8 Oxidative Phosphorylation Flashcards

1
Q

Acetyl CoA is produced from:

A

Multiple different catabolic processes

  • amino acids, monosaccharides and fatty acids all yield Acetyl CoA as a common molecule
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2
Q

Acetyl CoA enters into the _______ cycle and generates a lot of _________

In other words, the oxidation of the carbons in Acetyl CoA is associated with the reduction of _______ to _______

A

Acetyl CoA enters into the citric acid cycle and generates a lot of reduced cofactors (NADH/FADH2)
In other words, the oxidation of the carbons in Acetyl CoA is associated with the reduction of NAD+/FAD to NADH/FADH2

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3
Q

The high energy molecules (reduced cofactors NADH/FADH2) produced from the citric acid cycle can be oxidized back to NAD+/FAD via _________ and in the process, it leads to the reduction of ___ to generate H2O and leads to the formation of ______

A

The high energy molecules (reduced cofactors NADH/FADH2) produced from the citric acid cycle can be oxidized back to NAD+/FAD via Oxidative phosphorylation and in the process, it leads to the reduction of Oxygen to generate H2O and leads to the formation of ATP

  • Requires oxygen
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4
Q

What are the two purposes of Catabolic pathways?

A
  1. Breakdown of larger molecules into smaller building units
  2. Release and (temporary) storage of energy in high-energy molecules
    • ATP/NTPs
    • Reduced cofactors (NADH/FADH2)
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5
Q

Catabolic pathways are ______ (oxidative or reductive)

A

Catabolic pathways are Oxidative (oxidative or reductive)

  • Metabolites are oxidized as cofactors are reduced
  • Re-oxidation of cofactors is used to generate ATP (source of e- to drive e- transfer processes to make more ATP through Oxidative phosphorylation)
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6
Q

How are Reduced cofactors used in oxidative phosphorylation?

A

Re-oxidation of cofactors is used to generate ATP (source of electrons to drive e- transfer processes to make more ATP through Oxidative phosphorylation)

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7
Q

What are the two “separate but connected” processes of Oxidative Phosphorylation?

What links the two processes?

A
  1. Oxidation of reduced cofactors (NADH, FADH2)
    • NADH → NAD+ + H+ + 2e-
    • FADH2 → FAD + 2H+ + 2e-
    • 4H+ + 4e- + O2 → 2H2O (or 2H+ + 2e- + 1/2O2 → H2O)
      • Oxygen acts as terminal electron acceptor (reduced as cofactors are oxidized)
  2. Phosphorylation of ADP to ATP
    • ADP + Pi + H+ → ATP + H2O
  3. Processes are linked through a proton gradient across the mitochondrial membrane
    • ​​
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8
Q

Oxidative Phosphorylation consists of:

  1. Oxidation of _______ (NADH, FADH2) and reduction of ________
    • NADH → ___ + __ + __
    • FADH2 → ___ + ___ + ___
    • 4H+ + 4e- + O2 → ____ (or 2H+ + 2e- + 1/2O2 → ___)
      • Oxygen acts as a ____________
  2. Phosphorylation of ______
    • _____(reaction)_____
  3. Processes are linked through a ________ across the _______ membrane
A

Oxidative Phosphorylation consists of:

  1. Oxidation of reduced cofactors (NADH, FADH2) and reduction of molecular Oxygen (O2)
    • NADH → NAD+ + H+ + 2e-
    • FADH2 → FAD + 2H+ + 2e-
    • 4H+ + 4e- + O2 → 2H2O (or 2H+ + 2e- + 1/2O2 → H2O)
      • Oxygen acts as terminal electron acceptor (reduced as cofactors are oxidized)
  2. Phosphorylation of ADP to ATP
    • ADP + Pi + H+ → ATP + H2O
  3. Processes are linked through a proton gradient across the mitochondrial membrane
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9
Q

The proton gradient is a __________ transport process moving ______ up their concentration gradient using energy from __________

A

The proton gradient is a primary active transport process moving protons up their concentration gradient using energy from redox reactions within the electron transport chain

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10
Q

Proton gradient is a source of ______ energy

A

Proton gradient is a source of potential energy

  • converts potential energy into chemical energy in the form of a phosphoanhydride bond
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11
Q

Where does Oxidative phosphorylation occur?

