Summarise extremely briefly what happens to the pyruvate produced during glycolysis in the link and Krebs.
- Pyruvate produced during glycolysis in the cell cytoplasm is actively transported across the inner and outer mitochondrial membranes to the matrix.
- Changed into acetate (2C), during link reaction.
- Acetate is then oxidised during Krebs cycle.
Describe what happens in the link reaction. (4)
- Pyruvate dehydrogenase removes hydrogen atoms from pyruvate.
- Pyruvate decarboxylase removes a carboxyl group (COOH), which eventually becomes CO2, from pyruvate. Now it is acetate.
- NAD accepts the hydrogen atoms. (2 each). These will carry hydrogen to the inner mitochondrial membrane and will be used to make ATP during oxidative phosphorylation.
- Coenzyme A accepts acetate, to become acetyl coenzyme A. The acetate is carried by CoA to the Krebs cycle.
Give the equation that summarises the link reaction.
- 2pyruvate + 2NAD + 2CoA --- 2CO2 + 2reduced NAD + 2acetyl CoA
- 2 pyruvate molecules are considered here because 2 are derived from each glucose molecule.
- Note that no ATP is produced here.
The Krebs cycle, like the ___ reaction, takes place in the ___ ____.
What is the Krebs cycle, in brief? What does it produce?
- A series of enzyme-catalysed reactions that oxidise acetate to 2 molecules of CO2.
- It also produces (for each acetate)
- 1 ATP from substrate-level phosphorylation
- 3 reduced NAD
- 1 reduced FAD [these reduced coenzymes have potential to produce more ATP in oxidative phosphorylation].
Describe the steps involved in the Krebs cycle.
- 1. Acetate from acetyl CoA joins with oxaloacetate (4C), to form citrate (6C/ also called citric acid). CoA is released and becomes available to collect more acetate.
- 2. Citrate is decarboxylated (carboxyl group becomes one CO2 later) and dehydrogenated to form a 5C compound. The pair of hydrogen is accepted by molecule of NAD, becoming reduced NAD.
- 3. 5C compound decarboxylated and dehydrogenated to form 4C compound. Another reduced NAD. Carboxyl group becomes CO2.
- 4. 4C compound changed into another 4C compound. During this, a molecule of ADP phosphorylated to produce ATP. This is substrate-level phosphorylation.
- 5. The second 4C compound is changed into a third 4C compound. Pair of hydrogen removed and form reduced FAD. Dehydrogenation.
- 6. The third 4C compound is further dehydrogenated and regenerates oxaloacetate. Another reduced NAD released. This oxaloacetate will start the Krebs cycle again.
There is __ turn of Krebs cycle for each molecule of acetate, which was made from one molecule of ____. Therefore there are __ turns of cycle for each molecule of glucose.
So, what are the products of link reaction and Krebs for each molecule of glucose.
- Reduced NAD: 2
- Reduced FAD: 0
- CO2: 2
- ATP: 0
- Reduced NAD: 6
- Reduced FAD: 2
- CO2: 4
- ATP: 2
Familiarise yourself with the krebs cycle diagram, be sure to be able to rewrite it and check with p88 on textbook!
The link reaction and the Krebs cycle are ___ preocesses, unlike ___. Although oxygen is not used in these stages, they ___ occur in the absense of oxygen.
We have looked at the example of glucose, but what other substances can be respired?
- Fatty acids are broken down to acetates and can enter Krebs via CoA.
- Amino acids can be deaminated and rest may enter Krebs cycle directly or be changed to pyruvate or acetate, depending on type of amino acid.
The inner membrane of mitochondria is impermeable to reduced NAD. How then, is the hydrogen atoms in reduced NAD from glycolysis used in the oxidative phosphorylation?
Through a shunt mechanism whereby the hydrogen atoms are moved from the reduced NAD to the matrix side of inner mitochondrial membrane, where it is attached to another NAD, reducing it.
What is the definition of oxidative phosphorylation?
The formation of ATP by adding a phosphate group to ADP, in the presence of oxygen, which is the final electron accceptor.
How is the inner membrane adapted to enable oxidative phosphorylation?
Folded membrane into cristae, increasing the surface area for electron carriers and ATP synthase enzymes. (I know we've kinda covered this already)
Outline the process of oxidative phoshorylation.
- Reduced NAD and FAD (in matrix) are reoxidised when they donate hydrogen, which are split into protons and electrons, to electron carriers in inner membrane. Protons go into solution in matrix.
- First electron carrier (protein complex I), accept electrons.
- Electrons passed along chain of electron carriers and finally donaed to molecular oxygen, which is the final electron carrier.
- Electrons flow along electron transport chain. Energy released is used by coenzymes associated with some carriers to pump protons (H+) across to intermembrane space.
- This builds up proton gradient/pHgradient/electrochemical gradient. - potential energy created
- H+ cannot diffuse through lipid part of membrane, so flow through channel, which are associated with enzyme ATP synthase. - chemiosmosis.
What happens after the phosphorylation?
- The electrons are passed from last electron carrier in chain to molecular oxygen (in matrix I think), which is the final electron carrier.
- H+ also joins so that oxygen is reduced to water.
- 4H+ + 4e- + O2 --- 2H2O
How many reduced NAD and FAD is there from the preceding processes?
- 2reduced NAD from glycolysis; 2reduced NAD from Link, 6reduced NAD and 2reduced FAD from Krebs.
- Both reduced NAD and FAD donate electrons to electron transport chain, but only red.NAD provide hydrogen ions to proton gradient (red.FAD's H+ stays in matrix but can combine with O2 later to form water).
For each molecule of reduced NAD reoxidised, theoretically, up to how many molecules of ATP can be made? So, how many ATP can be theoretically be produced for 1 glucose molecule?
