Ox Phos

Question 1

What reactions occur in the different compartements of the mitochondrion?

Answer 1

Inner membrane → ETC, Ox Phos, Transport systems, Fatty acid transport

Matrix → PDC, CAC, Glutamate dehydrogenase, Fatty acid oxidation, Urea cycle, Transcription, Translation (of mitochondrial DNA)

Outer membrane → Fatty acid elongation, Fatty acid desaturation, phospholipid synthesis

Question 2

What is the difference between the outer and the inner mitocondrial membrane?

Answer 2

Outer membrane → porous, compounds can pass freely

Inner membrane → not porous, lots of transporter

Question 3

Complete the sentence:
The Elelctron transport chain transports electrons from — to — reduction potential.

Answer 3

The Elelctron transport chain transports electrons from LOW to HIGH reduction potential.

Question 4

How does complex I (NADH-Coenzyme Q Reductase) of the ETC function?

Answer 4

Composed of a hydrophobic transmembrane segment + Hydrophilic arm going in the matrix (where CAC)

1. NADH gives 2 protons to FMN (flavin) in the arm → NAD+
   NADH/NAD → FMN/FMNH2 → Fe(2+)-S/Fe(3+)-S → CoQ/CoQH2 (shuttling of protons)

*Pumps 4 net protons in the TM segment as a result of shuttling protons

Question 5

What is the role of complex II in the ETC?

Answer 5

It does NOT pump protons

Can feed electrons from succinate (SDH) into the Q pool
Contributed to membrane potential by feeding electrons to the Q pool, but does not pump protons

Structure: 3 hydrophobic subunits extend into the matrix

Succinate → FAD → Fe-S → Cyt b → CoQ

Question 6

What is the electron acceptor in the cytochromes?

Answer 6

Heme iron converts between Fe3+ and Fe2+

Question 7

Except for Complex I and II, what other pathways can reduce Coenzyme Q?

Answer 7

1) Fatty acyl CoA → Fatty enoyl CoA = Fatty acid b-oxidation
FAD → ETF (Electron transport flavoprotein)→ ETF-QO → CoQ

2) G3P → DHAP reduces FAD → CoQ

*Fuels the Ubiquinon pool

Question 8

How does complex III of the ETC function?

Answer 8

Q cycle → pumps 4 net protons

QH2 binds to Qo site → 1e- onto Cyt c, 1e- onto a new Q (gives QH)
QH2 binds Q1 site → 1e- onto Cyt c, 1e- onto a QH (gives QH2)

Balance:
- 2x QH2 are fully oxidized to Q
- 1x Q is reduced to QH2
- 2x Cyt c molecules are reduced (1e- each)

Question 9

What is the chain of electrons acceptor involved complex III of the ETC?

Answer 9

Cyt b 562 → Cyt b 566 → Fe-S → Cyt c 1

Question 10

What are the names of Q, QH, QH2?

Answer 10

Q = Ubiquinone
QH = Semi-quinone
QH2 = Ubiquinol

Question 11

What is the chain of electron acceptor in complex IV?
How many electrons are pumped in complex IV?

Answer 11

2 electron pumped

Cu2+ → Cyt a → Cyt a-Cu

Question 12

Is the electron transport chain in a linera arrangement in the mitochondria?

Answer 12

NO, it is much more closely assembled to allow better efficiency

Question 13

What is the net equation of free energy change of NADH to O2?

Answer 13

NADH + H+ + 1/2 O2 → NAD+ + H2O = -220 kJ/mol

These -220kJ/mol are the sum of the free energy released in all the smalle steps of increasing → makes much more energy than 1 big step

Question 14

What are the names of the 2 reactions allowing shuttling of cytosolic NADH to the mitochondria?

Answer 14

1. The dihydroxyacetone phosphate/glycerol-3-phosphate shuttle
2. The malate/aspartate shuttle

Question 15

How does the dihydroxyacetone phosphate/glycerol-3-phosphate shuttle work?

Answer 15

Allows shuttling cytosolic NADH to mitochondria:

1. Reduction of DHAP by NADH in the cytosol (DHAP → G3P)
   *DHAP and G3P can passe freely through the outer membrane
2. G3P binds to Glycerol-3-phosphate dehydrogenase (TM inner membrane protein) gives electrons to FAD and oxidizes G3P → DHAP
3. FADH2 → Q → Complex III
4. DHAP returns to the cytosol
   *Cycle

Question 16

How does the malate/aspartate shuttle work?

Answer 16

1. Reduction of oxaloacetate to malate (electrons coming from NADH)
2. Malate transported to the mitochondrial matrix
3. Reoxidation of Malate → Oxaolacetate which converts mitochondrial NAD → NADH2
4. Oxaloacetate converted to aspartate by transamination (coupled with Glutamate giving nitrogen → a-Ketoglutarate)
5. Asp and aKG are symported back to the cytosol
6. Asp and aKG are transaminated back in the cytosol to Oxaloacetate (to restart the cycle)

*Malate enters the Mitochondria through the Malate-Aspartate antiport

Question 17

What are the values of the P/O ratio?

Answer 17

How many ATPs are generated for every oxygen consumed
P/O ratio through Complex I, III, IV: 10/3.7 ~ 2.5
P/O ratio through Complex III, IV: 6/3.7 ~ 1.5

10 H+ pumped for every full turn (Complex I, III, IV)
8 H+ pumped back to the mitochondrial matrix for every full turn → make 3 ATP (for every ATP, add 1 H+ from H+/Pi symport) → 11H+ needed for a full turn → 11 H+/ 3 ATP made = 3.7 H+/ATP

P/O ratio of NADH = 10 H+/ 3.7 H+/ATP = ~ 2.5 ATP

Question 18

What is the structure of ATPase?

