Electron Transport System and Oxidative Phosphorylation Flashcards
1
Q
Principle of oxidative phosphorylation
A
- NADH and FADH2 contain energy obtained from catabolism - get reoxidised by transferring electrons to components of Electron Transport System (ETS)
- Energy is released as electrons are transferred along ETS
- This energy is used to convert ADP + Pi to ATP i.e. ATP synthesis.
2
Q
E0
A
- measure of tendency of a molecule in solution to give or take electrons, to or from an electrode under standard conditions relative to a standard hydrogen electrode
3
Q
How is free energy released from electron transport?
A
- ETS is effectively composed of a series of redox pairs - each has its own redox potential E0
- Tendency to donate electrons to other compounds = reducing agent
- Tendency to accept electrons from other compounds = oxidising agent
4
Q
Oxidation and reduction
A
- oxidation: lose electrons
- reduction: gain electrons
- OILRIG; Oxidation Is Loss, Reduction Is Gain
5
Q
Redox potentials and free energy
A
- In ETS, electrons are passed from one redox pair to the next – eventually to O2
- acceptor must have a more positive redox potential than the donor
6
Q
Nicotinamide Adenine Dinucleotide (NAD+)
A
- first electron carrier involved in the oxidation of many metabolites
- NAD+ is oxidised form, NADH is reduced form
7
Q
Inner mitochondrial membrane
A
- where enzymes of the ETS and ATP synthase are found
8
Q
Inner membrane cristae
A
- increase membrane surface area and its impermeability allows the establishment of chemical gradients
9
Q
Electron Transport System (ETS)
A
- function is to regenerate NAD+ and FAD, with free energy released used to generate ATP, and with the reduction of oxygen to water
- consists of 4 large protein complexes: I to IV, and 2 small, mobile carriers
- each complex contains various cofactors/coenzymes which are required for the transfer of electrons from one complex to the next
10
Q
Carriers of ETS
A
- flavoproteins (with FMN or FAD)
- non-haem iron (iron sulphur complex)
- quinone
- cytochromes (with haem groups)
11
Q
Flavin adenine dinucleotide (FAD)
A
- transfers electrons as hydrogen atoms
- When combined to appropriate proteins, FAD can accept two H atoms and is reduced to FADH2
- FAD is a prosthetic group = it is covalently attached to the apoenzyme
- Isoalloxazine ring-ribitol-phosphate-phosphate-ribose-adenine
12
Q
Flavin Mononucleotide (FMN)
A
- Prosthetic group – similar to FAD but without AMP
- No positive charge; isoalloxazine ring structure
- Accepts two protons and two electrons
- reduced to FMNH2 in two steps: H+ + FMN –> FMNH and H+ + FMNH –> FMNH2
13
Q
Non-haem iron
A
- Non-haem bound iron found in iron-sulphur clusters
- 4 types – classed on number of atoms of each present: [Fe-S], [2Fe-2S], [3Fe-4S], [4Fe-4S]
- Bound to protein via 4 cysteines.
14
Q
Cytochromes
A
- Electron-transporting proteins with haem as prosthetic group
- iron in haem is a single-electron carrier
- ETS contains several different cytochromes
- cytochromes b, c1, c, a and a3
15
Q
Cytochrome b
A
- transmembrane protein composed of two subunits - cytochrome bL and bH
16
Q
Cytochrome c1
A
- Found associated with non-haem iron and located in outer layer of phospholipid bilayer
17
Q
Cytochrome c
A
- Peripheral protein on cytosolic surface of inner membrane.
- Bound to cytochrome c1 and cytochrome oxidase - acts as ‘electron shuttle’ between them.
18
Q
Cytochrome a and a3
A
- Transmembrane protein composed of two distinct haem groups.
