Respiratory Chain and ATP synthase II

Question 1

Where is ATP synthase (aka complex V, or

F1FO ATPase) located?

Answer 1

inner mitochondrial membrane

Question 2

The F1 portion of ATP synthase can be removed from the inner mitochondrial membrane through application of what?

Answer 2

urea. It can also be added back after the urea is removed to make a F1FO ATPase again.

Question 3

What do the F1 and Fo subunits of ATP synthase contain?

Answer 3

F1- catalytic subunits (structure known)

Fo- the proton channel. (a, b, and d subunits unsolved)

Question 4

What happens when the F1 part of ATP synthase is removed from the membrane?

Answer 4

the electron transport chain can still work and oxygen can be reduced to water. The oxidative phosphorylation (synthesis of ATP) cannot work, and the ATP synthesis is said to be uncoupled from respiration

Question 5

What are some ways that mitochondria can become “uncoupled”?

Answer 5

1) their membranes can be leaky. Not with big holes, but it does not take much to let protons through and destroy the proton gradient
2) addition of a chemical uncoupler

Question 6

How do chemical uncouplers work?

Answer 6

These small molecules serve as proton shuttles that carry a proton across the membrane down the gradient. They are protonated on the cytosolic side where the pH is lower (H+ concentration is higher). Then they diffuse across the membrane as uncharged species and release the proton to the matrix, where the pH is higher. At this point the uncoupler is charged and cannot go back across the membrane for another proton, so these are one-way proton carriers that accumulate in the matrix

Question 7

What are some examples of chemical uncouplers?

Answer 7

CCCP, 2,4-dinitrophenol (DNP), FCCP

Question 8

T or F. If electrons do not flow, no ATP can be made because there would be no proton gradient

Answer 8

T. But the opposite is also true: if no ATP can be made then electrons cannot flow because of the tight coupling

Question 9

Can electrons still flow if ATP synthase is not active?

Answer 9

No! The reason they don’t is the proton
gradient. When electrons flow, protons are pumped against an existing proton gradient. Once this gradient is large enough, the
energy released by electron movements to higher reduction potential is just equal to the force applied by the gradient and no more protons can be pumped. If the gradient is relaxed by using it for synthesis of ATP, then more electrons can flow. If the gradient is relaxed by an uncoupler then the electrons can flow. Otherwise a steady state is reached and
electron flow ceases.

Question 10

When mitochondria are incubated with succinate as an electron donor, and ADP and Pi are present, we have rapid oxygen consumption. What is this called?

Answer 10

state 3 respiration (steep slope)

When the ADP runs out, the slope drops back to a much lower level called state 4 respiration (almost horiztonal, but not quite because the internal membrane is naturally leaky and there is always some natural turnover of ATP to ADP)

Question 11

What is the the respiratory control ratio?

Answer 11

The ratio of state 3 to state 4 respiration. It should be about 5-6 in good preps of isolated mitochondria

Question 12

Addition of an uncoupler to stage 4 respiration would result in what?

Answer 12

stage 3 like respiration

Question 13

What do mitochondria look like during stage 3 respiration? stage 4?

Answer 13

3- The actively respiring mitochondria have a large periplasmic space and the matrix appears condensed.

In state 4 they look more typical with very little periplasmic space

Question 14

How do newborn animals and re-awakening animals uncouple their mitochondria in brown fat tissue?

Answer 14

This is done by hormonal control of UCP1, a member of the mitochondrial carrier family called the uncoupler protein, also called thermogenin because it generates heat.

UCP1 only works in brown fat

Question 15

How does UCP1 work?

Answer 15

This protein transports fatty acid anions out across the inner membrane from the matrix in exchange for a counter ion like chloride or hydroxyl ion. Once the fatty acids are released on the outer positive side of the membrane they become protonated and flip flop back across the membrane as non-charged species. On the matrix side they deprotonate and become charged again. This is like a chemical uncoupler, except the fatty acids do not have to accumulate at the matrix side since the uncoupler protein can transport them out again. This process uncouples oxidative phosphorylation and wastes the proton gradient as heat.

Question 16

What is UCP1 activated by?

Answer 16

by free fatty acids that are produced by lipolysis in response to hormones

Question 17

What is a required cofactor of UCP1?

Answer 17

Ubiquinone

Question 18

T or F. UCP1 only exists in mammals

Answer 18

T. It is a new evolutionary development

Question 19

What do UCP2 and UCP3 do?

Answer 19

They are uncoupling proteins like UCP1 that are found in many tissues. UCP2 and UCP3 might be turned on in adults as a way to regulate weight by wasting energy through uncoupling oxidative phosphorylation.

Question 20

oxidative phosphorylation is aka?

Answer 20

the chemiosmotic hypothesis

Question 21

Is the inner mitochondrial membrane impermeable to protons?

Answer 21

Yes, This permits the controlled leak of protons down the gradient through the ATP synthase to make ATP

Question 22

What is a a reconstitution experiment?

Answer 22

Showing that the ATP synthase can be split into F1 and FO parts and these can be rejoined

Question 23

Describe the reconstitution experiment experiment performed by Efriam Racker and Walther Stoeckenius and what it proved.