A

Inside the mitochondria

  • In and Across the Inner mitochondrial membrane
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12
Q

Compare the pH on the inside of the inner mitochondrial membrane (within the matrix of the mitochondria) against the pH on the outside of the inner mitochondrial membrane (intermembrane space)

A
  • pH of intermembrane space is LOW because ↑[H+]
  • pH of matrix is HIGH because ↓[H+]
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13
Q

What are the major components of the Electron Transport Chain?

A
  • Complexes 1-4 (I-IV)
    • Integral membrane proteins
  • Coenzyme Q
    • lipid-soluble coenzyme
    • Cofactor (not a protein)
  • Cytochrome C (Cyt C)
    • Peripheral membrane protein
    • involved in transporting e- between complexes III and IV
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14
Q

What are 4 prosthetic groups (cofactors) that participate (ie are reversibly oxidized/reduced during e- transport) in Oxidative Phosphorylation.

Where would you find these prosthetic groups?

A
  1. Flavin mononucleotide
  2. Iron-sulfur clusters
  3. Copper (Cu2+)
  4. Cytochrome heme groups
  • Found in complexes I-IV as well as in Cytochrome C
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15
Q

Name the lipid-soluble cofactor that acts as a cosubstrate for complexes I, II, and III

A

Coenzyme Q

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16
Q

How does the affinity for electrons of each cofactor in the electron transport chain assist in transport?

A

Electrons move from cofactors with lower reduction potential to those with higher reduction potentials

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17
Q

What is the difference between the structures of FAD and Flavin Mononucleotide (FMN)

A

FAD/FADH2 has an Adenosine whereas FMN does not

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18
Q

What is the reduction reaction of Flavin Mononucleotide (FMN)?

How does it compare to the reduction of FAD to FADH2

A

FMN + 2H+ + 2e- ←→ FMNH2

Exactly the same as the reduction of FAD to FADH2

(Accepts 2 e- and 2H+)

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19
Q

Iron-sulfur clusters are a way of providing ______ that can be reduced

A

Iron-sulfur clusters are a way of providing iron atoms that can be reduced

Switch between 3+ and 2+ state by accepting a single e-

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20
Q

What are the components of Iron-Sulfur clusters?

A

Iron sulfur clusters contain

  • Iron
  • Sulfur
  • Cysteine (Cys) residues
    • provide sulfur atoms as part of overall structures
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21
Q

Heme is found as a _________ within a number of the complexes

  • complexes that involve the addition of Heme group, as well as cytochrome C, can undergo:
A

Heme is found as a prosthetic group within a number of the complexes

  • complexes that involve the addition of Heme group as well as cytochrome C can undergo: reversible reduction/oxidation to obtain the iron atom found in the heme groups)
    • Recall Heme in myoglobin and hemoglobin was non-covalently associated with protein structure
      • In this case, there is a covalent bond between the polypeptide chain and the prosthetic group
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22
Q

What is the most important difference between the heme in cytochrome c vs in hemoglobin/myoglobin?

A

Most important difference between Heme in cytochrome C vs in myoglobin/hemoglobin is in cytochrome C we

  • want the oxidation/reduction process to occur
  • whereas part of the role of, say, the proximal histidine in myoglobin/hemoglobin was to change the reduction potential associated with the iron atom to prevent the process of oxidation/reduction
  • Protein association allows the heme to act as a proton acceptor or donor in a redox reaction
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23
Q

Coenzyme Q is a cofactor that is very _____\_(contains a large _____\_ portion of its structure in the form of a repeating _____\_)

  • Undergoes reduction from _______ to _______ by accepting:
A

Coenzyme Q is a cofactor that is very hydrophobic (contains a large hydrophobic portion of its structure in the form of a repeating isoprene unit)

  • Undergoes reduction from ubiquinone (Q) to Ubiquinol (QH2)
    • Net conversion includes accepting 2H+ and 2e-
      • Q + 2H+ + e- ←→ QH2
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24
Q

Because Coenzyme Q is hydrophobic, how might we expect it to behave with the membrane?

A

Hydrophobic = lipid-soluble

  • readily soluble within the hydrophobic portion of the inner mitochondrial membrane
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25
Q

Coenzyme Q is involved with transport of electrons from _________ to _________

A

Coenzyme Q is involved with the transport of electrons from complexes I and II to Complex III

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26
Q

Redox reactions have a free energy change related to _______

A

Redox reactions have a free energy change related to reduction potential

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27
Q

What is Reduction Potential?