- Up to 2.6 ATP's per reduced NAD.
- For 10 reduced NAD (from glycolysis, link and Krebs), 26 molecules of ATP can, theoretically, be produced.
- Add this with the 2ATP made during glycolysis and 2ATP made during Krebs (by substrate-level phosphorylation), total yeild of ATP should be 30 per glucose molecule.
However, explain why the theoretical yield of ATP per glucose is rarely, if ever, achieved. (3)
- Some protons leak across mitochondrial membrane, reducing number of protons to generate the proton motive force.
- Some ATP produced is used to actively transport pyruvate into mitochondria.
- Some ATP is used for shuttle to bring hydrogen from reduced NAD made during glycolysis in cytoplasm, into mitochondria.
What experimental evidence for the theory of chemiosmosis? Start with evidence from intact mitochondria. (2)
- pH of intermembrane space lower than matrix (more H+ in space than matrix)
- Potential difference across inner membrane was -200mV, being more negative on matrix.
Experimental evidence for chemiosmosis. With mitoplasts.
- Mitochondria isolated and placed in solution of very high water potential. Outer membrane ruptured, releasing contents of intermembrane space. If further treating with detergent these mitoplasts (mitochondria with no outer membrane),they could release contents of matrix. Enzymes identified in the different compartments of mitochondria, to work out that link reaction and Krebs cycle took place here and also enzymes for electron transport chain embedded in membrane.
- Electron transfer in mitoplasts did not produce ATP, so concluded that intermembrane space involved.
Experimental evidence. Other evidence. (4)
- ATP not made if mushroom-shaped parts of stalked particles were removed from inner membrane of intact mitochondria.
- Artificial vesicles with proton pumps and ATP synthase. ADP and Pi added, and ATP was produced. Show that a proton gradient can be used to synthesis ATP.
- ATP not made in presence of oligomycin, which block flow of protons through ion channel part of ATP synthase.
- Uncouplers are substances that destroy proton gradient across inner membrane, and when this was added, along with redNAD, ADP and Pi, no ATP was made.
What is anaerobic respiration?
The release of energy from substrates, such as glucose, in the absence of oxygen.
If oxygen is absent, the ___ ___ ___ cannot function, so __ __ and the __ __ also stop. (lack of reoxidised NAD etc). This leaves only the __ process of ___ as source of ATP. How is this ATP made?
- electron transport chain
- Krebs cycle
- link reaction
- glycolysis - made by substrate-level phosphorylation.
What is essential to keep glycolysis going during anaerobic respiration?
- The reduced NAD, generated during oxidation of glucose, has to be reoxidised so that glycolysis can keep operating.
- [Remember, glycolysis produces 2ATP, 2reduced NAD, 2pyruvate.]
What are the two pathways, in eukaryotic cells, to reoxidise NAD during temporary adverse conditions when oxygen is absent? What kind of organism does which?
- Fungi (such as yeast), use ethanol fermentation. (Plant cells such as root cells under waterlogged conditions can also use this pathway).
- Animals use lactate fermentation.
Why does anaerobic respiration produce a much lower yield of ATP than aerobic respiration?
- Because only glycolysis occurs. The electron transport chain cannot occur, as there is no oxygen to act as the final electron acceptor.
- This means that Krebs stops, as there are no NAD - all are reduced. Also prevents link from happening.
- Anaerobic respiration takes reduces the pyruvate reduced, and this frees up the NAD, so glycolysis can continue, producing 2 molecules of ATP per glucose by substrate-level phosphorylation.
Describe the anaerobic path animals take.
- Lactate fermentation
- Pyruvate accepts hydrogen from ruduced NAD, reoxidising the NAD, making it available to accept more hydrogen atoms from glucose.
- The pyruvate is reduced to lactate.
- Enzyme catalysing this is lactate dehydrogenase.
What is the fate of the lactate molecule? What causes muslce fatigue?
- Lactate builds-up in muscles.
- Taken into blood and carried to the liver.
- When more oxygen is available, the lactate can be converted back to pyruvate, which may enter Krebs via link, or it may be recycled to glucose.
- It is not the build-up of lactase itself that causes muscle fatigue, but it is specifically the reduction in pH that will reduce enzyme activity in muscles (unless kept constant by buffer).
Describe the anaerobic respiration that takes place in yeast.
- Alcoholic fermentation
- Each pyruvate is decarboxylated (CO2 removed) and becomes ethanal. (catalysed by pyruvate decarboxylase).
- Ethanal accepts hydrogen from reduced NAD, reoxidising reduced NAD, and itself becoming reduced to ethanol. (catalysed by ethanol dehydrogenase).
- The reoxidised NAD can now accept more hydrogen atoms from glucose.
Define the term respiratory substrate.
An organic substance that can be used for respiration.
What makes one type of molecule have more energy values than another? Take me through the logistics of this. What does this also mean?
- The more protons through ATP synthase, the more ATP produced.
- So, the more hydrogen atoms in a molecule of respiratory substrate, (per mol), the more ATP can be generated when it is respired.
- It also follows that if there are more hydrogen atoms per mole of respiratory substrate, then more oxygen is needed to respire it.
Give the 3 types of molecule; carbohydrate, lipid and protein, in order of most energy value.
- Lipid (39.4 kJ/g)
- Protein (17kJ/g)
- Carbohydrate (15.8kJ/g)
Why have lipids got especially high energy values?
- Because they are triglycerides, and when they are hydrolysed, glycerol and fatty acids are produced.
- Fatty acids are a long hydrocarbon chain with many hydrogen atoms. These are a source of many protons for oxidative phosphorylation so they produce lots of ATP.
Can fats and proteins be respired anaerobically? Why not?
- They can't - can only be aerobic.
- Because they cannot undergo glycolysis.