- Also has copper associated – CuA and CuB
19
Q
Ubiquinone (Coenzyme Q - Q10)
A
- called because of ubiquitous “expression” and its 10 isoprenoid residue hydrophobic tail
- non-protein lipid soluble molecule – freely mobile in the phospholipid bilayer
- receive 2 protons and two electrons - quinone is reduced to hydroquinone
- link between Complex I or II and Complex III
- move protons from the matrix to inter-membrane space
20
Q
Complex I
A
- oxidize NADH and reduce CoQ
- Large membrane spanning complex, contains binding sites for NADH and CoQ
- Contains 6-7 Fe-S clusters through which e- are carried in a zig-zag pattern and transferred to CoQ
- Net movement of protons occurs – provide energy for ATP synthesis
21
Q
Complex II
A
- oxidize succinate via the generation of FADH2
- not enough energy to pump H+ across the membrane
- also not a membrane spanning complex, like complex I, so cannot act as a proton pump
- Contains binding sites for succinate and Coenzyme A
- FADH2 then reduces Coenzyme Q
22
Q
Complex III
A
- oxidize CoQ and reduce Cytochrome c
- similar to Complex I - large membrane spanning complex that contains binding sites for CoQ and Cytochrome c
23
Q
Q cycle
A
- Transfer of 2e- from CoQ to cytochrome c1
- Composed of 2 loops
- Results in pumping of 4H+ across the membrane
24
Q
Complex IV
A
- oxidize cytochrome c and reduce oxygen to water
- large membrane spanning complex, contains binding sites for cytochrome c and molecular oxygen
- bound copper facilitates the collection of the 4 electrons needed to reduce O2
25
Mitochondrial shuttles
- During Glycolysis and the TCA Cycle reducing equivalents in the form of NADH are produced
- essential that NADH is reconverted to NAD+ to allow continuation of metabolism
26
NADH to NAD+ conversion in anaerobic conditions
- converting pyruvate to lactate
27
NADH to NAD+ conversion in aerobic conditions
- NADH generated in the mitochondria is converted back to NAD+ via the ETS
- NADH generated in the cytosol cannot pass directly into the mitochondria to be reconverted to NAD+ by the ETS and is regenerated through the mitochondrial shuttles
28
Glycerol-3-phosphate shuttle
- major shuttle in most tissues
- enzyme in cytosol transfers e- from NADH to dihydroxyacetone phosphate
- enzyme is glycerol-3-phosphate dehydrogenase
- flavoprotein dehydrogenase transfers e- to ET chain
29
Aspartate-malate shuttle
- NADH produced in glycolysis is used to reduce oxaloacetate into malate
- malate moves into the intermembrane space, then enters the matrix via an antiporter transport system in exchange for an alpha-ketoglutarate
- malate is then oxidized back into oxaloacetate and the pair of electrons are collected by NAD+ to form NADH
- oxaloacetate cannot move across inner mitochondrial membrane and converted into aspartate
- aspartate can now flow out of inner membrane via an antiporter system in exchange for glutamate
- glutamate that moves into the matrix transfers an amino group onto oxaloacetate to form aspartate and a-ketoglutarate
- aspartate transported into the cytoplasm is deaminated to form oxaloacetate
30
Link between ETS and ATP synthesis
- ETS facilitates regeneration of NAD+ and FAD via transfer of electrons to O2
- Mitchell’s Chemiosmotic Theory explains the link between the ETS and oxidative phosphorylation (OxP)
- Two steps: generation of an electrochemical gradient, synthesis of ATP by ATP synthase
31
Assumptions of Chemiosmotic Mechanism
- inner mitochondrial membrane is impermeable to protons
- Protons can only cross inner membrane via carriers of the ET chain or ATP synthase
- Carriers of ET chain are vectorially arranged so protons move matrix to inter-membrane space
- ATP synthase is also vectorially arranged
32
Electrochemical Potential Gradient
- electrons are transferred from one redox pair to the next – release of energy
- 3 membrane spanning complexes – I, III and IV, use the energy released to pump H+ across the inner membrane
- H+ can't re-enter the matrix as the inner membrane is impermeable – H+ gradient
- have movement of charge - electrochemical potential (ECP)
- ECP and H+ gradient generate a force that wishes to re-establish an equilibrium – Proton Motive Force
33
ATP Synthase
- According to Mitchell’s Chemiosmotic Theory, the energy of the Proton Motive Force is used by ATP synthase to generate ATP.
