Unit 12 (2)

Question 1

Considering the fact that no CO2 is lost, how many moles of ribulose 5-P should be produced from 5 moles of G6P by these reactions?

Answer 1

• No CO2 means its the nonoxidative pathway

5 x 6 = 30
30/5=6
- 6 moles

*for these questions, figure out how many carbons you would get from whichever pathway you choose. Then divide that number by 5, and that should give you the amount of moles of ribulose you have because ribulose is 5 carbons

Question 1

What type of enzymes catalyze the nonoxidative reactions?

Answer 1

• Transaldolase
• Transketolase

Question 2

Notice that the branch on the right, labeled the oxidative branch is irreversible and involves oxidation reduction reactions and the loss of CO2. How many moles of ribulose 5-P are produced from 5 moles of G6P from these reactions.

Answer 2

5 G6P x 6 = 30 carbons
30 carbons - 5 = 25

25/5 = 5 moles of ribulose 5 Phosphate

Question 3

What does ribulose 5-P turn into? Why is it important?

Answer 3

Ribose 5 Phosphate

Important precursor for DNA, RNA, NAD, FAD, ATP,

Question 4

How many NADPH’s are produced per pentose phosphate pathway? What is NADPH used for?

Answer 4

• 2 per cycle (generated)
• Important for biosynthesis and reducing radical oxygen species (places that are constantly exposed to O2 like the eye)

Question 5

Which pathway will be favored more when ribose 5-P is needed more than NADPH?

Answer 5

Non-oxidative

Question 6

Which pathway will be favored more when NADPH is needed more than ribose 5 phosphate?

Answer 6

Oxidative pathway

Question 7

Which pathway enables ribose 5-phosphate to be metabolized for energy?

Answer 7

Non Oxidative pathway *

because it is reversible, therefore you can recycle the ribulose 5-P back to glucose 6 P so it can be used for glycolysis

Also ribulose 5 P –> ribose 5 P which is a precursor for ATP

Question 8

Draw out the diagram of the mitochondria and all the important things that occur in the different areas

Answer 8

Outer membrane: Freely permeable to small molecules and ions

Inner membrane:
- Impermeable to most small molecules and ions, including H+
Contains the:
- ETC proteins
- ATP Synthase
- Other membrane transporters

Matrix:
- Pyruvate dehydrogenase complex
- TCA Cycle
- Fatty acid ox
- Amino acid ox
- DNA ribosomes

Intermembrane space:
- Contains H+ (impermeable to inner membrane)

Citrae: Creates folds on the inner membrane to increase surface area

Question 9

Are mitochondria found in prokaryotic cells?

Answer 9

No

Question 10

On the mitochondrion, indicate the locations of the electron carriers of the respiratory chain and the reactions of the citric acid cycle

Answer 10

• The electron carriers of the respiratory chain are on the inner membrane
• The reactions of the citric acid cycle occur in the matrix

Question 11

Explain/Draw the ETC complex and how electron flow creates a proton gradient. Make sure to detail how many protons are pumped per NADH and per FADH2

Answer 11

• 10 H+ per NADH
• 6 H+ per FADH2

Question 12

Explain/Draw how the cytosolic NADH can enter the mitochondrial matrix and how many H+ ot can pump

Answer 12

• Malate-aspartate shuttle: 10 H+
  NADH2 reduces Oxaloacetate to Malate, malate can go through a transporter, and it will go back into oxaloacetate to generate an NADH on the opposite side
• Glycerol 3 Phosphate shuttle: 6H+ (per NADH). NADH gives electrons to dihydroxyacetone so it can be reduced into glycerol 3 phosphate. The glycerol 3 phosphate will give its electrons to FAD on the mitochondrial glycerol 2-phosphate dehydrogenase enzyme. This will generate 6H+ because it takes a similar route to FAD

Question 13

What is different about cytochrome C and coenzyme Q, compared with other carriers of the chain

Answer 13

They are more mobile and can freely diffuse WITHIN the membrane to transfer electrons between complexes

Question 14

Draw/Discuss how the electrons move for the Q cycle

Question 15

What is the net reaction of the Q cycle?

