Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOC1009 Simona > PRACTICAL E: Oxidative phosphorylation > Flashcards

PRACTICAL E: Oxidative phosphorylation Flashcards

1
Q

What is oxidative phosphorylation?

A

The process in the mitochondria by which ADP is phosphorylated to ATP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What compound is the final acceptor of electrons?

A

Oxygen (oxygen is reduced to water)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

To what process is the phosphorylation of ADP coupled to?

A

To the flow of electrons from NADH or FADH2 through a series of electron carriers in the inner mitochondrial membrane

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What happens as electrons move down the electron transport chain?

A

H+ ions are pumped across the membrane, and a hydrogen ion gradient is set up

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

H+ ions are pumped from the _________across the _____________ to the ____________.

A

Matrix

inner membrane

intermembrane space

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Can the concentration of H+ outside the inner membrane be increased indefinitely?

A

No, the H+ ion gradient reaches limiting values after which no more H+ can be pumped out of the matrix.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What intermembrane protein carries out the synthesis of ATP, and how?

A

ATP synthase. As the protons flow back down their concentration membrane, they do so through this protein, which acts as a kind of rotary motor and phosphorylated ADP.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Can ATP be synthesised in the absence of an H+ gradient?

A

Nope

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Can ATP be synthesised if no ADP is present?

A

Nope

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Phosporylation of ADP and _______ _________ are coupled.

A

Electron transport. This means that they ought to happen together.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How many protein complexes make up the electron transport chain?

A

Four

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How many mobile electron carriers are found in the ETC?

A

Two

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

The mobile carrier that carries electrons from Complex I, Complex II and other flavoproteins to Complex III is….

A

Coenzyme Q (ubiquinone)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

The mobile carrier that carries electrons from Complex III to Complex IV is….

A

Cytochrome C

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Which complex passes electrons to oxygen?

A

Complex IV

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Which complex is not a hydrogen ion pump?

A

Complex II

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Which complex reoxidises NADH?

A

Complex I

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Which complex reoxidises FADH2?

A

Complex II

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How many H+ does Complex I pump?

A

4

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How many H+ does Complex III pump?

A

4

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How many H+ does Complex IV pump?

A

2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What’s another name for Complex II?

A

Succinate dehydrogenase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What happens when succinate is the substrate?

A

Less H+ are pumped into the intermembrane space, i.e we get less ATP for each pair of electrons from FADH2 than from a pair starting on NADH

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What’s the P:O ratio for malate?

A

2.5

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What’s the P:O ratio for succinate?

A

1.5

26
Q

Which ox states does ubiquinone cycle between?

A

CoQ and CoQH2

27
Q

Which ox states does Cytochrome C cycle between?

A

Fe3+ and Fe2+

28
Q

How can the process of ox phos be monitored?

A

By measuring the oxygen used when isolated mitochondria are given oxidisable substrate and a known amount of ADP.

29
Q

How is [oxygen] measured when monitoring the process of ox phos?

A

Using an oxygen electrode, which measures oxygen electrochemically (flow of current tells us the oxygen concentration)

30
Q

How does an oxygen electrode work?

A

A constant polarising voltage is applied acroos a cell which is sufficient to reduce O2, and the current is measured.

Oxygen is reduced at the cathode and the flow of current tells us the oxygen concentration.

31
Q

In the standard system for the oxygen electrode, what is the cathode and its half cell reaction?

A

It is a platinum electrode.

O2 + 4e- + 4H+ —-> 2 H2O

32
Q

In the standard system for the oxygen electrode, what is the anode and its half cell reaction?

A

It is a silver-silver chloride anode.

4Ag+ + 4Cl- —–> 4 AgCl + 2 H2O

33
Q

What is the overall reaction of the oxygen electrode?

A

O2 + 4 Ag + 4Cl- + 4H+ ——-> 4AgCl + 2H2O

34
Q

What is the Clarke electrode?

What features does it have?

A

It is the oxygen electrode used for biochemical measurements.