A

“affinity for electrons”

  • Higher reduction potential → more negative delta G
    • Electrons move from compounds with lower reduction potentials to those with higher reduction potentials
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28
Q

Electrons move from compounds with ____\_reduction potentials to those with ____\_ reduction potentials

A

Electrons move from compounds with lower reduction potentials to those with higher reduction potentials

Higher reduction potential = more negative delta G

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29
Q

How is the formation of the proton gradient an example of primary active transport?

A

Negative free energy associated with the increase in reduction potential from the redox reactions is used to transport protons across the membrane

  • primary active transport process connected to the redox reactions of the electron transport chain
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30
Q

Why is oxygen the terminal electron acceptor?

A

Oxygen has a very high reduction potential

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31
Q

NADH is reoxidized by the activity of complex ____

A

NADH is reduced by the activity of complex I

2 e- are passed into complex one and NADH is reoxidized to NAD+

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32
Q

Reoxidation of NADH is associated with the reduction of which component of the electron transport chain?

A

Complex I is reduced (accepts 2e-)

  • Needs to become reoxidized in order to oxidize another NADH
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33
Q

Complex I needs to be reoxidized in order to reoxidize another NADH. How is the reoxidation of Complex I accomploshed?

A

Complex I interacts with Coenzyme Q (lipid soluble cofactor)

  • Coenzyme Q is reduced
  • Complex I is (re)oxidized
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34
Q

How does the now reduced Coenzyme Q go from Complex I to Complex III?

A

Coenzyme Q is lipid-soluble = can move within the bilayer

  • Simply “migrates” to Complex III
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35
Q

Once Coenzyme Q reaches Complex III, what happens?

A

Complex III is reduced (accepts the 2 e-)

Coenzyme Q is reoxidized

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36
Q

Complex III, once reduced, passes the 2e- to ______

A

Complex III, once reduced, passes the 2e- to Cytochrome C (Cyt C)

  • Cytochrome C is reduced
  • Complex III is reoxidized
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37
Q

Cytochrome C, as a ____________ protein, is able to migrate from complex ___ on the _____ surface of the inner mitochondrial membrane to Complex ___

A

Cytochrome C, as a peripheral membrane protein, is able to migrate from complex III on the exterior surface of the inner mitochondrial membrane to Complex IV

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38
Q

Complex IV is reduced by ______ and then passes the electrons to _____ to create _____

A

Complex IV is reduced by Cytochrome C and then passes the electrons to Oxygen to create water

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39
Q

For every 2 electrons that came into the electron transport chain (as _____), how many water molecules are formed?

A

For every 2 electrons that came into the electron transport chain (as NADH), how many water molecules are formed?

  • 1 water molecule is produced at complex IV
40
Q

Complex II is part of the reoxidation of ______

A

Complex II is part of the reoxidation of FADH2 (NOT NADH)

41
Q

How many protons are moved from the matrix to the intermembrane space in conjunction with the reoxidation of the a cofactor (eg NADH) during electron transport?

A
  • 4 H+ are moved out during the passage of electrons to Complex I
  • 4 H+ are moved out in conjunction with the reaction of coenzyme Q with complex III
  • 2 H+ are moved out when electrons are passed from Cyt C to oxygen through complex IV

TOTAL: 10 protons

42
Q

For every NADH reoxidized, ____ protons are moved out of the matrix

A

For every NADH reoxidized, 10 protons are moved out of the matrix

43
Q

What leads to the H+ translocation across the inner mitochondrial membrane during the electron transport chain?

A
  • Electron transport (reduction/oxidation) causes a conformational change which allows these complexes to pump H+ ions
  • Primary active transport using redox reactions as energy
44
Q

What happens to electron transport when the proton gradient across the inner mito membrane gets too high

A

The electron transport chain will stop when the proton gradient gets large enough because the energies are such that proton translocation is no longer energetically favourable even when combined with redox reactions

  • Therefore, electron transport slows as the proton gradient increases
45
Q

Complex II is a ____________ complex and is part of the ________ cycle

A

Complex II is a succinate dehydrogenase complex and is part of the citric acid cycle

46
Q

Complex II is an _________ protein and contains _____ as a prosthetic group

A

Complex II is an integral membrane protein and contains FAD as a prosthetic group

47
Q

Complex II participates in which reaction?

