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Molecules to Cells - Practicals > Oxidative Phosphorylation > Flashcards

Oxidative Phosphorylation Flashcards

1
Q

Name two oxidative processes ?

A
  1. Catabolism of glucose to pyruvate in glycolysis

2. Breakdown of pyruvate to CO2 and water in citric acid cycle

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2
Q

Where are the enzymes from carbohydrate oxidation transferred to and what do they form?

A
  1. NAD+ and FAD

2. NADH and FADH2

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3
Q

What are fatty acids and amino acids catabolised by and what do they use ?

A
  1. Oxidative pathways

2. NAD+ and FAD

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4
Q

What does the continued availability of NAD+ and FAD as electron acceptors depend on ?

A

The reoxidation of NADH and FADH2 by 02

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5
Q

What is oxidative phosphorylation ?

A

The process by which the free energy derived from glucose and other substrates and stored in NADH and FADH2 is used to generate ATP from ADP and an inorganic phosphate

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6
Q

Where is the major site of oxidative phosphorylation in mammalian cells ?

A

The mitochondria

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7
Q

How does the transfer of electrons from reduced coenzymes to O2 occur ?

A

By a stepwise flow of electrons through a chain of intermediate electron carriers

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8
Q

Where is the stepwise flow of intermediate electron carriers situated ?

A

The mitochondrial inner membrane

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9
Q

What do the electrons pass along ?

A

The electron transport chain to the cytochrome oxidase

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10
Q

What is the cytochrome oxidase also known as ?

A

Complex IV

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11
Q

What is the final electron acceptor ?

A

O2

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12
Q

What happens at the final electron accepter ?

A

O2 is reduced to water

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13
Q

What happens to protons as electrons pass along the electron transport chain ?

A

Protons are translocated from the mitochondrial matrix across the inner membrane into the intermembrane space

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14
Q

What does the translocation of protons set up ?

A

A proton electrochemical gradient across the mitochondrial inner membrane

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15
Q

What does delta p stand for ?

A

Proton electrochemical gradient

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16
Q

What drives the synthesis of ATP from ADP and an inorganic phosphate ?

A

The flow of protons back across the membrane through ATP synthase

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17
Q

What is ATP synthase also known as ?

A

Complex V

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18
Q

What is ATP synthesis by mitochondria coupled to and by what ?

A
  1. Electron transport

2. Electrochemical gradient

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19
Q

What is ATP synthesis driven by ?

A

The free energy derived from the oxidation of substrates

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20
Q

In mammals, what is the majority of the oxygen that we consume a result of ?

A

ATP synthesis

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21
Q

What is the ATP produced used to do ?

A
  1. Maintain ion gradients
  2. Transport substrates and proteins across membranes
  3. Protein synthesis
  4. DNA synthesis
  5. Muscle contraction
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22
Q

What can electron flow and the resulting reduction of oxygen by suspensions of isolated mitochondria be demonstrated in a laboratory by ?

A

An oxygen electrode

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23
Q

What does an oxygen electrode consist of ?

A

A silver/silver chloride reference half cell joined to a platinum/O2 cathode by a saturated KCl bridge

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24
Q

What is the electrode compartment isolated from ?

A

The reaction chamber

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25
Q

What is a reaction chamber which is isolated from the electrode compartment ?

A

Where the mitochondria and their substrates or inhibitors interact

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26
Q

What isolated the electrode compartment from the reaction chamber ?

A

A thin teflon membrane

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27
Q

What is a teflon membrane ?

A

A membrane which is permeable to oxygen and allows oxygen to reach the cathode

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28
Q

What happens once oxygen reaches the cathode ?

A

It is electrochemically reduced

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29
Q

What does the reduction of oxygen at the cathode allow ?

A

A current to flow

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30
Q

What is mitochondrial electron transport monitored by ?

A

Measuring the rate of oxygen consumption

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31
Q

What is an example of an oxygen donor/oxidisable substrate ?

A

Succinate

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32
Q

What does stirring result in ?

A

Ensures the mixture is fully oxygenated

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33
Q

What does the blank rate represent ?

A

Respiration and ATP synthesis using substrates and ADP that were present on the mitochondria when they were isolated

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34
Q

At what point in the electron transport chain do electrons from succinate enter ?

A

Complex II

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35
Q

What happens upon addition of succinate and why ?

A

The rate of oxygen uptake increases as electrons from the added succinate move down the electron transport chain to O2

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36
Q

What happens after ADP is added to the reaction chamber and why ?

A

A sudden burst of oxygen uptake as ADP is converted into ATP

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37
Q

What is the actively respiring state called ?

A

State 3 respiration

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38
Q

Why does the trace curve upwards after about 2.5 minutes ?

A

The rate of oxygen consumption is slowing down as oxygen becomes limiting

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39
Q

What is electron transport limited by ?

A

ATP synthesis

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40
Q

What is electron transport coupled to ?

A

ATP synthesis

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41
Q

What can electron transport also be known as ?

A

Respiration

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42
Q

What is the respiratory control ratio ?

A

State 3 rate : State 4 rate

43
Q

What does the respiratory control ratio indicate ?

A

The tightness of the coupling between respiration and phosphorylation

44
Q

Why might the coupling not be perfect ?

A

Mechanical damage to mitochondria during the isolation procedure

45
Q

What does the further addition of ADP restore the rate to ?

A

State 3

46
Q

Why is state 4 respiration usually faster than the original rate before the first addition of ADP (state 2)?

A

Some ATP is broken down by ATPase activities contaminating the preparation and the resulting ADP is then re-phosphorylated by the intact mitochondria

47
Q

Why does oxygen consumption decrease when ADP becomes limiting?

