2.7 - Cell Integrity Flashcards
1
Q
Substrate level phosphorylation
A
- 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
2
Q
Oxidative phosphorylation
A
- 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)
3
Q
Mitochondrial compartments
A
- 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
4
Q
The electron transport chain
A
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
5
Q
Succinate dehydrogenase (complex II)
A
- 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
6
Q
Redox couples in the ETC
A
- 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
7
Q
Standard redox potentials
A
- 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
8
Q
Passage of electrons along the ETC
A
- 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
9
Q
ATP synthase
A
- 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
10
Q
Oxygen electrode
A
- 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
11
Q
Oxygen electrode and ETC method
A
1) prepare a sample of mitochondria
2) place in electrode chamber
3) monitor oxygen concentration for a set time
12
Q
ETC oxygen electrode graph
A
- 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.
13
Q
Metabolic poisons - cyanide
A
- 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
14
Q
Metabolic poisons - malonate
A
- 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
15
Q
Metabolic poisons - dinitrophenol
A
- 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