A

Complex II catalyzes oxidation of succinate to fumarate as part of the citric acid cycle

48
Q

What happens to the electrons produced from the oxidation of succinate to fumarate at complex II?

A

The electrons from succinate are transferred, through an iron sulfate cluster (Fe-S), to coenzyme Q in the membrane to create QH2

(Ubiquinone to Ubiquinol)

49
Q

How many protons are moved across the membrane at Complex II?

A

None

50
Q

Which cofactor would we see involved in the oxidation of C-C single bonds to C=C double bonds?

A
  • FAD is typically associated with the oxidation of C-C to C=C
  • NAD would be associated with oxidation of C-O to C=O

Comes down to associated redox potential of the C-C and C-O processes

51
Q

The reoxidation of FADH2 at Complex II leads to the movement of ___ H+ across the membrane

A

The reoxidation of FADH2 at Complex II leads to the movement of NO H+ across the membrane

52
Q

Describe the path of electrons from FADH2 to Oxygen and include the associated proton movement.

A
  1. FADH2 is reoxidized at Complex II
    • passes 2 e- through complex II to Coenzyme Q
    • Coenzyme Q is reduced to QH2
  2. QH2 migrates through the bilayer (lipid-soluble) and is reoxidized at complex III
    • 4 protons are moved out of the matrix to the intramembrane space
  3. Electrons are passed from Complex III to Cyt C
  4. Cyt C takes electrons from Complex III to Complex IV
  5. Complex IV accepts the electrons and passes them to Oxygen to generate H2O
    • 2 protons are moved across the inner mito membrane
53
Q

For every FADH2 reoxidized, ___ protons are being moved out of the matrix

A

For every FADH2 reoxidized, 6 protons are being moved out of the matrix

54
Q

Reoxidation of which cofactor (FADH2 or NADH) at the electron transport chain will yield MORE ATP?

A
  • Reoxidation of NADH results in the movement of 10 H+ into the intermembrane space
  • Reoxidation of FADH2 results in the movement of 6 H+ into the intermembrane space
  • Those protons are used to synthesize ATP
  • Therefore fewer protons = fewer ATP synthesized
    • NADH will result in more ATP being produced
55
Q

What are the two aspects to the proton gradient?

A

Proton gradient is an Electrochemical gradient:

  • Includes a concentration aspect (↑[H+] in Intermembrane space (low pH))
  • Includes an electrical gradient (ie charge separation)
    • High H+ in Intermembrane space = more positive than in the matrix
56
Q

The proton gradient is a source of ________

A

The proton gradient is a source of potential energy

  • moving proton back into the matrix would be associated with the release of free energy = exergonic
    • Spontaneous and energetically favourable
    • energy can be harnessed
57
Q

Overall, the potential energy of the H+ gradient is converted to the chemical energy in ____________ of ATP

A

Overall, the potential energy of the H+ gradient is converted to the chemical energy in phosphoanhydride bonds of ATP

58
Q

What type of membrane protein is ATP synthase?

A

Integral membrane protein

  • Transmembrane protein
59
Q

What happens at ATP synthase?

A

Protons move from the inter-membrane space into the matrix (down electrochemical gradient)

  • 3H+ coming back through the transmembrane portion of ATP Synthase are associated with the synthesis of ATP
60
Q

How many protons moving through ATP synthase are required for the synthesis of one ATP molecule?

A

3H+

(excluding “tax” from other processes)

61
Q

If it takes the movement of 3H+ through ATP synthase to generate one ATP molecule, how many ATP molecules could be synthesized per NADH?

A

Reoxidation of NADH by the electron transport chain is associated with the movement of 10H+ into the intermembrane space

  • therefore one NADH could produce ~3 ATP (excluding tax from other processes)
62
Q

What are the two portions of the protein ATP Synthase?

A
  1. FO (“O” is for Oligomycin)
    • Transmembrane portion
    • Protons pass-through
    • Triggers conformational change in F1
  2. F1
    • Catalytic portion
    • Synthesis of ATP from ADP and Pi
  • It is the rate of ATP synthesis that determines proton movement (and ultimately oxygen consumption)
63
Q

What is the Fo portion of ATP synthase?