Answer 18

F0 is the TM part, formed of c subunits which all pump 1H+

F1 has many subunits that synthesize ATP from ADP + Pi

Question 19

What would be the P/O ratio of NADH if the ATPase had 20 C subunit if every full turn makes 5 ATP?

Answer 19

How many ATPs are generated for every oxygen consumed?

10 H+ pumped for every full turn (Complex I, III, IV)
20 H+ pumped back to the mitochondrial matrix for every full turn → make 5 ATP (for every ATP, add 1 H+ from H+/Pi symport) → 25H+ needed for a full turn → 25H+/ 5 ATP made = 5 H+/ATP

For every NADH that goes to the complex (every O consumes), 5 ATPs are produced

Question 20

How can the free energy generated by the ETC be calculated?

Answer 20

NADH + H+ + 1/2O2 → NAD+ + H2O = -220kJ/mol

Can calculate the -220kJ/mol based on the sum of the differences in reduction potentials + how many electrons go through

Question 21

What were the 2 theories explaining the ATP synthesis in the ETC?

Answer 21

Chemical coupling hypothesis (Eward Slater):
ATP is synthesized from a high energy intermediate of the respiraotry chain during oxidation (same as in glycolysis)

Chemi-osmotic theory (Peter Mitchell):
Energy is stored in membrane potentials and passed through coupling sites
(*Each complex is a coupling site, not yet thought of)

Question 22

What are the 6 evidences of the chemi-osmotic coupling theory?

Answer 22

1. The Resp. Chain can function in absence of phosphate → O2 can still be consumed without makign ATP
2. The # moles of ATP generated through NADH oxidation is not an integer (2.5/NADH)
3. Intact IMM is required for OXPHOS (tested with detergents)
4. Key ETC proteins are in that critical IMM
5. Uncouplers (DNP) inhibit ATP synthesis
6. Generating artificial proton gradient (pH) permits ATP synthesis without electron transport
   *Electrons generate the gradient, but if the gradient is generated artificially, they are not needed for ATP synthesis

Question 23

What experiment was done to understand how much energy Complex I alone provides to the membrane potential?
What was the found P/O ratio?

Answer 23

• Blocked Complex III (and after) with Antimycin A (electrons can’t pass through → O2)
• Electrons are accepted by Ferricyanide
  *measure how many protons where pumped
  P/O ratio = 1
  (10 H+ → 2.5, 4H+ → 1)

Question 24

What experiment was done to understand how much energy Complex IV alone provides to the membrane potential?
What was the found P/O ratio?

Answer 24

Gave electrons direclty to cyt c through: ascorbate → TMPD → cyt c of complex IV → electrons accepted by Ferricyanide
*measure how many protons where pumped
P/O ratio = 1 (but pumps 2 protons?)

Question 25

What experiment was done to understand how much energy Complex III alone provides to the membrane potential?
What was the found P/O ratio?

Answer 25

• Complex IV was inhibited with Cyanide
• Electrons entered through Succinate → FAD → CoQ → complex III
• Added exogenous cyt c into the mitochondria for electron accepting
  *measure how many protons where pumped

P/O ratio = 0.5

Question 26

What happens to the P/O ratio in presence of uncouplers?

Answer 26

It decreases → less ATP produced/more O2 consumed

Question 27

How do mitochondrial chemical uncouplers functions?

Answer 27

DNP = weak acid which can diffuse through the IMM
Takes H+ on its O-, diffuses through the IMM (down [H+] gradient) → releases H+ in the matrix → diffuses back to intermembrane space → repeats

Question 28

What can be added to inhibit complex IV?

Answer 28

Cyanide, azide and carbon monoxide inhibit complex IV by reaction with cytochrome a3 (highest reduction potential of all before giving electrons to O2)

Question 29

Name an endogenous uncoupler?

Answer 29

UCP1 → in adipose cells

Question 30

How did the mitochondrai first get integrated into proto-eukayote cells

Answer 30

Endosymbiotic relationship → aerobic bacterium entered a cell and provided energy, in exchange, the cell provided the bacteria with nutrients

Eventually, some of the bacteria’s DNA migrated to the nucleus DNA

Question 31

How many base pairs form the mtDNA?
How many ETC proteins are encoded in it?

Answer 31

Human mtDNA = 16.5 kbp
13 protein critical for ETC (out of the 2000 proteins of the ETC)

*The cell nucleus encodes for the whole transcriptional machinery of the mitochondrial DNA

Question 32

Explain respiratory control of the ETC.

Answer 32

Regulatory mechanism that controls the rate of electron transport in the respiratory chain according to need via a direct influence of the electrochemical proton gradient.
Reflects a balance between free energy change for electron pumping and free energy change for electron transport → affects directionality of ATP synthase
*tight coupling between conversion of ADP → ATP and transfer of electrons

Question 33

What is the effect of Glutamate, ADP, DNP on O2 consumption?

Answer 33

Glutamate increases O2 consumption moderately → by transmination → a-ketoglutarate → enter CAC → NADH consumed

ADP + Pi added to the mediate increases O2 consumption significantly → promotes ATP synthesis → slows down when ADP concentration goes down and ATP build up

DNP increases drastically the rate of O2 consumption (NADH oxidation) without increase the rate of synthesis of ATP (uncoupled)