2.7 - Cell Integrity

Question 1

Substrate level phosphorylation

Answer 1

• the production of ATP by the direct transfer of a high-energy phosphate group from an intermediate substrate to ADP
• in contrast to oxidative phosphorylation, where ATP is produced using energy derived from the transfer of electrons in an electron transport system

Question 2

Oxidative phosphorylation

Answer 2

• ATP is produced using energy derived from the transfer of electrons in an electron transport system
• within the mitochondria, the reduced coenzymes NADH and FADH2 are re-oxidised by molecular oxygen:
• NADH + H+ + 1/2 O2 –> NAD+ + H2O (delta G -220 kJ/mol)
• FADH2 + 1/2 O2 –> FAD + H2O (delta G -167 kJ/mol)
• delta G for ATP hydrolysis is -31 kJ/mol
• energy released from re-oxidation of cofactors can generate several phosphoanhydride bonds = make ATP from ADP
• common causes of failure of OxPhos - lack of oxygen e.g. hypoxia (diminished), anoxia (total)

Question 3

Mitochondrial compartments

Answer 3

• oxidative phosphorylation takes place in the inner membrane (vs Krebs cycle in matrix)
• numerous folds within the cristae increase the surface area for oxidative phosphorylation to take place

Question 4

The electron transport chain

Answer 4

Membrane proteins:

• complex I (NADH dehydrogenase)
• complex II (succinate dehydrogenase)
• complex III (Q-cytochrome C oxidoreductase)
• complex IV (cytochrome c oxidase)
  (complexes I, III & IV accept electrons, and in doing so protons from the aqueous solution. As electrons pass through each complex, protons are pumped into the intermembrane space)

Mobile carriers:

• co-enzyme Q (ubiquinone)
• cytochrome C

Question 5

Succinate dehydrogenase (complex II)

Answer 5

• enzyme of TCA cycle that sits in the inner membrane
• uses FAD as a cofactor and communicates directly with coenzyme Q (ubiquinone)
• as electrons pass from FADH2 –> coenzyme Q, it also picks up a proton pair, regenerating FAD and forming QH2
• electrons from FADH2 bypass complex I - directly into complex II
• since complex I is bypassed, fewer protons are pumped into the intermembrane space when FADH2 is re-oxidised to FAD compared with NADH
• therefore, less ATP is made from the reoxidation of FADH2

Question 6

Redox couples in the ETC

Answer 6

• redox couples undergo electron transfers involving a reduced substrate (oxidised) which donates electrons to an oxidant (reduced)
• substrate can exist in both reduced and oxidised forms
• NAD+ / NADH
• FAD / FADH2
• Fe3+ / Fe2+
• 1/2 O2 / H2O
• the ability of a redox couple to accept or donate electrons is known as the reduction / redox potential

Question 7

Standard redox potentials

Answer 7

• negative E0 implies the redox couple has a tendency to donate electrons = more reducing power than hydrogen
• positive E0 implies the redox couple has a tendency to accept electrons = more oxidising power than hydrogen

Question 8

Passage of electrons along the ETC

Answer 8

• transfer of electrons from one complex to another is energetically favourable
• as electrons progress along the ETC, they lose energy - this energy is used to pump protons into the intermembrane space

Question 9

ATP synthase

Answer 9

• multimeric enzyme with two parts:
• F0 membrane bound (a, b and c subunits)
• F1 projecting into the matrix space (alpha, beta and gamma subunits)
• rotates to drive transition states, with altering affinities for ADP and ATP –> conformational energy flows from the catalytic subunit into ADP + Pi to make ATP
• protons in matrix drives rotational movement of the F1 part of ATP synthase
• a,b,g subunits of F1 have different affinities for ATP, ADP and Pi
• direction of proton flow determines whether it generates ATP (ATP synthesis) or consumes it (ATP hydrolysis)
• conformational energy is converted into chemical energy in the phosphoanhydride bonds of ATP

Question 10

Oxygen electrode

Answer 10

• measures oxygen concentration in solution, allowing OxPhos to be measured (by oxygen consumption)
• base is formed by an oxygen permeable Teflon membrane
• underneath is two electrodes - platinum cathode and silver anode
• oxygen diffuses through the membrane and is reduced to water at the cathode
• O2 + 4H+ + 4e- –> 2H2O (platinum cathode)
• 4Ag + 4CL- –> AgCl + 4e- (silver anode)
• resulting current is proportional to the oxygen concentration
• circuit completed by silver anode - oxidised to AgCl due to KCl electrolyte

Question 11

Oxygen electrode and ETC method

Answer 11

1) prepare a sample of mitochondria
2) place in electrode chamber
3) monitor oxygen concentration for a set time

Question 12

ETC oxygen electrode graph

Answer 12

• basal respiration - initially, gradual decrease in [O2] as it is consumed by the mitochondria - absence of additives
• ADP added - sudden burst in O2 consumption. If quantity of ADP added is known, ADP:O2 index can be calculated to measure the efficiency of the mitochondrial phosphorylation system
• once all ADP consumed, the mitochondria return to basal respiration rate and O2 continues to decrease until it is all used up
• respiratory control - O2 uptake by mitochondria is controlled by ADP + Pi, to match the consumption to energy requirements.

Question 13

Metabolic poisons - cyanide

Answer 13

• metabolic poisons interfere with the flow of electrons down ETC / protons through ATP synthase, and interrupt ATP synthesis
• cyanide (CN-) and azide (N3-) bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex (IV)
• blocks the flow of electrons through the respiratory chain = stops production of ATP

Question 14

Metabolic poisons - malonate

Answer 14

• closely resembles succinate so acts as a competitive inhibitor of succinate dehydrogenase (complex II)
• succinate dehydrogenase is located in the inner mitochondrial membrane and passes electrons directly to ubiquinone via FAD
• malonate slows down the flow of electrons from succinate to ubiquinone by inhibiting oxidation of succinate to form fumarate

Question 15

Metabolic poisons - dinitrophenol

Answer 15

• proton ionophore which can shuttle protons across the inner membranes
• weight loss drug by transporting protons across the mitochondrial membrane, bypassing ATP synthase
• decouples ATP production from proton pumping
• increases metabolic rate and body temperature (due to more H+ pumped), increasing the fuel metabolised to make ATP

Question 16

Non-shivering thermogenesis

Answer 16

• UCP-1 is activated in response to a drop in core body temperature
• UCP-1 is a channel that can transport H+ from intermembrane space into matrix, bypassing ATP synthase so ATP is not produced
• ATP synthase is bypassed and much of the energy within the H+ gradient is dissipated as heat
• regulated uncoupling of OxPhos seen in newborn babies and hibernating animals in non-shivering thermogenesis

Question 17

Metabolic poisons - rotenone

Answer 17

• isoflavone found in roots and seeds of some plants
• inhibits transfer of electrons from complex I to coenzyme Q

Question 18

Metabolic poisons - oligomycin

Answer 18

• antibiotic produced by Streptomyces
• inhibits OxPhos by binding to the stalk of ATP synthase and blocking the flow of protons through the enzyme