TCA Cycle and Mitochondria Lecture Aug 30

Question 1

Describe the structure of Mitochondria?

What is the composition of the two membranes?

Which membrane is permeable and which isn’t?

Answer 1

There is an outer membrane, intermembrane space, inner membrane, and matrix.

The outer membrane is permeable to most molecules under 6 kDa. Its composition is similar to plasma membrane: ~45% cholesterol.

The inner membrane is very impermeable– molecules get in or out through regulated passages. The inner membrane contains cardiolipin almost no cholesterol.

Question 2

Describe the proton gradient of mitochondria.

Answer 2

The electron transport chain pumps protons out of the matrix and into the intermembrane space, so…

Intermembrane [H+] = high

Matrix [H+] = low

Question 3

In terms of DNA, why are mitochondria special?

Answer 3

Mitochondria have their own small genome, which encodes for 13 genes that make components of the electron transport chain, 23 tRNAs for translation of mitochondrial genes, and 2 ribosomes.

Question 4

Where are the majority of mitochondrial proteins encoded?

Answer 4

Most are encoded by nuclear DNA and need to be transported into the mitochondria after translation.

Question 5

How do Mitochondria reproduce?

How are they inherited?

Answer 5

Mitochondria reproduce by fission.

They are inherited maternally.

Question 6

Why is mitochondrial DNA more likely to be damaged then nuclear DNA?

(3 reasons)

Answer 6

1. Mitochondrial DNA is proximal to the ETC, where there is high potential for ROS to be generated.
2. mDNA is more “naked’ than nDNA because it’s not associated with histone/chromatin, it’s associated with a single protein that doesn’t provide as much protection.
3. Mitochondrial DNA polymerase isn’t as good at repair as nuclear DNA polymerase, so errors aren’t fixed as readily.

Question 7

How do mitochondria get around the problem of mtDNA hypermutatability?

Answer 7

mtDNA is heterogenous, meaning there are multiple copies of mtDNA in each mitochondria and there are multiple mitochondria in each cell.

The higher the energy needs of the cell, the more copies there will be and the more mitochondria there will be.

This means that a single mutation to one of the mtDNA genomes is unlikely to knock out an enzyme because there are still plenty more genomes and mitochondria without the mutation.

Question 8

Why are mitochondrial diseases heterogenous?

In other words, by do mitochondrial diseases present in such variable ways?

Answer 8

Normal and mutant mtDNA can be present within the same individual (and within the same cell) at different ratios.

This leads to a range of phenotypes for mitochondrial diseases.

Question 9

Why do mitochondrial diseases almost always get progressively worse?

Answer 9

Because mutations of mtDNA caused by ROS have a snowballing effect. Mutations will impinge on mitochondrial function, which will in turn create even more ROS, which will then cause even more damage.

Question 10

What causes myoclonic epilepsy and ragged red fiber disease (MERRG)?

Answer 10

point mutation in mtRNA^Lys

In mitochondria that have this point mutation there will not be appropriate protein synthesis because any protein requiring Lysine would not be translated.

Question 11

What are the three general functions of the TCA cycle?

In other words, what are they able to synthesize/produce?

Answer 11

The TCA cycle takes acetyl CoA from metabolism of glucose, fatty acids, and some amino acids, and:

1. oxizides the 2 carbons from acetate to CO2
2. Creates reduction equivalents by redues NAD+ to NADH and FAD to FAD2H, also forming GTP
3. Generates precursors for biosynthesis.

Question 12

What enzyme is the link between glycolysis and the TCA cycle?

“The secretary of glucose conservation”

Answer 12

Pyruvate Dehydrogenase

Question 13

Where does PDH “live”? In the cytosol, where glycolysis happens, or in the mitochondria where the TCA cycle happens?

Answer 13

PDH is in the mitochondria

Question 14

How is pyruvate transported into the mitochondria?

Answer 14

The MITOCHONDRIAL PYRUVATE CARRIER (MPC)

MPC acts as a heterodimer of two subunits:

MPC1 and MPC2

Question 15

What would happen in an inherited point mutation in MPC1?

What would build up?

Answer 15

The patient would have hyperpyruvatemia (a buildup of pyruvate)

A buildup of pyruvate would increase lactate production, leading to lactic acidosis

Question 16

PDH is a multi-protein enzyme with four subunits. What are the enzymes, and what cofactors do they utilize?

Answer 16

E1: pyruvate decarboxylase; thymine pyrophosphate
E1 α subunits
E1 β subunits

E2: transacetylase; lipoate

E3: dihydrolipoyl dehydrogenase; FAD, NAD+

X

Question 17

What happens to PDH in order for it to become inactive, resulting in a switch to fatty acid metabolism instead of glucose metabolism?

Answer 17

THe E1 alpha subunit of the PDH complex is inactivated when its serine residues become phosphorylated.

The enzyme that carries out this phosphorylation and inactivation is PDH KINASE

Question 18

PDH Kinase is the enzyme phosphorylates the E1 subunit of the PDH complex, inactivating it. How is PDH Kinase regulated?

Answer 18

Think first about when you would want PDH Kinase active. PDH kinase shuts off PDH, so you would want it active when the cell has an energy surplus. Now think of the products you get with energy surplus/PDH activity…NADH and acetyl CoA.

So PDH Kinase is activated by acetyl CoA and NADH.

Now think of the reverse. You would want PDH active during times of energy deficit, so you need PDH Kinase to be inactivated. In this case, ADP is increased. Additionally, you would want to have PDH active when there is an abundance of carbs around because you want to use those before you use fatty acids. When there is a lot of glucose available, pyruvate concentration will increase.

PDH Kinase is inhibited by ADP and pyruvate.

Question 19

What enzyme will work in opposition of PDH kinase and activate PDH?

Answer 19

PDH phosphatase regulates PDH by phosphorylating and dephosphorylating serine residues in the E1 alpha subunit.

Question 20

How is PDH phosphatase regulated?

Answer 20

PDH phosphatase is activated by Ca²⁺ because Ca2+ is an indicator that there is work being donw (remember Ca2+ mediates muscle cell contraction)

If there is work being done in the cells, you want to maximize energy production, thus you want PDH active and you need it in the dephosphorylated form.

Question 21

Glycolysis is not the only source of acetyl CoA.

What are some other sources?

Answer 21

The fatty acid palmitate

The ketone body acetoacetate

The amino acid alanine can be converted to pyruvate to make acetyl CoA

Ethanol

Question 22

O2 is never used as an electron acceptor in the TCA cycle. So why can’t the TCA cycle occur under anaerobic conditions?

Answer 22

Because O2 is the ultimate electron acceptor in the electron transport chain. If you don’t have it, NADH and FAD2H build up and you don’t get regeneration of NAD+ and FAD. These are required substrates of the TCA cycle and without them the cycle cannot function.

Question 23

What is the key rate limiting enzyme in the TCA cycle?

What does it do?

Answer 23

Isocitrate dehydrogenase

It decarboxylates isocitrate to form alpha-ketoglutarate

This reaction has a large negative deltaG, and the energy, which is used to reduce NAD+ to NADH, giving us the first reducing equivalent in the cycle

Question 24

There are two steps in the TCA cycle that are actually energetically unfavorable. What are they?

Answer 24

Citrate’s conversion to isocitrate, catalyzed by aconitase

Malate’s conversion to oxaloacetate, catalyzed by malate dehydrogenase

Question 25

If you have an issue with isocitrate dehydrogenase, what will build up? In other words, what would be the actual product of aconitase in that situation?

Answer 25

You would think that isocitrate would buildup because that's what isocitrate dehydrogenase is converting to alpha keotglutarate. But remember that the conversion of citrate to isocitrate by aconitase is actually energetically unfavorable, so the real buildup would be CITRATE, not isocitrate.