  • Anode and cathode are combined in a single assembly,
  • bathed in saturated KCl,
  • and separated from the test solution by an oxygen permeable PTFE membrane.
35
Q

In the Clarke electrode, what is the function of the magnetic stirrer?

A

It ensures that the concentration of oxygen at the electrode is the same as that in the bulk sample.

The volume of electrolyte under the membrane is very small.

36
Q

What is state 2 respiration?

A

Mitochondria with substrate and no added ADP are said to be in state 2 respiration

In this state, there is little to no oxidation of substrates/consumption of O2

37
Q

What is state 3 respiration?

A

State III respiration is defined as ADP-stimulated respiration. We put isolated mitochondria into state III when we add ADP to intact, unpoisoned mitochondria in the presence of excess substrate.

If ADP is added to the mitochondria, we can see that oxygen is used until all of the ADP is phosphorylated and oxygen consumption ceases. When ADP is added the initial rate of oxygen use is proportional to the amount of ADP added.

38
Q

What is state 4 respiration?

A

When the ADP is exhausted, the rate of oxygen use drops to zero. This is state 4 repiration.

39
Q

What assumptions where made?

A

That there is plenty of Pi and ample substrate.

40
Q

What are uncouplers?

A

In short:

Uncouplers are weak acids that transport the protons back into the matrix directly, bypassing the ATP synthetase

More detailed explanation:

Compounds that can dissolve in the mitochondrial membrane and carry hydrogen ions back across the membrane.

They short-circuit the normal controlled route through ATP-synthase: oxygen is reduced regardless of whether or not ADP is present; electrons flow down to oxygen as long as there is substrate available and oxygen present.

They break the obligatory coupling of ADP phosphorylation to electron transport.

41
Q

What are the two groups of coenzymes that react directly with substrates in the ETC?

A
  • Nicotinamide nucleotides
  • Flavin coenzymes
42
Q

In addition to nicotinamide nucleotides and flavin coenzymes, what electron carriers does the ETC contain?

A
  • Ubiquinone
  • Cytochrome C
  • Non-haem iron proteins
43
Q

Nicotinamide nucleotides, flavins and ubiquinone all undergo….

A

Oxidation / reduction reactions reactions involving transfer of both hydrogen ions and electrons

44
Q

Cytochromes and non-haem iron proteins undergo…

A

Oxidation / reduction reactions reactions involving transfer of electrons only.

45
Q

What is 2,4-dinitrophenol and what does it do?

A

It is an uncoupler.

Water soluble when deprotonated and lipid soluble when protonated, it will diffuse across the crista membrane down its concentration gradient. This means that the rate of oxidation of substrates, and utilisation of oxygen, is no longer controlled by the availability of ADP, and in the presence of an uncoupler there is rapid and more or less complete utilisation of oxygen, regardless of the amount of ADP present.

In the inter-membrane space there is an excess of protons, so dinitrophenol is protonated, dissolves in the crista membrane and diffuses across down a concentration gradient. In the crista space there is an excess of hydroxyl ions, so the dinitrophenol gives up its proton, and is trapped inside the crista. This means that dinitrophenol transports protons from the intermembrane space into the crista space, so discharging the proton gradient that would otherwise drive ATP synthesis.

46
Q

Which enzyme oxidises malate?

A

Malate dehydrogenase, which is NAD+ dependent

47
Q

Which enzyme oxidises succinate?

A

Succinate dehydrogenase, which uses FAD

48
Q

What is the P:O ratio?

A

It is the ratio between ADP phosphorylated and oxygen atoms used.

49
Q

What is oxygen saturation?

A

It is the concentration of oxygen that is established as 100%, and it is the concentration of oxygen of air saturated experimental medium at the temperature of the experiment.

50
Q

How is the oxygen electrode calibrated?

A

By noting the distance between 0% oxygen and 100% oxygen, which is a known concentration of oxygen

51
Q

Why are the state 2 and state 4 rates of oxygen consumption not zero?

A
  • Mitochondria turn over a little ATP themselves
  • Mitochondria get damaged in preparation.

