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ETC and ATP Synthesis (Electron Flow and Work
Amount of work that can be…
ETC and ATP Synthesis
Electron Flow and Work
- Amount of work that can be carried out is given by the tendency of NADH to give up electrons and oxygen to accept eletrons
- Tendency to give up or accept electron is given by the standard reduction/oxidation (redox) potential E0'
- E0’ is measured in volts: strong oxidising agents have a positive redox potential
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- ETC and oxidative phosphorylation to make ATP
- This is different from making if ATP via substrate level phosphorylation occurs in glycolysis
OILRIG
Oxidation is loss
Reduction is gain
- In ETC NADH is oxidised (loses electron and these are passed to oxygen which is reduced to water
- Substrate level ATP formation takes place in glycolysis
- Oxidative phosphorylation takes place as a product of the ETC following Krebs cycle
ETC is exergonic
- The difference between the given values reflects the work that can be obtained (E0' net)
- Amount of work is given by ΔG0’ = ‐nF E0’net
- n = number of electrons transferred
- F = a Faraday’s constant (96 kJ/mol)
- E0’net = net potential difference.
- 4 protein complexes within or on inner mitochondrial membrane
- NADH or FADH2 feed electrons into these complexes
- Electrons flow from high energy state to low energy state, eventually bind to oxygen
- Energy released is used to pump protons across the membrane
Nature of the ETC
- Found in the inner mitochondrial membrane
- Components are organised into large complexes in membrane
- Components are vectorially organised
- Complex I (NADH dehydrogenase accepts e- from NADH
- Complex II (succinate dehydrogenase) accepts e- from FADH2
- Comple III (ubiquinone-cytochrome c oxidoreductase)
- Complex IV (cytochrome c oxidase
- NON OF THESE COMPLEXES MAKES ATP
- Electrons flow through and between complexes
- Electrons shuttled between the complexes by 'mobile' components
- Electrons from Complex I and II shuttled by Coenzyme Q to Complex III
- Electrons from Complex III shuttled by Cytochrome C to complex IV
- Energy generated by electron flow (DG0') used to pump proteons across innter mitochondrial membrane - from matrix to intermembrane space against the concentration gradient
Flavin Ring
- Flavoproteins are proteins with FAD tightly bound to it
- Complex I and II are a flavoproteins
- The flavin ring accepts 2 electron and 2 protons
Iron-sulphur
- Fe/S proteins (non-haem iron proteins) have an iron ion that can be ocidised or reduced (Fe2+/Fe3+)
- The iron is bound with sulphur atoms
- Complex I, II, III have these
Quinones
- Most common is ubiquinone (Coenzyme Q)
- Mobile electron carries in membrane bilayer
- Shuttles between complex I, II, III
- Highly hydrophobic, so lipid soluble molecules
- They accept 2 electrons and 2 protons
Cytochromes
- are haem proteins with a central iron ion
- Classified based on the structure of the haem group (Types a,b,c,d)
- Undergo 1 electron reduction
- Complex II, III, cytochrome c and Complex IV
Cytochrome oxidase
- Cytochrome oxidase, this is complex IV
- Terminal oxidase has cytochrome a/a3 and 2x copper centres
- This is the last component of the chain
- Reacts with oxygen to form water
- A structually complex enzyme with many subunits
- Pumps protons across the membrane
Net Yield of ATP from oxidation of one molecule of glucose
- 1 molecule of NADH generate 2.5 ATP and 1 molecule of FADH2 generate 1.5 ATP
- Oxidation of NADH causes 10 protons to be pumped out
- Oxidation of FADH2 causes 6 protons to be pumped out
- 4 protons need to flow back in to make one ATP molecule
Therefore:
- Oxidation of NADH is 10+ out/4 H+ in = 2.5 ATP
- Oxidation of FAH2 is 6H+ out/4H+in = 1.5 ATP
ATP Production in Mitochondria
- ATPase (ATP synthase) is separate from electron transport chain
- Makes ATP: from ADP and phosphate
- (Alternatively it can use ATP and hydrolyse it to release energy)
- It is found on the inner face of the inner mitochondrial membrane as stalked particles
Chemiosmosis
- Electron transport and oxidative phosphorylation are couple by process of Chemiosmosis
- Proposed by Peter Mitchell
- Respiratory chain and ATP synthase are vectorially organised, they have a particular orientation in space
- Inner mitochondrial membrane is impermeable to H+, essential for existence of H+ gradient
- Primary energy conserving event is movement of H+ across the membrane
Generating the proton gradient
- Proton gradient is also known as the proton motive force (PMF)
- Complexes I, III, IV of electron transport chain pump proton from matrix to inter-membrane space
- As electrons are passed from NADH to oxygen
- In plants photosynthesis can create the proton gradient
- In some bacterial specific H+ pumping proteins
Using the proton gradient
- Flow of H+ down the gradient (into matrix) drive ATP synthesis
- Believed formation of ATP from ADP and Pi occurs spontaneously on ATP synthase headpiece
- so no major input of free energy
- Protein conformation changed allow release of ATP
- Protein conformation cause by rotation; this is driven by H+ gradient
Evidence for chemiosmosis
- Can detect protons as they are pumped out of the inner membrane
- If a pH gradient is created across the membrane ATP is synthesised
- Evidence for vectorial organisation of the ETC
- ATPase is on inner side of membrane, cytochrome c is on other side
- A closed compartment is necessary for ATP synthesis (to maintain the proton gradient) Ruptured mitochondria do not make ATP
- Reconstitution experiments using different biological systems
- Action of uncoupling agents
Reconstitution experiments
- Using the proton pumping protein bacteriorhodopsin found in the purple membrane of bacterium halophile Halobacterium
- Bacterirhodopsin will pump protons when activated by light
- Isolate ATP synthase from cow heart tissue
- Put the 2 proteins together in artificial membrane
- The researcher control what components present
Uncoupling agents
- Artificial substances (DNP) that reversibly bind H+ and return it back through inner membrane and into matrix of mitochondrion
- Breaks the proton gradient
Thermogenin
- Brown adipose tissue has specialised mitochondria containing thermogenin (UPC1)
- Acts as uncoupling agent; uses proton gradient and generates heat
- Important in newborn animal, hibernating animals and mammal adapted to the cold
- Similar system in some plants allows them to melt the snow covering them, enabling them to receive more light
Inhibitors of chemiosmosis
- Cyanide - bind to complex IV (cytochrome oxidase) and stops electron transport
- Rotenone - pesticide - binds to complex I and stops electron transport
- Mitochondrial diseases - reduced ATP production
- Show in energy demanding tissues such as muscle and neurons
- Cardiomyopathy, sporadic myopathy affect Complex I and II
- LHON, MELAS affect Complex I
Other uses of the proton gradients
- Central roles in many processes that involve energy
- Making ATP
- Generating heat
- Causing movement (the bacteria flagellum rotates as the gradient is used up)
- Transports ion across membranes
- Involved in photosynthesis
In aerobic condition 1 moleclue glucose makes 32 ATP
- Anaerobic 2 ATP
- Oxidative phosphorylation much more efficient
- However rate in anaerobic glycolysis can be 100x faster
- This is why some muscle fibres are specialised for anaerobic glycolysis