Question 26

If there is a shutoff anywhere in the TCA cycle, what would you ultimately get an accumulation of?

Answer 26

You would think it would be oxaloacetate, since that's the "end" of the cycle. However, remember that the conversion of malate to oxaloacetate by malate dehydrogenase is energetically unfavorable. So MALATE is actually what builds up in TCA cycle problems, not oxaloacetate.

Question 27

What is the functional significance of having the two energetically unfavorable reactions in the TCA cycle? Wouldn't it make more sense for them all to be favorable?

Answer 27

While it seems odd to have unfavorable reactions in the TCA cycle, it is actually very important that it be this way so that intermediates can be diverted away from the TCA cycle at times when you don't need to be oxidizing acetyl CoA for energy. Remember the two intermediates that build up at the other end of the unfavorable reactions? 1. Citrate can leave the cycle and be used as a substrate for fatty acid synthesis 2. Malate will build up and can be diverted from the TCA cycle to the liver where it is used as a gluconeogenesis substrate

Question 28

What is the first step of the TCA Cycle?

Answer 28

The acetyl CoA is joined to oxaloacetate by **citrate synthase** to form citrate, removing the CoASH. Oxaloacetate + Acetyl CoA --------------\> Citrate

Question 29

What enzyme catalyzes the isomerization of citrate to isocitrate? Why is this step important?

Answer 29

Aconitase This step is important because it's one of the energetically unfavorable steps, allowing citrate to be diverted out when needed.

Question 30

What step in the TCA cycle gives you your first reducing equivalent?

Answer 30

The decarboxylation of isocitrate to alpha-keotglutarate using isocitrate dehydrogenase. This is the key regulatory step.

Question 31

In the isocitrate dehydrogenase reaction converting isocitrate to alpha-ketoglutarate, you get the loss of CO2 and the first reduction of NAD+ to NADH. Is the carbon that leaves as CO2 one of the carbons that came into the cycle on acetyl CoA?

Answer 31

No. It's one of the carbons that was on oxaloacetate when it the acetyl CoA was added.

Question 32

What happens to alpha ketoglutarate in the TCA cycle?

Answer 32

It undergoes the second decarboxylation to form succinyl CoA through the enzyme alpha-ketoglutarate dehydrogenase, reducing NAD+ to NADH This is where the second NADH comes from in the TCA cycle. Again, the carbon on the CO2 that gets kicked off is NOT one of the carbons that enteres the TCA as acetyla CoA.

Question 33

What happens to succinyl CoA in the TCA cycle? What enzyme acts on it? What is produced?

Answer 33

Succinyl CoA undergoes a reaction where **Succinate thiokinase** cleaves the thioester bond, releasing **succinate** from the CoA. Remember that the link between CoA and anything is very high energy, so the enzyme uses the energy to catalyze **substrate level phosphorylation of GDP to make GTP.**

Question 34

What enzyme oxidizes succinate to fumarate? Why is this such an important enzyme for the steps that follow the TCA cycle?

Answer 34

Succinate dehydrogenase. First of all, this is the step in the TCA cycle that reduces FAD to FAD2H, providing a reducin equivalent. However, SDH is also a component of the electron transport chain, involved in creating a proton gradient across the inner mitochondrial membrane for the generation of ATP.

Question 35

What happens to fumarate in the TCA cycle? Which enzyme is responsible?

Answer 35

**Fumarase** (also called fumarate hydratase) addes a proton and hydroxyl from H20 to the double bond to form **malate.**

Question 36

How are succinate dehydrogenase and fumarase tumor suppressors?

Answer 36

They avoid any buildup of fumarate. Remember, fumarate can act as a competitive inhibitor of succinate as a substrate for HIF1 prolyl hydroxylate (HPH) in O2 sensing. If HPH is inhibited, HIF builds up and acts as a transcription factor, making it easier to cells (cancer cells in this case) to utilize glucose for energy.