Answer 23

bacteriorhodopsin (a light driven proton pump) was reconstituted into phospholipid vesicles with the intact F1FO ATPase. When light was applied, the bacteriorhodopsin pumped protons into the vesicles. Fortunately for the experiment, the orientation of the pump was not random and there was net pumping into the vesicles not out from them. With this light generated proton gradient, the ATP synthase was able to make ATP.

This experiment showed there was no high-energy intermediate (as was suspected at the time), or no specific interaction with the electron transport complexes. Just a simple proton gradient could power ATP synthesis.

Question 24

Describe the structure of the Fo unit of ATP synthase.

Answer 24

The membrane part and the F1 part are connected by a 50 angstrom stalk. The number of subunits in the FO part varies depending on the source, but there are only 3 FO subunits in E coli. They are called a, b and c and they exist in the ratio 1:2:10-12.

Question 25

Describe the structure of the c subunit of the Fo unit of ATP synthase.

Answer 25

It has only two transmembrane segments and one of these has a critical aspartate residue in the middle of the helix. This aspartate is required for the proton channel.

Question 26

What is dicyclohexyl carbodiimide (aka DCCD binding protein) and what does it do?

Answer 26

A chemical that reacts with this aspartate side chain in a c subunit of the Fo unit and kills the ATP synthase enzyme

Question 27

Why is aspartate vital to ATP synthase (particularly the pumping action of Fo)?

Answer 27

The carboxyl that exists on aspartate (also on glutamate which can substitute for aspartate**) is protonated at some point during the transport of protons through the subunit a channel and this is required to make the protein work. (DCCD blocks this process)

Question 28

Are the subunits of Fo encoded by the nucleus or mitochondrial genome?

Answer 28

mitochondria. This is another example of the mitochondrion keeping control of these very critical proteins and not giving them up to the nuclear genome

Question 29

Does the stalk that connects the Fo and F1 units arise from the Fo or the F1 unit?

Answer 29

contributed by BOTH

Question 30

Where can you find oligomycin

sensitivity conferring protein (OSCP)?

Answer 30

the stalk connecting F1 and Fo

Question 31

What does OSCP do?

Answer 31

OSCP is required for sensitivity to oligomycin, an antibiotic inhibitor of the ATPase.

(However, oligomycin binds to the Fo part of the protein, not to the OSCP)

Question 32

Describe the subunit structure of the F1 unit of ATP synthase.

Answer 32

The subunit composition of F1 is alpha 3, beta 3, gamma, delta, epsilon. Alpha and beta alternate like 6 slices of an orange to make a ball

Question 33

Which subunits of F1 have catalytic ability?

Answer 33

only the beta subunits. The alpha subunits

have binding sites for nucleotide, but they are not catalytic

Question 34

Describe the gamma subunit of F1.

Answer 34

gamma has two long helices that run through the center of the ball made by alpha and beta. This long helical gamma subunit sticks out the bottom of the ball formed by alpha and beta to form part of the stalk.

It is known that the gamma subunit rotates relative to the F1 ball during ATP synthesis, so that gamma is like a molecular axle and the central hole in F1 is like a molecular bearing

Question 35

Is the gamma subunit symmetrical?

Answer 35

No. It is very important to this model that the gamma subunit is not symmetrical. It has significant differences in its interactions with the three catalytic beta subunits, and this causes distortions or conformational changes in the beta subunits.

Question 36

What are the postulates of the binding change mechanism?

Answer 36

1. The first postulate is that the energy of the proton gradient is not used to form ATP but to release ATP from a very tight binding site where it forms spontaneously. The release of this tightly bound ATP requires the energy of the proton gradient converted into the mechanical energy of protein conformational changes probably brought about by interactions with the gamma subunit and cooperative changes with the other alpha and beta subunits.
2. The second postulate says that the three catalytic sites are each in a unique conformation and the conformations are interconvertible. They represent three different stages of the catalytic cycle. One site L is loose, with ADP and Pi bound. ATP forms without added energy input. Afterwards the contacts with the newly formed ATP become very strong. This tight conformation T then needs the energy from the proton gradient to release the ATP. The third state is the open or O state before nucleotide is bound.
3. The third postulate is that conformational changes at the three sites are driven by rotation of the asymmetric gamma subunit relative to the F1 ball.

Question 37

What provided evidence that the first postulate was right?

Answer 37

O18 water exchange experiments

Question 38

How did the O18 water exchange experiments work?

Answer 38

If the ATP synthase is incubated with ADP, Pi and O18 water, Paul Boyer showed that O18 gets into free phosphate. The only way this can happen is by forming ATP and then rehydrolyzing it. The O18 incorporation occurred without a proton gradient, therefore, ATP was formed without energy input from the gradient. ATP was not released unless the gradient was applied, so the energy is needed to release the ATP not to form the ATP.

Question 39

What provided evidence that the second postulate was right?

Answer 39

The crystal structure immediately supported the second tenet, because the three catalytic sites were each in a different conformation.

Question 40

What provided evidence that the third postulate was right?

