Ox Phos Flashcards by sophie courville

What reactions occur in the different compartements of the mitochondrion?

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

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

Outer membrane → porous, compounds can pass freely

Inner membrane → not porous, lots of transporter

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

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

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

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

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

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What is the role of complex II in the ETC?

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

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What is the electron acceptor in the cytochromes?

Heme iron converts between Fe3+ and Fe2+

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

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

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How does complex III of the ETC function?

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)

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

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

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What are the names of Q, QH, QH2?

Q = Ubiquinone
QH = Semi-quinone
QH2 = Ubiquinol

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

2 electron pumped

Cu2+ → Cyt a → Cyt a-Cu

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

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

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

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

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

The dihydroxyacetone phosphate/glycerol-3-phosphate shuttle
The malate/aspartate shuttle

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

Allows shuttling cytosolic NADH to mitochondria:

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

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How does the malate/aspartate shuttle work?

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

*Malate enters the Mitochondria through the Malate-Aspartate antiport

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What are the values of the P/O ratio?

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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

What is the structure of ATPase?

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

F1 has many subunits that synthesize ATP from ADP + Pi

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

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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

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

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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

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

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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)

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

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The Resp. Chain can function in absence of phosphate → O2 can still be consumed without makign ATP
The # moles of ATP generated through NADH oxidation is not an integer (2.5/NADH)
Intact IMM is required for OXPHOS (tested with detergents)
Key ETC proteins are in that critical IMM
Uncouplers (DNP) inhibit ATP synthesis
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

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

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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)

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

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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?)

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

- 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

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

It decreases → less ATP produced/more O2 consumed

How do mitochondrial chemical uncouplers functions?

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

What can be added to inhibit complex IV?

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

Name an endogenous uncoupler?

UCP1 → in adipose cells

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

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

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

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

Explain respiratory control of the ETC.

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

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

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)

Wich 3 carriers are important for maintenance of the gradient on both sides of the inner mitochondrial membrane?

ATP synthase: H+ from Intermembrane space → Mitochondrial matrix Adenine Nucleotide Translocase: ATP → intermembrane space // ADP → Mitochondrial matrix (for ATP synthesis) Phosphate carrier: H+ and Pi enter → mitochondrial matrix *None of them are part of the ETC

What are the electrical charges on both sides of the Inner Mitochondrial matrix?

Inner Membrane space → +ive charge (many H+) IMM Mitochondrial matrix → -ive charge (attracts H+) *The electrical gradient is the major force driving the ETC and ATP synthase (Chemical gradient is nto as important) ADP3- tends to go towards matrix ATP 4- tends to go towards IMS

What is the structure of Adenine Nucleotide Translocase? How does it function?

1. Empty c-state faces the intermembrane space (open facing inner membrane space) 2. ADP binding c-state 3. c → m transition (cup flips facing sides) 4. ADP leaving m-state 5. empty m-state 6. ATP binding m-state 7. m → c transition 8. ATP leaving c-state

What equaton allows us to calculate the energy released from the transfer of electrons from lower to higher reduction potential?

∆G˚' = -n(F)(∆E˚') n = # of electrons F = faraday constant = 96.4kJ/V/mol ∆E˚' = (E˚' acceptor - E˚' donor) in V

What is the equation for electron transfer in complex I ?

NADH + H+ + Q → NAD+ + QH2 *Coenzyme Q receives the electrons

What is the equation for electron transfer in complex II ?

FADH2 + Q → FAD+ + QH2 *Coenzyme Q receives the electrons

What is the equation for electron transfer in complex III ?

QH2 + cytc(oxidized) → Q + cytc(reduced) Cyt c receives only 1 electron, but CoQ has 2 so one goes to recycling step

What is the equation for electron transfer in complex IV ?

cyt c(reduced) + 2H + 1/2 O2 → cyt c(oxidized) + H2O *The jumps/increase in reduction potentials increase

What is the # of ATP molecule synthesized through the ATP synthase complex?

of ATP molecules is proportional to the # of protons driving their synthesis

Ox Phos Flashcards

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