Question 37

Where does the third reduction of NAD+ to NADH occur in the TCA cycle? What enzyme does this? Why is this reaction of particular importance?

Answer 37

**Malate Dehydrogenase** oxidizes malate to oxaloacetate, reducing NAD+ to NADH in the process. This reaction is important because it's a reversible reaction, allowing for malate buildup if TCA is shut down, with malate being diverted to the liver for gluconeogenesis.

Question 38

What are the net substrates and the net products of the TCA cycle?

Answer 38

Net Substrates: 2C acetyl group, two H20, 3 NAD+, 1 FAD, 1 GDP Net Products: 2 CO2, 3 NADH + H, 1 FAD2H, and 1 GTP

Question 39

WHen energy is consumed, NAD+ increases, and NADH decreases. What effect does this have on oxaloacetate? What effect does this have on isocitrate?

Answer 39

With increased NAD+, you an increase of malate conversion to oxaloacetate, and an increased conversion of isocitrate to alpha-keotglutarate. So, oxaloacetate levels increase when energy is consumed, and isocitrate levels decrease as energy is consumed.

Question 40

When energy is abundant, NAD+ decreases and NADH increases. What effect does this have on malate? On isocitrate? On citrate?

Answer 40

When NADH is abundant, the malate dehydrogenase favors the conversion of oxaloacetate to malate, so malate builds up. In terms of citrate and isocitrate, an increase of NADH would actually results in an increase in citrate, but not much change in the levels of isocitrate because the aconitase conversion of citrate to isocitrate would rather go in the opposite direction, convertion isocitrate to citrate.

Question 41

What does citrate essentially tell the body?

Answer 41

It tells the body that fatty acids aren't being used for energy.

Question 42

How does alcohol poisoning affect the TCA cycle?

Answer 42

Ethanol metabolism produces NADH. This causes the TCA cycle to lock up because the ethanol metabolism produces NADH faster than the body can oxidize it back to NAD+, and you lose that important substrate required for 3 different reactions in the TCA cycle.

Question 43

What affect does ADP have on isocitrate dehydrogenase's Km?

Answer 43

ADP binding decreases IDH's Km, making it a more active enzyme.

Question 44

What affect does NADH have on isocitrate dehydrogenase's Km?

Answer 44

NADH is an allosteric inhibitor of IDH, so NADH binding will increase IDH's Km.

Question 45

Why is the TCA cycle considered amphibolic?

Answer 45

It serves both catabolic and anabolic purposes. Catabolic: TCA cycle reduces NAD+ and FAD for the generation of ATP via the electron transport chain Anabolic: TCA cycle intermediates are feedstock for other biosynthetic pathways.

Question 46

During the FED state, three different TCA intermediates are diverted out for biosynthesis. What are these three intermediates, and what are they used for?

Answer 46

During the FED state, fatty acid metabolism is not needed, so you use the intermediates to build things. 1. Citrate is diverted out for fatty acid biosynthesis 2. alpha-ketoglutarate is diverted out to make glutamate, which can be used to make GABA 3. Succinyl CoA ( from the cycle and from the beta oxidation of odd chain fatty acids) can be diverted for heme biosynthesis.

Question 47

During the FASTED state, there are two intermediates that divert out of the TCA. What are they and what are they used for?

Answer 47

Malate is diverted out of the TCA to the liver to be a substrate for gluconeogenesis. Oxaloacetate can be diverted out to be used in amino acid biosynthesis.

Question 48

For the TCA cycle, what is the purpose of an anapleurotic reaction?

Answer 48

It's a way to keep the intermediates or "electron buckets" available in the cycle. This is important because some intermediates are diverted out for biosynthesis, but if they all left, the TCA cycle would shut down.

Question 49

Pyruvate usually enters the TCA cycle through a catabolic process, but there is a second way pyruvate can enter the cycle. WHat is it?