Answer 15

QH2 + 2 cyt c (oxidized) + 2H+ ==> Q + 2 cyt c (reduced) + 4H+

Question 16

Name the cofactors in the Q cycle that occurs in the cytochrome bc1 complex *

Answer 16

• heme bL
• heme bH
• QH2
• Q
• 2Fe-2S
• heme c1
• Cytochrome C

Question 17

What is the function of the Q cycle?

Answer 17

To transport protons across the membrane (even if this is unfavorable, it uses the energy of the redox reactions in order to do so)

Question 18

Name the two components of the proton motive force

Answer 18

1) The chemical potential energy (concentration) due to the difference in concentration of a chemical species
2) The electrical potential energy that results from the separation of charge when a proton moves across the membrane without a counterion

Question 19

In the respiratory chain, which complexes contribute to the proton motive force

Answer 19

I, III, IV because they all pump H+ into the intermembrane space

Question 20

Explain the main points of the experiments to demonstrate that the ETC is coupled to ATP synthesis

Answer 20

• O2 consumption was monitored as samples were tested
• Addition of ADP and Pi alone results in little or no increase in either O2 consumption (black) or ATP synthesis (red)
• When succinate is added, respiration begins immediately, and ATP is synthesized.
• Addition of cyanide, which blocks electron transfer between Complex IV and O2, inhibits both respiration and ATP synthesis
• Mitochondria provided with succinate respire and synthesize only when ADP and Pi are added
• Addition if oligomycin, inhibitor of ATP synthase, blocks both ATP synthesis and respiration.
• DNP is an uncoupler, allowing respiration to continue without ATP synthesis

Question 21

Why is succinate added in these experiments

Answer 21

Because succinate –> fumarate will provide FADH2 which needs to give its electrons to the ETC and cause respiration

*Provides the electrons

Question 22

What is oligomycin and what does it do?

Answer 22

Oligomycin is a ATP synthase inhibitor. It will bind to the Fo subunit and prevent H+ on the intermembrane side from binding and entering

By blocking this, there will be no ATP synthesis. This is a problem because if there is no ATP synthesis, the ATP synthase isn’t taking the H+ from the intermembrane space and putting them in the matrix. This will cause a buildup of H+ on the outside which makes it unfavorable for the ETC to continue and push H+ on the intermembrane space.

Question 23

What is an uncoupler? How do they act?

Answer 23

Intermembrane molecules that disrupt the proton gradient.

This means they shuttle protons into the mitochondrial matrix without passing through ATP synthase. This allows respiration to continue without ATP synthesis because now it is favorable to continue to pump out H+

Question 24

Explain how the Fo subunit of ATP synthase works

Answer 24

- Can either be an aspartate or glutamate On the intermembrane side, there is a high amount of H+ ions. They can enter the half-channel on that side and attach to an Asp or Glu residue to turn their negative charge neutral This Asp or Glu residue was creating a salt bridge with the nearby Arg. Now that there is not more negative charge, the Arg can dissociate and swing back to another Asp residue with a negative charge. This Asp with a negative charge is closer to the matrix. This is why its allowed to easily remove its bound H+. This rotation causes the C subunits to shift by one. Each H+ that comes in will shift the C subunits by one.

Question 25

How is the energy transferred from Fo to F1? Describe the experimental evidence for this

Answer 25

The rotation of the c subunits, allow for the gamma subunit to rotate. THe rotation of the gamma subunit will give each alpha beta pair a confirmation change, making it more or less likely to bind to/make ATP Experiment: - In one experiment, they fluorescently attached actin to a c subunit of the Fo subunit. When there is a lot of ATP in the environment, the ATP synthase can actually work to hydrolyze the ATP if it rotates CLOCKWISE as opposed to counterclockwise (remember, enzymes can work in reverse so it can hydrolyze ATP as well as create it). As the ATP hydrolyzed, the actin filament on the Fo subunit rotates as well, showing that the movement of the Fo powers the F1 - In the second experiment, they just attached a fluorescent molecule to the gamma subunit to see it rotate. This explains that it is the linker between the two domains

Question 26

Discuss the binding change model for ATP synthesis

Answer 26

The F1 subunit of the ATP synthase is composed of a trimer of two alpha-beta subunits. Each pair of alpha beta subunits has a conformation that makes it bind tightly, loosely, or not at all with ATP. Therefore, each conformation has a preference for what it wants bound to it at a given time. As the Fo subunit shifts its C subunits, the gamma subunit will rotate. This rotation will change the conformation of the subunit and change its preference. This allows for the synthesis of ATP from ADP + Pi. It also facilitates the release of it. The amount of H's required to generate the 3 ATPs is dependent on the c subunits. *ADP + Pi --> ATP --> release of ATP

