins?
NAD+/NADH as it is involved in the malate-aspartate shuttle
The glycerophosphate shuttle can transfer the FAD/FADH between the cytosol and the matrix
What is a disadvantage of aerobic respiration?
If O2 isn’t completely reduced it can form O2-, a reactive oxygen species or a superoxide radical
This can be protonated to eventually form H2O2 - hydrogen peroxide
However, most of these radicals have a short half life