Answer 49

**Pyruvate carboxylase** can convert pyruvate directly to oxaloacetate. So the pyruvate can enter the TCA cycle as ocaloacetate instead of as acetyl CoA.

Question 50

What are 4 examples of amino acids being converted into TCA intermediates in anaplerotic reactions/

Answer 50

Alanine and Serine can be converted to pyruvate, which can then enter the TCA cycle as oxaloacetate. Glutamate is converted back and forth to alpha-ketoglutarate (with glutamate dehydrogenase and transaminase) Valine and Isoleucine can be converted to priopionyl CoA, which is then converted to succinyl CoA Aspartate can be converted to malate

Question 51

During exercise, oxaloacetate can be a limiting factor for the production of energy through the TCA cycle. What pathway supplies oxaloacetate to the cycle in this situation?

Answer 51

Muscle contraction uses ATP and converts it to ADP. **Adenylate kinase** will then take one phosphate from an ADP and stick it on a second ADP in order to regenerate ATP again quickly. The result of this is 1 ATP and 1 AMP from the original 2 ADP. The AMP is used in biosynthesis of oxaloacetate! How clever! **AMP Deaminase** will convert AMP to IMP, which can join aspartate to form adenylosuccinate. Adenylosuccinate can be converted to fumarate, which is easily hydrated to form malate, which is then converted to oxaloacetate with malate dehydrogenase.

Question 52

What happens in myoadenylase deaminase deficiency (AMP deaminase deficienty)?

Answer 52

There is an inherited mutation in the gene coding the muscle specific AMPD1 isoform of AMP deaminase. This results in an inactive enzyme. This means you can't convert the AMP to IMP, and you don't get the anaplerotic supply of oxaloacetate to the muscles during exercise. This means your muscles get energetically starved during exercise, forcing glycolysis to increase, leading to lactic acidosis in the muscles. Symptomatically, this results in muscle pain and burning/excercise intolerance. Long term symptoms are muscle breakdown and myoglobinuria.

Question 53

What is the lab hallmark of AMP deaminase deficiency?

Answer 53

Remember that AMP deaminase converts AMP to IMP by releasing a molecule of ammonia. In normal exercise, the blod levels of ammonia will increase slightly. Without AMP deaminase, however, this ammonia concentration increase will NOT occur. In addition, there would be no AMPD1 present in muscle biopsy, because the patient wouldn't have it.

Question 54

There are 4 complexes in the electron transport chain. What are they? Which complexes does NADH use? WHich complexes does FAD2H use? What is sometimes considered the 5th complex?

Answer 54

Complex I, II, III, IV. NADH uses I, III, and IV FAD2H uses II, III, IV Atp synthetase is sometimes considered the 5th.

Question 55

What is complex I in the electron transport chain?

Answer 55

NADH dehydrogenase. Remember that it uses FMN, Fe-S clusters and eventually CoQ to transfer electrons from NADH down energy levels, harnessing energy to pump H+ across the membrane into the intermembrane space. [![]()](https://s3.amazonaws.com/brainscape-prod/system/cm/048/437/721/a_image_thumb.png?1378070398)

Question 56

What is the final electron acceptor in the NADH dehydrogenase complex (which is complex I of the electron transport chain)?

Answer 56

Coenzyme Q

Question 57

Describe CoQ. What about CoQ makes it a major source of ROS in the electron transport chain?

Answer 57

CoQ is lipid soluble and exists in the inner mitochondrial membrane. Unlike CoA, it is not bound to protein. It can accept one e- to form semiquinone. It then accepts another e- to form the fully reduced dihydroquinol (CoQH2) Single electron transfers from CoQ to molecule oxygen is a major source of ROS.

Question 58

Where are the electrons from CoQH2 released in the electron transport chain?

Answer 58

In Complex III--the cytochrome b-c complex--the electrons on CoQH2 are transferred to cytochrome-B.

Question 59

How does the Cytochrome b-c complex handle electron transfers?