A
  • FO (“O” is for Oligomycin - drug that interferes with this process)
    • Transmembrane portion
    • Protons pass-through = mechanical “turbine”
    • Triggers conformational change in F1
    • transport
64
Q

What is the F1 portion of ATP synthase?

A
  • F1
    • Catalytic portion
    • Synthesis of ATP from ADP and Pi
65
Q

What determines proton movement and oxygen consumption?

A

The rate of ATP synthesis determines proton movement and (ultimately) oxygen consumption

  • Translocation of H+ is needed to provide the energy for ATP synthesis, but
  • the rate of ATP synthesis is determined by the availability of the substrates (ADP and Pi)
66
Q

If the movement of H+ across ATP synthase is responsible for providing the energy needed for ATP synthesis, why is the rate of ATP synthesis NOT determined by proton movement?

A
  • The rate of ATP synthesis determines proton movement and (ultimately) oxygen consumption
    • Translocation of H+ is needed to provide the energy for ATP synthesis, but
    • the rate of ATP synthesis is determined by the availability of the substrates (ADP and Pi)
      • NOT by the size of proton gradient
67
Q

How would an increase in the proton gradient effect the rate of ATP synthesis at ATP synthase?

A

It wouldnt.

A large H+ gradient would suppress the electron transport chain but is not responsible for determining the rate of ATP synthesis.

  • rate of ATP synthesis depends on the availability of ADP and Pi
68
Q

What is the protein in the image?

A

ATP synthase

69
Q

On what side of the membrane (Matrix or Intermembrane space) would you find the F1 portion of ATP Synthase?

A

F1 portion of ATP Synthase is on the Matrix side of the membrane

70
Q

How many active sites are in the F1 portion of ATP Synthase that make ATP?

A

3 active sites make ATP

One complete turn of the central shaft of ATP Synthase is associated with the generation of three ATP

73
Q

What happens to newly synthesized ATP (from ATP synthase)?

A

Newly synthesized ATP is exported from the mitochondrial matrix into the intermembrane space and then into the cytosol where it can be used to “drive” the many energy-requiring processes in the cell.

The ADP and Pi produced in the cytosol are then transported back into the mitochondrial matrix

74
Q

How is the ATP made in the matrix translocated into the cytosol (for use by the cell)?

A

Via the Adenine Nucleotide Translocase

75
Q

The Adenine Nucleotide Translocase transports ______ and _____.

Is this symport or antiport?

A

The Adenine Nucleotide Translocase transports ATP4- (out of the matrix into the intermembrane space) and ADP(3-). (into the matrix from the intermembrane space)

Is this symport or antiport?

Antiport

76
Q

Adenine Nucleotide Translocase is an antiport that is associated with the import of ____ and the Export of ___

A

Adenine Nucleotide Translocase is an antiport that is associated with the import of ADP and the Export of ATP

77
Q

What makes the export of ATP and the Import of ADP across the inner mitochondrial membrane via Adenine Nucleotide Translocase energetically favourable?

A

ATP carries a charge of 4-

ADP carries a charge of 3-

  • Recall that inside the matrix, we have a higher pH and a more negative environment (due to lower [H+])
  • Therefore moving ATP with a charge of 4- (more negative than ADP) towards a more positive (matrix) environment = down its voltage gradient = energetically favourable
78
Q

What transports Pi from the intermembrane space into the matrix?

A

The Pi-H+ Symport

79
Q

The import of Pi requires the associated import of ____

A

The import of Pi requires the associated import of a H+

80
Q

What happens to the ADP and Pi brought into the matrix by the Adenine Nucleotide Translocase and the Pi-H+ Symport respectively?

A

The ADP and Pi will come together at ATP synthase (the F1 portion in the matrix) to make a new molecule of ATP

81
Q

In total (taking into consideration both the needs of ATP synthase and the needs of Pi-H+ symport) how many H+ are needed for each ATP synthesized in the matrix?

A

4 H+ are required

3 H+ for ATP synthase and 1 H+ for Pi-H+ symport

82
Q

How are oxidation and phosphorylation coupled?

A

The rate of oxygen consumption is connected (coupled) to the rate of ATP synthesis

  • The rates of reoxidation of NADH, electron transport, and oxygen consumption are coupled to the rate of consumption (and synthesis) of ATP through the magnitude of the H+ electrochemical gradient
83
Q

If ATP synthase is not working to make ATP what results?