Question 27

Explain how one proton is used to drive the uptake by mitochondria of ADP and Pi in exchange for ATP DRAW THIS OUT INCLUDING THE ATP SYNTHASE

Answer 27

-Remember, Fo is on the top and F1 is on the bottom. - In order to power our binding change model, we need to have ADP and Pi in the mitochondrial matrix. To do this, we use the adenine nucleotide translocase to antiport ATP 4- out of the mitochondria and ADP3- into the cell. This is favored because it will put a net negative charge on intermembrane side which is good because it has so many H+. - Now that we have ADP in the cell we have to get phosphate in as well. The phosphate comes in by the phosphate translocase. It is symport with H+. This uses the power of H+ going down its electrochemical gradient because H+ wants to be inside the matrix. - Now you have the materials to undergo the creation of 1 ATP. If you rotate 360, you would need 3H+ to bring in 3 Pi. - Additionally, remember that the ATP synthase itself is bringing in one H+ to facilitate the movement of oen subunit. Depending on the number of subunits will determine how many times it will rotate.

Question 28

What would happen to the ETC if we could no longer take up H+ with the ATP synthase?

Answer 28

If we could no longer take up H+ from the intermembrane space with H+, the concentration of H+ would remain high on the outside. At first, the Pi would still be able to come in to the matrix with H+ but evenually the concentration of Pi would get so high because there is not ATP being formed and moving out of the cell. Therefore, it will make it unfavorable to bring in anymore Pi, and thus H+. With such a high concentration of H+ on the intermembrane space, it would be very unfavorable for the ETC to keep pumping out electrons into this area, therefore, due to Le Chatelier's principle, it would slow down and stop

Question 29

Uncouplers must be permanent in both the protonated and ionized forms, which is unusual. Why do you think that is? The DNP anion is permanent because the negative charge is delocalized by resonance structures

Answer 29

This has to be true because they need to be able to grab an H, pass through the membrane (which is fine), but then they have to release the H and leave. This would normally be unfavorable because ionized compounds cannot pass through membranes, but in this case, since there is a high amount of resonance, its okay.

Question 30

Define the term P/O

Answer 30

The number of moles of ATP formed in oxidative phosphorylation per 1/2 O2 reduced

Question 31

Why is the P/O ratio of ATP synthesis from NADH and from succinate to oxygen approximately 2.5 and 1.5? Discuss these values in terms of the proton numbers transported by the various components

Answer 31

To make 3 ATP (full 360 of atp synthase), we need 3 Pi that require 3H+ to flow in. We also need 1 H+ for every subunit. Let's assume we have 9 subunits. Therefore: 3H+ + 9H+ = 12 H+/3 ATP --> 4H+ per ATP We know that in total we have: 2 NADH (from glycolysis) 2 ATP (from glycolysis) 6 NADH (from TCA) 2 ATP (from TCA) 2 FADH2 (from TCA) We know that NADH will pump out 10H+ into the intermembrane space and FADH2 will pump out 6H+ Therefore: 10H+/4H+ per ATP --> 2.5 ATP from NADH 6H+/4H+ per ATP --> 1.5 ATP from NADH ------------------------------------------------------ Now we can multiply the number of ATP per each cofactor by the number of cofactors: 6 NADH (from TCA) x 2.5 2 ATP (from TCA) 2 FADH2 (from TCA) x 2.5 For the NADH in glycolysis, depending on which shuttle it uses, it could generating 10H+ or 6H+ 2 NADH (from glycolysis) x 2.5 or 1.5 = When you add all your ATP, you get 30-32 ATPs

Question 32

What is the yield of ATP from the complete oxidation of glucose?

Answer 32

30-32 depending on which shuttle you use

Question 33

Do review problem 9-12

Question 34

Regulation of oxidative phosphorylation compared to other pathways has a lack of regulation. As long as mitochondria has __ available, they are always ready to made ___ when ___ and __ are added

Answer 34

Regulation of oxidative phosphorylation compared to other pathways has a lack of regulation. As long as mitochondria has substrates available, they are always ready to made ATP when ADP and Pi are added *substrates like succinate etc