Answer 59

1. Transfers electrons from CoQH2 to Cytochrome B 2. CYtochrome B transfers its electrons to an Fe-S cluster 3. THe Fe-S cluster transfers the electrons to Cytochrome C.

Question 60

What makes up complex IV of the electron transport chain? How does it handle electron transfers?

Answer 60

Complex IV is cytochrome C oxidase. 1. It transfers electrons from CytC (from complex III) to CytA 2. Electrons from cytA are transferred to CytA3 3. Electrons from CytA3 are transferred to the ultimately electron acceptor--molecular oxygen--to form H20.

Question 61

For one molecule of NADH, how many protons are pumped across the membrane? Provide specific numbers for each complex involved.

Answer 61

A total of 10 protons are pumped across the membrane per molecule of NADH. NADH dehydrogenase: 4 protons Cytochrome b-c complex: 4 protons Cytochrome C oxidase: 2 protons

Question 62

FADH2 is handled differently than NADH in the electron transport chain? How so? What complex does it travel through first?

Answer 62

It skips NADH dehydrogenase (for obvious reasons) and enters complex II first -- Succinate Dehydrogenase. Remember from last week that the SDH complex converts succinate to fumarate by reducing FAD to FADH2, after which is puts the electrons on CoQ to form CoQH2.

Question 63

After passing through the SDH complex, where do the electrons from FADH2 go in the electron transport chain?

Answer 63

Since SDH eventually transfers the electrons from FAD2H onto CoQ, just like NADH dehydrogenase, the remainder of the electron transport chain from FADH2 is identical to NADH electrons. THey will be transferred to CytC in the cytochrom b-c complex, then onto oxygen in the cytochrom c oxidase complex.

Question 64

How many protons are pumped across the inner membrane when FAD is used as the acceptor? Why is this less than when NAD is used as the acceptor?

Answer 64

Only 6 protons are pumped across for FADH2 (4 from the cytochrome b-c complex and 2 from cytochrome c oxidase). This is less than the 10 from NADH because succinate dehydrogenase does not cross the inner mitochondrial memebrane, so it isn't capable of pumping protons across (unlike NADH dehydrogenase, which can).

Question 65

What two steps of the electron transport chain can result in ROS formation?

Answer 65

CoQ can sometimes donate 1 electron to O2, causing superoxide formation. CytA3 can also donate 1 electron to O2, causing superoxide formation.

Question 66

What does superoxide dismutase do to ROS? What happens after that?

Answer 66

It converts them to hydrogen peroxide H2O2. If catalse is present, it can convert H2O2 to water and you don't have an issue. If catalase is not present, however, H2O2 can make the hydroxyl radical, which is the most dangerous of the ROS and can damage proteins and lipids with a subsequent "snow balling effect" where more radicals are created to cause more damage [![]()](https://s3.amazonaws.com/brainscape-prod/system/cm/048/441/372/a_image_thumb.png?1378072513)

Question 67

What are the two ways that hydroxyl radical can be made?

Answer 67

The Haber-Weiss reaction with superoxide and hydrogen peroxide. The Fenton reaction with hydrogen peroxide and iron. [![]()](https://s3.amazonaws.com/brainscape-prod/system/cm/048/441/640/a_image_thumb.png?1378072732)[![]()](https://s3.amazonaws.com/brainscape-prod/system/cm/048/441/640/a_image_thumb.png?1378072761)

Question 68

What cofactors do each of the electron transport chain complexes use?

Answer 68

NADH Deydrogenase: FMN and Fe-S Succinate Dehydrogenase: FAD and Fe-S Cytochrome b-c complex: Fe-S and heme Cytochrome c oxidase: heme, copper, zinc

Question 69

What is the general structure of ATP synthase?

