Biochemistry- formation of ATP Flashcards
Describe the link between ATP usage and ATP production
The average human only possesses about 250 g of ATP. This will be recycled 300 times a day in order to meet energy needs
(from ADP to ATP).
Although the equilibrium between ATP and ADP is heavily in favour of ADP, the actual ATP:ADP ratio in the cell is 5:1.
Describe substrate level phosphorylation
There are only a few catabolic pathways that directly produce ATP. The reaction needs to be exothermic enough to drive
the endothermic synthesis of ATP.
Normally this involves taking a phosphorylated substrate, removing it and attaching it to ATP.
In glycolysis, 1,3-bisphosphoglycerate removes a phosphate to form 3-phosphoglycerate and phosphorylates ADP to ATP.
In the citric acid cycle, the reaction of succinyl CoA to succinate is exothermic enough to drive the phosphorylation of GDP
to ATP.
Describe oxidative phosphorylation
Most of the ATP is produced through oxidative phosphorylation.
Reduced enzymes NADH and FADH2 are converted to NAD (coenzyme) and FAD (cofactor) which is a very exothermic
process, and is used to produce ATP.
Describe the structure of mitochondria
Double membrane
o Outer membrane: permeable and has proteins forming large pores
which allow ions to freely move into the membrane.
o Inner membrane: less permeable and has specific transporters which
are selective to what travels into the matrix.
Intermembrane is similar to the cytosol (pH and ions). This means that people
commonly refer the intermembrane space as part of the ‘outside’.
Matrix has a lower [H
+] than the intermembrane space and cytosol.
What processes act as sources for reduced coenzymes
Fatty acid oxidation: occurs in the mitochondria. Reduced coenzymes are made
where they need to be.
Amino acid oxidation: occurs in the mitochondria. Reduced coenzymes are
made where they need to be.
Citric acid cycle: takes products from other catabolic pathways and further
metabolises them to CO2
. All the reduced coenzymes are produced where they
need to be.
Glycolysis: takes place in the cytosol (outside the mitochondria). Produces 2 NADH per molecule of glucose. NADH cannot
enter the mitochondrial matrix where all the oxidative phosphorylation occurs. Shuttles can be used to indirectly transport
NADH (transports the reducing power of NADH).
Describe the electron transport chain
Electrons and hydrogen ions produced by the oxidation of
reduced coenzymes are passed to the electron transport chain.
The transfer of electrons in the ETC is extremely exothermic as
electrons are transferred from an electron carrier that has a
lower affinity for electrons to one that has a higher affinity for
electrons. Oxygen has a very high affinity for electrons.
Complexes catalyse the transfer of protons across the inner
mitochondrial membrane from the matrix to the
intermembrane space (against their concentration gradient).
This leads to a high [H
+] in the intermembrane space and a low concentration in the matrix, allowing for ATP synthase to
work.
An exception is complex II as the difference in electron affinity between FADH2 and coenzyme Q is very small so no energy is
made available for the transfer.
The next reaction is very exothermic which allows the reaction at complex II to occur.
Describe oxidative stress
The respiratory chain (especially complex I) produces reactive oxygen species at low levels, which vary according to
conditions. These have been implicated in ageing, diabetes etc. but are also required for useful functions and act as
signalling molecules.
Oxidative stress occurs when production of ROS exceeds removal.
Aerobic exercise transiently increases production of ROS but also leads to the increased expression of defence against
oxidative stress. People used to believe that because exercise causes oxidative stress, that this would be a downside to
exercise.
Some cells will deliberately produce superoxide molecules to kill bacteria.
Cells can remove reactive oxygen species to prevent oxidative stress (e.g. with superoxide dismutase).
Antioxidant supplements appear to prevent some of the beneficial effects of exercise. Studies have shown that people
doing aerobic exercise and supplements did not gain any benefits (i.e. muscle strength/mass etc.).
Give examples of electron carriers and how they work
The ETC contains several types of electron carriers, each with a
different affinity for electrons. Some are hydrogen, some are electron
carriers, and each transport different numbers.
Uniquinone (coenzyme Q) (electron or hydrogen carrier): most
flexible of the electron carriers as it is capable of taking up electrons
and protons and hydrogen atoms.
Flavin mononucleotides (hydrogen carrier): accepts or donates 2 H
+
(hydrogen).
Cytochrome (electron carrier): contains a haem group that is
covalently or tightly held with an iron ion in the centre that can
transfer or donate 1 electron at a time.
Iron sulphate protein (electron carrier): any molecule that has iron
ions and sulphur atoms complexed within its structure. Always transfers or donates 1 electron at a time.
Summarise the electron transport chain (Describe role of Complex 1-4 and ATP synthase)
NADH can transport more H
+ to the intermembrane space
than FADH2
, because the difference between FADH2 and
oxygen in terms of their electron affinity is smaller than the
difference between NADH and oxygen.
When NADH passes electrons to oxygen, more energy is
available than when FADH2 passes electrons to oxygen.
1. Complex 1 (NADH:uniquinone oxidoreductase)
Transfers 2 e
− from NADH to coenzyme Q. Hydrophobic
chemical so it is dissolved in the inner mitochondrial
membrane.
The energy given out pumps 4 H
+ out into the mitochondrial matrix.
2 H
+ is lost from the matrix and reduces coenzyme Q (as it is taking 2 e
−) to form QH2
.
2. Complex 2 (succinate dehydrogenase)
Complex 2 has an FAD molecule.
Complex 2 is actually part of the citric acid cycle, and its FAD gets reduced during this process.
Part of the electron transport chain and citric acid cycle.
Succinate is oxidised and FAD is reduced as it accepts hydride to form FADH2
.