A
  • H+ accumulate in the inter-membrane space
  • Becomes energetically prohibitive to move more protons out of the matrix into the inter-membrane space
  • Electron Transport Chain Stops
    • Oxygen consumption stops
    • NADH/FADH2 reoxidation stops
84
Q

What is the P:O Ratio?

A

The amount of ATP made (P) per oxygen atom reduced in water (O)

  • 1 water is made for each NADH or FADH2 reoxidized (each 2e-)

P:O ratio may vary with uncoupling

85
Q

What are the P:O ratios for NADH reoxidized and FADH2 reoxidized?

A
  • P:O ratio is ~2.5/NADH reoxidized
  • P:O ratio is ~1.5/FADH2 reoxidized
86
Q

Consider Coupling in Oxidative Phosphorylation and follow what would happen if we have HIGH ENERGY USE

A

High Energy Use (lots of ATP used)

↑[ADP], [Pi]

↑ ATP Synthase Activity (increased synthesis of ATP; because more ADP and Pi available; recall, rate of ATP synthase is dependent on available ADP and Pi)

↓H+ Gradient (because more protons being used by ATP synthase)

↑ e- transport (to replenish proton gradient) === initial coupling event: increased ATP = Increase O2 consumption

[NADH] and [FADH2] decrease === Activation of CAC, PDH

CAC = Cyclic Acid Cycle

PDH = Pyruvate Dehydrogenase

87
Q

How is ATP synthesis coupled to O2 consumption in oxidation phosphorylation?

A

Increased ATP synthesis = Increased O2 consumption

Increased ATP synthesis

Decrease H+ gradient

Increased e- transport

Increased O2 consumption

88
Q

Consider Coupling in Oxidative Phosphorylation and follow what would happen if we have LOW ENERGY USE

A

Low Energy Use (not using much ATP)

↓[ADP], [Pi]

↓ ATP Synthase Activity (decreased synthesis of ATP; because less ADP and Pi available; recall, rate of ATP synthase is dependent on available ADP and Pi)

↑H+ Gradient (because fewer protons being used by ATP synthase)

↓ e- transport (large proton gradient) === coupling event: decreased ATP = decreased O2 consumption

[NADH] and [FADH2] increase === Inhibition of CAC, PDH

CAC = Cyclic Acid Cycle

PDH = Pyruvate Dehydrogenase

89
Q

Analyze the graph in the image

(substrate refers to something that is making reduced cofactors Citric acid cycle)

A
  • Initially we have a Coupled Environment
    • Oxygen is not consumed because there is no ATP synthesis
  • Oxygen consumption increases ([O2] decreases) when we add ADP (because ADP stimulates ATP synthesis)
  • Once we run out of ADP, proton transport associated with ATP synthase stops = e- transport stops = oxygen consumption stops
90
Q

What is uncoupling?

A
  • Something creates a hole in the inner mitochondrial membrane
  • Protons can enter the matrix without ATP synthesis (ie without going through ATP synthase)
91
Q

What happens, energetically, when protons move across the inner mitochondrial membrane without the use of ATP synthase?

A

The energy produced from the movement of H+ down its electrochemical gradient is converted into kinetic energy (heat) (instead of into chemical energy in the phosphoanhydride bond in ATP)

92
Q

Why might mammals want to uncouple oxidative phosphorylation?

A

To intentionally generate heat

93
Q

What happens to oxygen consumption in the presence of an uncoupling protein?

A

Oxygen consumption increases in the presence of an uncoupler whether or not we are making ATP

  • electron transport occurs without ATP synthesis
  • Proton gradient is dissipated faster = Increased rate of electron transport = increased O2 consumption
    • decreases rate of reoxidation of reduced cofactors and increases rate of citric acid cycle
94
Q

What is 2,4-dinitrophenol?

A

DNP

A compound that acts as an uncoupler by binding to H+ on the matrix side, diffusing across the membrane and releasing the H+ in the cytosol

  • used as a “diet pill”
95
Q

Analyze the graph

A
  • The graph shows that with the addition of 2,4-DNT (Dinitrophenol) oxygen is consumed even without ATP synthesis
  • DNT uncouples the system