ETC and OP - ATP synthase and beyond Flashcards
What are the two functional subunits of ATP synthase?
F1 and F0
Which subunit of ATP synthase is water soluble, which is water insoluble?
F0 is water insoluble and is a transmembrane protein
F1 is water soluble and is a peripheral protein
What does DCCD do?
Inhibits H+ transport through F0 subunit.
What does oligomycin do?
Also binds to F0 and inhibits H+ transport
How many subunits does F1 have? What are they?
3 alpha, 3 beta, gamma, delta, eta
9
What is DNP?
An uncoupler which destroys the proton gradient.
How do oligomycin and DCCD work?
Not uncouplers, block ATP synthesis.
Only block transport through F0 unit.
Will eventually lead to oxidation being blocked
How can F1 be isolated?
Just add increasing salt content since not present in the membrane.
What does the delta unit do?
Small delta unit is bound through transmembrane protein called b2, keeps F1 from falling away.
Translocation of H+ carried out by ___, formation of phosphoanhydride bond is catalyzed by ___.
F0
F1
What is the L-state?
What is the T-state?
What is the O-state?
L-state: binds ADP and Pi loosely
T-state: binds ADP, Pi, and ATP tightly
O-state: does not bind at all –> releases ATP
How is ATP synthesized by ATP synthase?
Proton translocates through F0, when the proton rushes through F0 this leads to movement of cylinder.
This causes movement of gamma which changes the conformation of the F1 alpha-beta unit.
The alpha-beta is present in three conformations: L, T, O.
When gamma not touching, ADP and Pi are bound.
When gamma turns around, loose site becomes tight site, brings the two closer to make ATP.
When gamma turns again, forces it open and spits ATP out.
What site is ATP synthesized at?
T
ATP is synthesized on _ site on one subunit while ATP dissociates from _ site on another subunit. Free energy supplied by ______-gradient?
T
O
proton
What is the P/O ratio?
Amount of ATP generated for oxygen consumed.
So, what is the P/O ratio of NADH?
1 oxygen atom consumed, and 3 ATP produced.
So, 3 (theoretical)
Practically, 2.5
What is the P/O ratio for FADH2?
2 theoretically
1.5 practically
Why do the practical and theoretical values of ATP synthesis differ?
Gradient is not only used for ATP synthesis, also used to drive other reactions such as transport of other molecules and some of the gradient is lost to leakage.
In ATP synthase, ATP is not needed for ________ but is needed for _________.
Synthesis
release
Describe how ATP synthesis doesnt need energy in ATP synthase but needs it to be released.
In the tight conformation, ADP and Pi are forced to react, unless energy is provided, ATP won’t be released. Energy needs to be provided for release.
Describe the experiment with isolated F1 in regards to hydrolysis and synthesis of ATP.
Labeled oxygen in water, if hydrolysis of ATP occurs, oxygen will get incorporated into Pi.
But, all 4 oxygens got labeled.
If the reaction is favoured in 1 direction, i.e. hydrolysis, only get one oxygen labeled but, if all 4 are labeled, this means that deltaG for this reaction is nearly zero and thus reversible.
Thus, the reaction can flip-flop between hydrolysis and synthesis leading to all 4 oxygens in Pi getting labelled.
(This only occurs if no change in conformation occurs)
What are some of the other uses of the proton gradient?
Proton gradient also drives transport of various metabolites such as Pi, pyruvate, ADP in and ATP out, etc.
The aspartate-malate shuttle is uses in which tissues?
Liver, kidney and heart
Why is the aspartate-malate shuttle needed?
Since cytosolic NADH and mt NADH do not mix.
Need a way to transport the electrons from glycolysis formed NADH to the mt.
Describe the aspartate-malate shuttle?
NADH from glycolysis is used to convert oxaloacetate to malate (opposite of CAC) using malate DH. Exergonic reaction. (Two electrons from NADH transferred to oxaloacetate to generate malate)
Malate is then transported into the mt through the malate-a-ketoglutarate transporter; a-ketoglutarate goes into the cytosol.
Malate is then converted to oxaloaceate using malate DH, generating NADH (same as CAC).
Oxaloacetate in the mt is then converted to aspartic acid through aspartate aminotransferase.
To generate the amino acid, need an amino group. This is supplied by glutamate which is converted to a-ketoglutarate which is shuttled through the malate-a-ketoglutarate antiporter.