Succinate dehydrogenase takes up 2e
− at a time to form FADH2 and transfers them (via Fe− S) centres to coenzyme Q
which takes up 2 H
+ from the matrix to produce QH2
- Complex 3 (cytochrome complex)
Reduced coenzyme Q (QH2
) passes electrons to cytochrome C which is loosely attached to the outer surface of the inner
mitochondrial membrane. Cytochrome C can only take 1 e
− at a time so 2 cytochrome Cs are required.
4 H
+ are released into the intermembrane space.
4. Complex 4 (cytochrome c oxidase) 2 H \+ pumped out of the matrix. 2 H \+ from cytochrome C are added to O2 to form H2O. This is the final electron acceptor.
- ATP synthase
Large protein with several subunits and uses the return of H
+ to the mitochondrial matrix to
produce ATP.
𝛂 subunit (within the mitochondrial inner membrane): contains 2 half channels (only go half
way through the membrane) which allow H
+ partly across the membrane.
C subunit: H
+ bind to binding site for H
+ in the middle of a ring of subunit C. This causes
subunit C to rotate and H
+ can leave ATP synthase into the matrix when it reaches the α
subunit.
𝛄 subunit stalk: connected to subunit C. Turns as the subunit C turns.
𝛃 subunit (catalytic subunit): binds to ADP and Pi. Changes shape as the γ stalk turning interacts with the β subunit. There
are three shapes that a β subunit can take up and each one of the three β subunits are at different points in the cycle:
o Open: releases ATP.
o Loose: binds ADP and phosphate and cannot release them.
o Tight: combines ADP and phosphate to form ATP but cannot release ATP.
If the β subunits were to turn the opposite direction, they would in fact break apart ATP to ADP and Pi.
One full rotation of the ATP synthase causes the production of 3 ATP molecules.
Describe coupling of the electron transport chain and ATP synthase
The electron transport chain and ATP synthase are coupled. This is because without ATP synthase, H
+ cannot return to the
matrix from the intermembrane space. Inhibitors of the electron transport chain means that there is no H
+ gradient so ATP
synthase cannot work.
If ADP molecules are removed, ATP synthase cannot work. This means that H + cannot flow through ATP synthase, causing a
build-up or [H+]. This makes it harder to the reduced coenzymes to transfer their electrons through the ETC. This is because
the ETC cannot stand a change in [H+] of a greater pH difference than 2.
What is the oxygen electrode trace and what are P:O ratios
Oxygen electrode traces allow us to see how much oxygen is
required by the electron transport chain to use up all the ADP
under different circumstances.
Oxygen usage of the mitochondria increases when ADP is added
as it allows ATP synthase to work.
Malate is oxidised by NADH.
Succinate is reduced by 𝐅𝐀𝐃𝐇𝟐/succinate is oxidised by FAD.
NAD pumps out 𝟏𝟎 𝐇
+ whilst FAD only pumps out 𝟔 𝐇
+ using the
same amount of energy.
4 H
+ diffusing through ATP synthase is required to make 1
molecule of ATP.
Therefore, the P:O ratio (how many phosphorylations per O atom reduced) for NADH is 2.5, but for FADH2 is 1.5.
ATP synthesis is normally tightly coupled to the function of the ETC so an oxygen decrease will normally only be seen when both substrate and ADP are present
What are electron transport chain inhibitors
Some compounds inhibit ETC complexes or ATP synthase.
Because the respiration of the ETC cannot occur unless ATP
synthesis is also occurring, inhibiting ATP synthase with
oligomycin will also prevent use of oxygen by the ETC.
Inhibition of complex I will allow the mitochondria to use
succinate.
Inhibition of complex II will allow the mitochondria to use malate
What are uncouplers
Weak acids, or proton channels allow H
+ to enter the matrix
without doing any work (leading to generation of heat instead of
ATP).
Can be weak acids.
This results in the electron transport chain working even faster
but with less of the energy being used to make ATP.
More oxygen is needed to use all the ADP.
The proton channel thermogenin is highly expressed in the
mitochondria of brown adipose tissue, found in human babies.
When activated, heat is generated, contributing to temperature
homeostasis.
How does dinitrophenol work
Weak acid that can dissolve through membrane. Weak acid is hydrolysed, which can then pass into the cell.
Originally used to make dynamite during WW1.
In the 1930s, doctors prescribed it for weight loss, but due to significant side effects (death) it
was banned in the US (illegal to sell in the UK).
As it transports protons into the matrix the rate of electron transfer across the ETC increases,
with little ATP being produced.
This causes a rapid increase in consumption of metabolic fuels and oxygen. The resultant
increases heat production, combined with the decrease in ATP production, can kill the cell.
Bad drug because therapeutic dose > lethal dose.
What are mitochondrial diseases
Heterogeneous group of disorders that affect oxidative phosphorylation.
Inherited from the mother. Mother might not be affected but offspring can be. Each mitochondria can be genetically
different (mitochondrial DNA). Some mitochondria may be normal, and some will be mutated.
Can be caused by mutations in the respiratory chain, transport of ATP, synthesis of ubiquinone, PDH (pyruvate
dehydrogenase), mitochondrial translation etc.
Extremely difficult to diagnose, can effect multiple organ systems or only one.
Age of onset varies, severity varies and inheritance is not straightforward.
Common symptoms include elevated lactic acid or alanine in blood, exercise intolerance and seizures.
Tests can include molecular genetic testing, electron microscopy of muscle tissue, oxygen electrode traces, MRI to detect
brain lesions.
Fatty mitochondria may need to be at 60-80% of a sample for biochemical testing to be effective.