Answer 69

There are two main parts: F1 and F0. F) is the part in the membrane, and F1 is the part within the matrix. THe F0 unit has 12 C subunits, each with 1 channel for a proton. This means that through 1 360-degree of rotation by the rotor, 12 protons will pass through from the intermembrane space into the matrix. The F1 unit has three catalytic subunits (each with an alpha and beta portion) which can generate 1 ATP. So...for 1 full 360-degree rotation there are 12 protons used to make 3 ATP. In other words, 4 protons are needed to make 1 ATP. [![]()](https://s3.amazonaws.com/brainscape-prod/system/cm/048/441/889/a_image_thumb.jpg?1378076622)

Question 70

How much harnessable energy in kcal is generated per mol of NADH? How much energy is released as heat because it wasn't harnessed to make ATP?

Answer 70

18. 25 kcal/mol 34. 75 kcal/mol is released as heat and used for transport across membranes. This is why we're warm blooded.

Question 71

Under what situations does ATP synthase activity stop?

Answer 71

If a person is at rest and the ratio of ATP to ADP is high. In hypoxia where there is no final acceptor of electrons, there is insufficient proton motive force to run ATP synthase.

Question 72

What is uncoupling? What are the three types of uncoupling?

Answer 72

Uncoupling occurs when the transfer of eletrons from NADH to O2 occurs without the generation of ATP. In other words, the electron transport chain is uncoupled from ATP synthase. THree types: 1. Adaptive thermogenesis (this is good) 2. Chemical uncouping (this is bad) 3. Mechanical uncoupling (this is bad)

Question 73

WHat happens in adaptive thermogenesis

Answer 73

This is a normal response to cold. 1. norepinephrine is released 2. norepinephrine activates a lipase which forms free fatty acids from triacylglycerol in brown fat cells 3. A proton channel called thermogenin (a.k.a. UCP1) is actuvated 4. This allows fat to be utilized for heat, independently of ATP consumption In other words, a baby on its back won't be doing much work, so ATP synthase won't be running. But the baby still needs to be able to generate heat! So the UPC1 chanels allow protons to flow back down their concentration gradient without ATP synthase being on. Thus, the TCA cycle doesn't get bogged down and the electron transport chain can sill operate and generate heat.

Question 74

What happens in chemical uncoupling?

Answer 74

Lipid soluble molecules with a pKa near neutral can bind a proton in the intermembrane space, where [H+] is high. It can then diffuse across the inner membrane with the proton. Once it's in the matrix where [H+] is low, it will release the electron This means the gradient will gradually dissipate and ATP synthase won't work because the protons will have no gradient to pass down. The classic chemical uncoupler is dinitrophenol (DNP)

Question 75

What is the protoypical chemical uncoupler? Why are they rare? What's one we should be concerned about as future doctors?

Answer 75

Dinitrophenol (DNP) They're rare because the inner mitochondrial membrane is only impereable to very few things. Salicylic in aspirin can also act as a chemical uncoupler, which is one mechanism of aspirin toxicity.

Question 76

What is mechanical uncoupling?

Answer 76

Mechanical uncoupling occurs in membrane damage. Mitochondrial damage can occur due to peroxidation of membrane lipids by ROS Mitochondrial swelling due to influx of water Protons leak through holes in the membrane, which causes the concentration to dissipiate and ATP synthase won't be able to function. In severe cases, this can even cause ATP synthase to work backwards, synthesizing ADP and Pi from ATP.

Question 77

What 4 molecules is the inner mitochodnrial membrane permeable to? What must everything else use?

Answer 77

O2, CO2, NH3, and H2O can pass through the inner mitochondrial membrane. All other molecules must use specific transport channels.

Question 78

Where does the energy for transport across the inner mitochondrial membrane come from? (2)

Answer 78

1. **the electrochemical gradient**: the matrix side of the membrane is negatively charged compared to the intermembrane space side 2. **The pH gradient**: THe [H+] is lower in the matrix than in the intermembrane space, so the pH is lower in the intermembrane space

Question 79

What are the three basic types of transport systems?

Answer 79

Antiporters Symporters Uniporters

Question 80

How do antiporters work?