- re-entry of H+ into the matrix is facilitated by the F0 subunit of ATP synthase which forms a pore through the inner membrane
- “discharges” the pH and electrical gradients across the membrane, with the energy released being used by the F1 subunit of ATP synthase to drive ATP synthesis from ADP + Pi
34
ETC and ATP synthase
- ETC complexes are associated into supercomplexes
| - ATP synthase forms dimers or oligomers (associations of dimers) – in yeast, mammals, plants and fungi
35
Uncoupling of oxidative phosphorylation
- Uncoupling occurs when H+ re-enter mitochondrial matrix without going through ATP synthase i.e. the electrochemical gradient is dissipated without the generation of ATP
- Can be brought about by: chemicals and physiologically by uncoupling proteins
- Results in increased oxygen consumption and the release of energy as heat
36
Chemical uncouplers
- aka proton ionophores – lipid soluble and dissociable H+
| - Examples: 2,4-Dinitrophenol, Valinomycin, Gramicidin A
37
2,4-Dinitrophenol
- crosses inner mitochondrial membrane (IMM) and releases protons in matrix – reduces the proton gradient
38
Valinomycin
- antibiotic that makes the IMM permeable to K+ and dissipates the membrane potential (no effect on proton gradient)
39
Gramicidin A
- a channel forming drug – makes the IMM permeable to protons and reduces the proton gradient
40
Physiological uncouplers
- involves uncoupling proteins (UCPs)
- UCPs, 5 isoforms – form channels through the inner membrane which conduct H+ back into the matrix
- UCPs increase the amount of energy from oxidation released as heat, when activated
41
Types of uncoupling proteins
- UCP1: (thermogenin) expressed in brown adipose tissue.
- UCP2: expressed in most cells.
- UCP3: expressed principally in skeletal muscle.
- UCP4 & 5: expressed in the brain
42
Inhibition of ETS
- common end result is reduction or inhibition of electron flow, proton pumping and reduction or cessation of ATP synthesis
- Absence of oxygen, anoxia will also result in inhibition of the ETS – no terminal acceptor
- Iron deficiency, anaemia, result in inhibition of the ETS – no iron for cytochromes or Fe-S
43
Examples of ETS inhibitors
- Rotenone, Amytal, Piericidin A (Complex I)
- Thenoyltrifluoroacetone (Complex II)
- Antimycin (Complex III)
- Cyanide, Azide, Hydrogen sulphide, Carbon monoxide (Complex IV)
44
Oligomycin (ATP synthesis inhibitor)
- site of action: F0 ATPase
| - effect: Inhibition of H+ channelling, no ATP generation, failure to dissipate H+ gradient, shut down of ETC
45
Inhibitors of ATP/ADP transport
- Atractyloside, Bongkrekic acid
- site of action: ANT
- effect: Deletion of intramitochondria ADP, inhibition of ATPase, failure to dissipate H+ gradient shut down of ETC
46
Uncouplers
- 2,4-DNP, Valinomycin, Gramicidin A
- site of action: IM
- effect: Dissipate H+ gradient, no ATP generation
47
Aerobic breakdown of glucose (equation)
- Glucose + 6O2 + 30ADP + 30Pi --> 6CO2 + 6H2O + 30ATP
48
Anaerobic breakdown of glucose (equation)
- Glucose + 2ADP + 2Pi --> 2 lactate + 2ATP
49
Diseases associated with mitochondrial DNA (mt DNA)
- Mutation rate of mt genome is 10x that of nuclear genome
- Defects of OxP likely to arise from mutations in mt genome
- Mitochondria are maternally inherited and thus are associated disorders– effect both sexes
- Mitochondria replicate by fission during cell division – variation in amounts of normal and mutant mt DNA within cell – Heterplasmy
- Disease severity/pathology worsens with age due accumulation of mutations – inability of mt DNA to be repaired
50
Leber Hereditary Optic Neuropathy (LHON)
- Cause: mutation in NADH dehydrogenase (Complex I)
- Late onset loss of bilateral vision due to neuroretinal degeneration – optic nerve
- High energy demand, depends almost entirely on oxidative phosphorylation
- Affected mother will pass LHON to all offspring