This background is compensated by only seeing the oxygen used as a result of ADP added (see card on P:O ratio calculation)

52
Q

How is the P:O ratio calculated?

A

Things to remember:

  • Measure distance between 0 and 100% oxygen, which represents 1000nmol
  • P= nmol of ADP added
  • O = nmol of oxygen consumed (calculated from graph)
53
Q

What does amytal do?

A

It acts on Complex I.

In the presence of amytal there is no consumption of oxygen when malate is the substrate, and adding the uncoupler (dinitrophenol) has no effect. This means that the electron transport chain has been inhibited, so there is no proton gradient to be discharged by the dinitrophenol. Amytal has no effect on oxygen consumption when succinate is the substrate, therefore it must be acting on complex I.

54
Q

What does antimycin A do?

A

It binds to the Qi site of cytochrome c reductase, thereby inhibiting the oxidation of ubiquinol in the electron transport chain. It disrupts formation of the proton gradient, inhibiting formation of ATP.

In the presence of antimycin A there is no consumption of oxygen when either malate or succinate is the substrate, and adding the uncoupler (dinitrophenol) has no effect. This means that the electron transport chain has been inhibited, so there is no proton gradient to be discharged by the dinitrophenol. Because both substrates are affected, antimycin A must be acting on part of the electron transport chain that is common to both substrates – either complex III or complex IV. Without performing different experiments there is no way to distinguish between inhibition of complex III or complex IV.

55
Q

What does oligomycin do?

A

Acts on ATP synthase and prevents state 3 respiration.

In the presence of oligomycin there is very slow consumption of oxygen, whichever substrate (malate or succinate) is being oxidised. This suggests that the electron transport chain itself is unaffected, and addition of the uncoupler dinitrophenol confirms this. When dinitrophenol is added together with oligomycin there is rapid and more or less complete consumption of oxygen, since substrates can now be oxidised in an uncontrolled manner. Without the addition of dinitrophenol, there is only a slow leakage of protons back across the crista membrane, and as the proton gradient builds up it inhibits further electron transport. This is because oligomycin inhibits ATP synthase, and if the enzyme is inhibited, protons cannot cross the “stalk” of the enzyme back into the crista space. Remember that the whole process is controlled by the availability of ADP, or the ability to catalyse formation of ADP from ATP.

56
Q

What does KCN do?

A

It inhibits Complex IV.

In the presence of cyanide there is no consumption of oxygen when either malate or succinate is the substrate, and adding the uncoupler (dinitrophenol) has no effect. This means that the electron transport chain has been inhibited, so there is no proton gradient to be discharged by the dinitrophenol. Because both substrates are affected, cyanide must be acting on part of the electron transport chain that is common to both substrates – either complex III or complex IV. Without performing different experiments there is no way to distinguish between inhibition of complex III or complex IV.

57
Q

What does rotenone do?

A

It inhibits Complex I.

In the presence of rotenone there is no consumption of oxygen when malate is the substrate, and adding the uncoupler (dinitrophenol) has no effect. This means that the electron transport chain has been inhibited, so there is no proton gradient to be discharged by the dinitrophenol. Rotenone has no effect on oxygen consumption when succinate is the substrate, therefore it must be acting on complex I.

58
Q

How many moles of O2 are used for every mol of NADH?

A

0.5

59
Q

What is the PO ratio?

A

Ratio of ADP phosphorylated to atoms of oxygen used up.

60
Q

What happens if the stirrer is turned off in the Clarke electrode?

A

[O2] decreases in the vicinity of the electrode.

61
Q

Outline the experimental procedure in 3 steps

A
  1. Calculation of P:O ratio: two experiments were carried out with malate and differing amounts of ADP (500nmol, 1000nmol), and repeat with succinate.
  2. Investigation of the effects of DNP: two one of the experiments carried out in 1, DNP was added. This was tried with and without ADP.
  3. The effects of different respiratory poisons were investigated: amytal, rotenone, antimycin A, oligomycin and potassium cyanide. Each was tried with both substrates, with and without addition of DNP in order to draw conclusions about where each acts.

BIOC1009 Simona (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions