ETC and OP - ATP synthase and beyond

Question 1

What are the two functional subunits of ATP synthase?

Answer 1

F1 and F0

Question 2

Which subunit of ATP synthase is water soluble, which is water insoluble?

Answer 2

F0 is water insoluble and is a transmembrane protein

F1 is water soluble and is a peripheral protein

Question 3

What does DCCD do?

Answer 3

Inhibits H+ transport through F0 subunit.

Question 4

What does oligomycin do?

Answer 4

Also binds to F0 and inhibits H+ transport

Question 5

How many subunits does F1 have? What are they?

Answer 5

3 alpha, 3 beta, gamma, delta, eta

9

Question 6

What is DNP?

Answer 6

An uncoupler which destroys the proton gradient.

Question 7

How do oligomycin and DCCD work?

Answer 7

Not uncouplers, block ATP synthesis.
Only block transport through F0 unit.
Will eventually lead to oxidation being blocked

Question 8

How can F1 be isolated?

Answer 8

Just add increasing salt content since not present in the membrane.

Question 9

What does the delta unit do?

Answer 9

Small delta unit is bound through transmembrane protein called b2, keeps F1 from falling away.

Question 10

Translocation of H+ carried out by ___, formation of phosphoanhydride bond is catalyzed by ___.

Answer 10

F0

F1

Question 11

What is the L-state?
What is the T-state?
What is the O-state?

Answer 11

L-state: binds ADP and Pi loosely
T-state: binds ADP, Pi, and ATP tightly
O-state: does not bind at all –> releases ATP

Question 12

How is ATP synthesized by ATP synthase?

Answer 12

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.

Question 13

What site is ATP synthesized at?

Answer 13

T

Question 14

ATP is synthesized on _ site on one subunit while ATP dissociates from _ site on another subunit. Free energy supplied by ______-gradient?

Answer 14

T
O
proton

Question 15

What is the P/O ratio?

Answer 15

Amount of ATP generated for oxygen consumed.

Question 16

So, what is the P/O ratio of NADH?

Answer 16

1 oxygen atom consumed, and 3 ATP produced.
So, 3 (theoretical)
Practically, 2.5

Question 17

What is the P/O ratio for FADH2?

Answer 17

2 theoretically

1.5 practically

Question 18

Why do the practical and theoretical values of ATP synthesis differ?

Answer 18

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.

Question 19

In ATP synthase, ATP is not needed for ________ but is needed for _________.

Answer 19

Synthesis

release

Question 20

Describe how ATP synthesis doesnt need energy in ATP synthase but needs it to be released.

Answer 20

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.

Question 21

Describe the experiment with isolated F1 in regards to hydrolysis and synthesis of ATP.

Answer 21

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)

Question 22

What are some of the other uses of the proton gradient?

Answer 22

Proton gradient also drives transport of various metabolites such as Pi, pyruvate, ADP in and ATP out, etc.

Question 23

The aspartate-malate shuttle is uses in which tissues?

Answer 23

Liver, kidney and heart

Question 24

Why is the aspartate-malate shuttle needed?

Answer 24

Since cytosolic NADH and mt NADH do not mix.

Need a way to transport the electrons from glycolysis formed NADH to the mt.

Question 25

Describe the aspartate-malate shuttle?

Answer 25

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.

Question 26

What pathway is used in the skeletal muscles and the brain?

Answer 26

In the brain and in muscles, the NADH from glycolysis is not transported into the mt. Use Glycerol 3 phosphate shuttle.

Question 27

Describe the glycerol 3 - phosphate shuttle

Answer 27

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.

Question 28

What is an uncoupler?

Answer 28

Any mechanism that can dissipate H+ gradient causing ATP generation to be uncoupled with electron transport.

Question 29

What are two uncouplers?

Answer 29

FCCP and DNP

Question 30

How do DNP and FCCP work?

Answer 30

Both are lipid soluble compounds which can transport H+ across the inner membrane.

Question 31

When DNP and FFCP are used, what occurs to ET?

Answer 31

Increases

Question 32

Why was DNP used as a diet pill?

Answer 32

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.

Question 33

What was the issue with using DNP as a diet pill?

Answer 33

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.

Question 34

Describe brown fat and thermogenin.

Answer 34

Thermogenin is a physiological protein in newborn babies. | Thermogenin is in brown fat on the back of babies, allows for heat generation.

Question 35

Describe the action of thermogenin.

Answer 35

In brown fat, in mt, express the UCP protein which allows protons to enter the mt and generate heat.

Question 36

Describe the skunk cabbage.

Answer 36

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.

Question 37

How is heat produced in the skunk cabbage without using the proton gradient?

Answer 37

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)

Question 38

Describe the control of the ATP producing pathway.

Answer 38

ADP will increase ATP generation | however, if the gradient is not used, ATP synthase inhibited, inhibit ETC

Question 39

What is IF1? | Why is it used?

Answer 39

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.

Question 40

What are the advantages of aerobic metabolism? | What are the disadvantages

Answer 40

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

Question 41

What is the reason for toxicity due to low oxygen?

Answer 41

Due to low pH leading to cell death and low ATP so membrane potential collapses, leading to rupture of membrane and cell death

Question 42

What do free radicals react with? | What do they cause?

Answer 42

Lipids, proteins, DNA | Cause damage, mutation and cell death.

Question 43

What are the different anti-oxidative mechanisms?

Answer 43

1 - Superoxide dismutase 2 - Catalase 3 - Glutathione with glutathione peroxidase

Question 44

If the anti-oxidative mechanisms fail, what other mechanism is there?

Answer 44

ubiquitin protease or autophagy

Question 45

What does superoxide dismutase do?

Answer 45

Converts superoxide to hydrogen peroxide

Question 46

What does catalase do?

Answer 46

Converts hydrogen peroxide to water and oxygen

Question 47

What does reduced glutathione with glutathione peroxidase do?

Answer 47

Converts hydrogen peroxide to oxidized glutathione and water.

Question 48

What does cytochrome p450 do?

Answer 48

Detoxifies, hydroxylates toxic material to get it water soluble and excreted. used by liver

Question 49

What does NADPH do for managing oxidative stress?

Answer 49

Reduces glutathione.