Answer 40

Hiroyuki Noji and collaborators engineered ten histidines onto the N-terminal of the the ATPase beta subunit and expressed these his-tagged proteins in bacteria. The F1 part of the ATPase was purified and attached to a glass slide that was coated with nickel.

The ATPase beta subunits in the F1 complex bound to the slide through their his tags and immobilzed the F1 particle upside down with the gamma subunit facing up. The gamma subunit was mutated so it had only one cys residue near its end. The cys was used as a site for biotinylation and attachment of streptavidin. Finally, a biotinylated flourescent actin filament was added to the complex and it bound to the streptavidin. Fluorescence of the actin filament tagged to the gamma subunit was observed in a flourescence microscope. When ATP was added to the slide, the filaments spun around. It moved at about 4 cycles per second, slower than expected for the free enzyme, (about 20 cycles per second) but it has a big drag on it from the actin filament. A more recent experiment repeats this strategy with intact F1FO and couples the actin fiber to the c subunits. The result is the same, showing that the c subunits rotate

Question 41

How many protons are pumped during the ETC complex all the way to reduction of O2 to H2O?

Answer 41

The best estimates are 4 protons per electron pair at complex I, 4 protons per electron pair at complex III (2 for each turn of the Q cycle) and 2 protons per electron pair at cytochrome oxidase (This amounts to 4 protons for complete reduction of O2 to water).

Question 42

The estimate for protons consumed per ATP synthesized is 4. There is an interesting theory about the ATPase that might explain this number. It is called ?

Answer 42

the elevator model

Question 43

Describe the elevator model

Answer 43

Remember that the FO part had 10-12 c subunits, each with a single DCCD binding aspartate. Single mutations in subunit A stop transport, so subunit a probably forms part of the proton channel. The interesting fact is that mutation of even 1 of the 10-12 subunits of C will also stop transport. This suggests that subunit c ring rotates relative to the A subunit to form a complete proton pathway.

The elevator model suggests that protons start their passage through the membrane in subunit a. At some point they cannot continue unless they move onto one of the c subunits which then carries the proton as the ATPase rotor moves 1/12th (or 1/10th) of a full circle. This opens up the proton path in subunit a once again for another proton to leave its c subunit and exit. By this model, the rotor would have to turn 1/12th (or 1/10th) of a circle for every proton, or 10-12 protons could pass through per revolution. We already know that three ATPs are made per complete revolution of gamma relative to F1, so that gives 3/12 = 1 ATP per 4 protons. If the number of c subunits were variable, then there might be a variable stoichiometry. If there were 10 c subunits the math would be 3.3 protons per ATP, It does not have to be an integer number if the number of c subunits is variable

Question 44

Is there hard evidence for the elevator model?

Answer 44

No. This elevator model is a speculative model that is not supported with hard evidence like the actin filament experiment.

Question 45

Supplemental explanation of the elevator model and the movement of H+ through Fo

Answer 45

H+ enters from the periplasmic space outside the inner membrane. After traveling all the way around, H+ exits to the matrix space

Question 46

What forms the ‘rotor’ of ATP synthase?

Answer 46

Experiments have shown that the c subunit
ring (Fo) is attached to the epsilon and gamma subunits (F1). These three subunits form the rotor of this molecular motor

Question 47

What forms the ‘stator’ of ATP synthase?

Answer 47

the a and b subunits of Fo, and the

alpha, beta and delta subunits of F1.

Question 48

For one molecule of glucose, what is the yield of glycolysis?

Answer 48

2 ATP and the 2 NADH have to be used by lactate dehydrogenase to reoxidize NADH to NAD

Question 49

For one molecule of glucose, what is the yield of oxidative phosphorylation?

Answer 49

two ATP and you can keep the two NADH from glycolysis. In addition you get 8 NADH and 2 FADH2 and 2 GTP from two turns of the TCA cycle, one for each pyruvate made from glucose. If we use 4 protons/ATP for the efficiency of the ATPase then 1 NADH generates 10 protons (4 at complex I, 4 at complex III and 2 at complex IV). 1 FADH2 generates 6 protons (4 at complex III and 2 at complex IV).

From one glucose we get 10 NADH x 10 protons each, plus 2 FADH2 x 6 protons each = 112 protons = 28 ATP. We can then add the 2 ATP from glycolysis and the 2 GTP from TCA to get 32 ATP equivalents for one glucose. This is 16 times more energy recovered from the oxidation of glucose to CO2 than from glucose to lactate.

Question 50

How many protons are lost by using the glycerol 3-phosphate shuttle?

Answer 50

The electrons from NADH get into the chain at ubiquinone bypassing complex I and losing 4 protons

Question 51

What happens to ATP once it’s made/release?

Answer 51

once ATP is made it must be carried out of the mitochondria by the ADP/ATP carrier. It is not useful to the cell inside the mitochondria. All of the ATP in your body turns over about 200 times per day. This amounts to about 80 pounds of ATP. Because this has to go in as ADP and back out of the matrix as ATP each cycle, the flux of adenine nucleotide through the ADP/ATP carrier is close to your body weight every day.