Aspartate is then transported through the glutamate-asparate transporter.
Glutamate is synthesized in the cytosol.
What pathway is used in the skeletal muscles and the brain?
In the brain and in muscles, the NADH from glycolysis is not transported into the mt.
Use Glycerol 3 phosphate shuttle.
Describe the glycerol 3 - phosphate shuttle
It is instead used to reduce dihydoxyacetone phosphate, converted to glycerol-3-phosphate.
- the enzyme is cytosolic glycerol-phosphate DH
There is a similar enzyme (mt G3P DH) on the mt membrane which oxidizes glycerol-3-phosphate back to dihydroxyacetone phosphate and reduces FAD to FADH2.
FADH2 passed onto complex 2, etc.
What is an uncoupler?
Any mechanism that can dissipate H+ gradient causing ATP generation to be uncoupled with electron transport.
What are two uncouplers?
FCCP and DNP
How do DNP and FCCP work?
Both are lipid soluble compounds which can transport H+ across the inner membrane.
When DNP and FFCP are used, what occurs to ET?
Increases
Why was DNP used as a diet pill?
DNP functions to uncouple ATP synthesis with electron transport.
It basically causes wasting of NADH, by transporting protons from the outside of the mt back in, dissipating the gradient.
Lots of fat gets burned since oxidation of fatty acids is increased to generate NADH.
What was the issue with using DNP as a diet pill?
Not making enough ATP.
CAC is running but no ATP synthesis and, the brain needs ATP so, without it will faint and can possibly die.
Describe brown fat and thermogenin.
Thermogenin is a physiological protein in newborn babies.
Thermogenin is in brown fat on the back of babies, allows for heat generation.
Describe the action of thermogenin.
In brown fat, in mt, express the UCP protein which allows protons to enter the mt and generate heat.
Describe the skunk cabbage.
Only found in cold temperatures and blossoms in the cold/snow.
The plant gets pollinated by heating up to evaporate its smell to attract insects.
It does this by generating heat without using the electron gradient.
How is heat produced in the skunk cabbage without using the proton gradient?
Uses short circuit of oxidation.
Two enzymes - external NADPH DH and alternative oxidase.
Electrons go to NADPH DH, gives electrons to coq10, some of it is grabbed by alternative oxidase, takes oxygen from coq10 and gives it to oxygen
- energy released is converted to heat
(Direct transfer of electron, short circuit produces heat which is utilized to evaporate aroma to attract insects)
Describe the control of the ATP producing pathway.
ADP will increase ATP generation
however, if the gradient is not used, ATP synthase inhibited, inhibit ETC
What is IF1?
Why is it used?
IF1 is a small protein, inhibitory factor that is synthesized when cells are feeling oxygen deprived.
The reason for this, is that in low oxygen, gradient decreases and leads to ATP synthase’s other activity - hydrolysis so, this protein is expressed to prevent loss of ATP by shutting down ATP synthase.
Binds to both F1 subunits.
What are the advantages of aerobic metabolism?
What are the disadvantages
Advantage
- more energy generation (oxygen most EN molecule physiologically)
Disadvantage:
1 - oxidative radicals - run risk of oxidative damage in molecules
2 - if there is lack of oxygen, we will die
- if there is no oxygen, glycolysis goes faster, more acidic, activates lysosomal enzymes which digest proteins and DNA
What is the reason for toxicity due to low oxygen?
Due to low pH leading to cell death and low ATP so membrane potential collapses, leading to rupture of membrane and cell death
What do free radicals react with?
What do they cause?
Lipids, proteins, DNA
Cause damage, mutation and cell death.
What are the different anti-oxidative mechanisms?
1 - Superoxide dismutase
2 - Catalase
3 - Glutathione with glutathione peroxidase
If the anti-oxidative mechanisms fail, what other mechanism is there?
ubiquitin protease or autophagy
What does superoxide dismutase do?
Converts superoxide to hydrogen peroxide
What does catalase do?
Converts hydrogen peroxide to water and oxygen
What does reduced glutathione with glutathione peroxidase do?
Converts hydrogen peroxide to oxidized glutathione and water.
What does cytochrome p450 do?
Detoxifies, hydroxylates toxic material to get it water soluble and excreted.
used by liver
What does NADPH do for managing oxidative stress?
Reduces glutathione.