Answer 80

They transport one molecule in and another molecule out. It's a swap. One example is adenine nucleotide translocase (ANT), which swaps ADP for ATP.

Question 81

How does a symporter work? Example?

Answer 81

A symporter transports two molecule in or out together. An example of this is pyruvate and inorganic phosphate--they're brought in together. In this case, the H+ is just travelling down its concentration gradient and the pyruvate tags along.

Question 82

How does a uniporter work?

Answer 82

A uniporter just transports one molecule in or out. An example is calcium channels.

Question 83

What is a VDAC

Answer 83

A VDAC is a voltage dependent anion channel This allows anions to flow down the electrochemical gradient

Question 84

What is a mitochondrial permeability transition pore? WHen do they form? What's in the complex? What happens?

Answer 84

An MPTP is a large complex between ANT, VDAC, and other proteins. It's a nonspecific pore that will cause depolarization of the mitochondrial membrane and disruption of the proton gradient. It happens under hypoxic conditions. Acitiation of an MPTP can lead to apoptosis or necrosis because the membrane becomes depolarized.

Question 85

What 2 drugs will block complex 1 (NADH dehydrogenase)?

Answer 85

Rotenone (a pesticide) Amytal (a barbituate)

Question 86

What drug will block complex III (cytochrom b-c1 complex)?

Answer 86

Antimycin A (an antibiotic)

Question 87

What 3 drugs will block complex IV (cytochrome c oxidase)?

Answer 87

Cyanide (from smoke and industrial chemicals) Carbon monoxide (combustion product) Azide (antimicrobial agent, AZT)

Question 88

What drug will block ATP synthase

Answer 88

Oligomycin (an antibiotic)

Question 89

What drug will block DNA polymerase gamma, the mitochondrial DNA replication enzyme?

Answer 89

AZT (zidovudine)--an anti-retroviral drug

Question 90

The effects of PDH deficiency are primarily neurological, affecting brain development. WHy?

Answer 90

Because the brain can't use fatty acids and needs glucose for fuel. But if you don't have PDH, you can't use glucose. It can and does use ketone bodies for fuel, but it oesn't like to and development will be hurt.

Question 91

What is Leigh's disease? What are its symptoms? When is it usually diagnosed?

Answer 91

Leigh's disease is PDH deficiency -- a metabolic disease, one cause of which is a mutation in a gene on the X chromosom which encodes for the E1alpha subunit of PDH. When Leigh's disease is caused by this, it's known as x-linked Leigh's disease. It's usually diagnosed in infants or young children. Symptoms include an ability to control movements, irritability (continuous crying in infants), seizures, etc.

Question 92

In Leigh's Disease, why is there a broader range of mutant PDH phenotype in girls compared to boys?

Answer 92

Boys with X-linked Leigh's disease typically form a PDH which is enzymatically active, but with an increased Km for pyruvate compared to normal PDH, making a less efficient enzyme. Because they only get one X, the boys with mutations that completely knock out PDH would not have been viable embryos and wouldn't hav ebeen born. In girls, there is more variation in mutant E1 phenotype, ranging from enzymes with lower efficiency to completely inactive enzymes. This is because they have 2 Xs and thus have a higher range of phenotype.

Question 93

What is one possible treatment for PDH deficiency caused by mutations that lower the effiiency of the E1 PDH subunit? Why does it work?

Answer 93

Thiamine Thiamine is the vitamin precursor for TPP, which is one of the cofactors in the PDH complex. So if you flood it with cofactor, the enzyme will get closer to its Vmax

Question 94

What drives ADP entrance and ATP exit through the inner mitochondrial membrane?

Answer 94

They go through an antiporter It's drive by the charge differential across the inner membrane. THe matrix side is negative (due to the pumping of H out into the matrix by the ETC) and the intermembrane space is positive (due to the high proton concentration). This makes the more negative ATP want to leave the matrix and the less negative ADP to enter the matrix.