A

the flow of protons through ATP synthase cannot occur because movement of protons through the complex into the matrix is obligatorily coupled to the synthesis of ATP

48
Q

What happens to the proton electrochemical gradient in the absence of ADP ?

A

The gradient quickly reaches and is maintained at maximum value

49
Q

What happens if the proton electrochemical gradient is already at a maximal value ?

A

The system is fully charged and further proton pumping cannot occur

50
Q

What happens if the proton electrochemical gradient is at a maximum value and further proton pumping cannot occur ?

A

Proton electrochemical gradient applies an energetic back pressure preventing further proton pumping by the proton transport chain

51
Q

What are chemical uncouplers ?

A

Molecules that can ferry protons from the inter membrane space across the mitochondrial inner membrane and into the matrix

52
Q

What does an uncoupler facilitate ?

A

Protons moving back into the matrix without going through ATP synthase

53
Q

What happens when protons are pumped without any back pressure from the gradient, ie protons are pumped out and returned via an uncoupler ?

A

Electron flow through the electron transport chain increases but the free energy available is not linked to production of ATP but is lost as heat

54
Q

What is an example of an uncoupler ?

A

2,4-DNP

55
Q

What happens upon the addition of 2,4-DNP and why ?

A

Respiration increases as there is no longer a limiting supply of ADP and state 4 is not achieved

56
Q

What is work done on transporting electrons being lost as ?

A

Heat

57
Q

What is malate ?

A

A source of NADH

58
Q

What is rotenone ?

A

A poison/inhibitor

59
Q

When does rotenone block electron transport ?

A

When NADH is the source of electrons but not when succinate is the source of electrons

60
Q

Where do electrons from succinate enter the electron transport chain ?

A

Complex 2

61
Q

What does rotenone block ?

A

Electron transport between complex I and complex II.

62
Q

Which electrons can by pass the rotenone block ?

A

Electrons from succinate

63
Q

Where does antimycin block and why ?

A

Complex III, succinate cannot pass the antimycin block therefore it must be beyond complex II

64
Q

What is bongkrekic acid ?

A

An inhibitor of the translocator

65
Q

What does atractyloside do ?

A

Limits ADP in matrix

66
Q

What does oligomycin do ?

A

Binds to the F0 and inhibits F1F0ATPase- blocks proton channel

67
Q

What is calcium uptake driven by ?

A

Membrane potential

68
Q

What happens when calcium ions are added ?

A

Rate of oxygen consumption increases

69
Q

Why does the rate of oxygen consumption increase on addition of calcium ?

A

Positive ions are taken uptake via the calcium ion uniporter driven by difference in membrane potential

70
Q

What is ruthenium red ?

A

An inhibitor of the uniporter

71
Q

What is the malate-aspartate shuttle ?

A

How NADH is transferred across the mitochondrial inner membrane by the shuttle involving glutamate and malate

72
Q

What do redox centers contain ?

A

Metal ions such as iron, copper and manganese

73
Q

How many cytochromes are there in the respiratory chain of eukaryotes ?

A

4

74
Q

What are the cytochromes involved in the respiratory chain of eukaryotes ?

A
  1. aa3
  2. b
  3. c
  4. c1
75
Q

Is cytochrome C part of a complex ?

A

No

76
Q

What is cytochrome C since its not in a complex ?

A

Mobile

77
Q

What are some ubiquinones ?

A
  1. Coenzyme Q10
78
Q

What is an ubiquinone ?

A

A lipophilic mobile redox center

79
Q

How many complexes is in the electron transport chain ?

A

4

80
Q

What is a respirasome ?

A

A super complex

81
Q

What enters complex I ?

A

Electrons from NADH

82
Q

How do electrons get from complex I to complex III ?

A

Via a coenzyme Q

83
Q

How do electrons move from complex III to complex IV ?

A

Via cytochrome C

84
Q

What happens to electrons at complex IV ?

A

The electrons are transferred to oxygen which is reduced to water

85
Q

Where do electrons from succinate enter the chain ?

A

Complex II

86
Q

What do the electrons from the oxidation of succinate, which enter the chain at complex II do ?

A

Feed the electrons into complex III via CoQ

87
Q

What is another name for complex I ?

A

NADH-ubiquinone oxidoreductase

88
Q

What does complex I contain ?

A

Multiple electron carriers, flavin mononucleotide plus iron sulphur centers

89
Q

What is the immediate electron acceptor from NADH in complex I

A

Flavin mononucleotide

90
Q

What inhibits electron transfer in complex i ?

A

Rotenone

91
Q

What is another name for complex II ?

A

Succinate-ubiquinone oxidoreductase

92
Q

What is the inhibitor in complex II ?

A

Malonate - inhibits succinate oxidation

93
Q

What inhibits electron transfer in complex III ?

A

Antimycin A

94
Q

What is another name for complex IV ?

A

Cytochrome c oxidase

95
Q

What inhibits electron transfer to O2 ?

A

Cyanide, CO and azide

96
Q

How many electrons does complex IV accept ?

A

4

97
Q

What is another name for ATP synthase ?

A

F1F0-ATPase

98
Q

What is ATP synthase ?

A

Oligomer

99
Q

What is the function of ATP synthase ?

A

ATP synthesis

100
Q

What inhibits ATP synthase?

A

Oligomycin

101
Q

What always generates a proton electrochemical gradient ?

A

Metabolic reactions that energise the membrane

102
Q

What does energy coupling require ?

A

A topologically closed structure

103
Q

What do uncouplers allow protons to do ?

A

To move down the electrochemical gradient, across the inner membrane back into the matrix without going through the ATP synthase

104
Q

What are uncouplers ?

A

Lipophilic weak acids

Molecules to Cells - Practicals (5 decks)

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