Week 10 Hitchcock Lecture 1 Flashcards
Briefly summarise what happens in glycolysis?
The 6-carbon sugar glucose is broken down to two molecules of triose sugar phosphate G3P, requires hydrolysis of two molecules of ATP. The two molecules of G3P then enter the second part of the pathway, where they are sequentially converted to pyruvate, generating 2 molecules of NADH and 4 ATPs by substrate level phosphorylation.
- Overall yield per glucose = 2 ATP + 2 NADH
- Does not require oxygen
Briefly summarise what happens in krebs/citric acid cycle?
The pyruvate generated by glycolysis is converted into acetyl-CoA by pyruvate dehydrogenase, which removes the carboxyl group as CO2 and in the process generates NADH. The 2-carbon acetyl-CoA feeds into the Krebs cycle where it is combined with the 4C oxaloacetate to generate the 6C citrate. Citrate is then sequentially converted back to the four-carbon oxaloacetate in a series of reactions that release 2 of the carbons as carbon dioxide and generates further NADH, ATP and FADH2. And because 2 pyruvates are generated for each glucose in glycolysis, the process happens a second time, meaning our total yield is 2 ATP equivalents, 8 NADH and 2 FADH2 molecules per mol of glucose.
What is the total output yield of both glycolysis and the krebs cycle, and what are the products used for?
generated 4 ATPs which can be hydrolyzed by the cell to power energy requiring reactions, and 10 NADH and 2 FADH2, which can be re-oxidized by the electron transport chain in order to generate a proton gradient that can be used to drive further synthesis of ATP by oxidative phosphorylation
How much ATP is produced by oxidative phosphorylation of the 10 NADH and 2 FADH2?
In oxidative phosphorylation:
Each NADH produces 3 ATP therefore 10 NADH = ~30 ATP
Each QH2 produces 2 ATP therefore 2 QH2 = ~4 ATP
Plus the 4 ATP already produced
Therefore total yield of ATP per mol of glucose oxidised = ~38-30
What does the electron transport chain do?
-What are the different components used in aerobic and anaerobic respiration?
The respiratory electron transport chain oxidises the NADH and FADH2 produced by glycolysis and the Krebs cycle to generate an electrochemical proton gradient that is used to drive ATP synthesis. The NADH and FADH2 act as electron donors. The electrons pass through the electron transport chain in a series of redox reactions where a donor is oxidised and an acceptor is reduced. Electron transfer is coupled to the translocation of protons across a membrane (mitochondrial inner membrane or inner/cytoplasmic membrane) which generates a proton motif force (PMF). The PMF is used to drive ATP synthesis as protons flow back down the gradient from the p side to the n side
-In aerobic respiration the terminal electron acceptor is oxygen, which is reduced to water. In anaerobic respiration the terminal electron acceptor is something other than oxygen
How does oxidative phosphorylation work?
the NADH we generated in glycolysis and the Krebs cycle is oxidized by complex I, and the FADH2 generated in the Krebs cycle during succinate oxidation is oxidised by complex II. The electrons liberated by NADH and succinate oxidation are used to doubly reduce quinones to quinols, and in the case of complex I the energy released is used to translocate protons from the n side of the membrane, the mitochondrial matrix, to the p side of the membrane, the inter membrane space, building up the proton gradient. These lipophilic quinols migrate through the membrane and are re-oxidised at complex III. Complex III in turn reduces this soluble electron carrier cytochrome c. The bifurcation of the two electrons from the quinol into the high and low potential chains by the proton-motif Q cycle means one quinol is generated for every two that are oxidized, amplifying the number of protons transferred per quinol oxidised, and that proton release and uptake on opposite sides of the membranes enhances the PMF. Next the reduced cytochrome c then deposits its electron at complex IV where the terminal electron acceptor, oxygen, is reduced to water, in a reaction that also moves protons into the p side of the membrane to build up the proton motif force. Finally, the movement of protons back across the membrane through the ATP synthase complex, and in the mitochondrial chain is complex number V, is used to generate ATP. The number of protons required to make an ATP varies in different ATP synthases depending on the size of the c ring.
Why is a ‘downhill’ electron transfer favoured?
Electron transfer ‘downhill’ from a donor with a negative midpoint potential to an acceptor with a positive one is thermodynamically favorable and releases energy to drive ATP synthesis
What is needed for electron transfer to be coupled with proton translocation?
ΔE (redox potential change) must exceed the proton motif force (Δp))
What is the equation for calculating the theoretical number of protons that can be translocated per electron?
H+/e-= ΔE/Δp
What is the typical gram-negative arrangement of barriers?
cytoplasm, inner membrane, periplasm and outer membrane
What are the electron donors in aerobic respiration of E. coli?
-How do they work?
NADH DH (dehydrogenase) and succimate DH.
- NADH is oxidised by NADH DH, which is a large multi subunit complex with a Flavin mononucleotide cofactor that accepts electrons from NADH and passes them though a wire of 7 FeS clusters to reduce quinone to quinol. The protons for quinone reduction are taken up from the cytoplasm and overall one proton is released during NADH oxidation thus net we use up one proton on the n side of the membrane. And the complex also pumps four protons per NADH oxidized from the cytoplasm to the periplasm, generating a proton gradient.
- Quinone reduction by the SDH complex during succinate oxidation does not directly contribute to the proton gradient as the two protons taken up for quinone reduction in the cytoplasm are replaced by the two removed from succinate oxidation, however this complex still feeds electrons into the electron transport chain to generate quinols
What are the terminal oxidases used in aerobic respiration of E. coli?
- How do Cyo and Cyd work?
- -How do they contribute to the PMF and what are they made out of?
E. coli lacks complex III/cytochrome bc1 and cytochrome c oxidase (complex IV) - instead it has two different respiratory terminal oxidases, Cyo and Cyd, which directly oxidise quinols
-Cyo
–Haem-Cu oxidase (haem b plus binuclear heme o3 - Cu centre)
–Redox loop – releases two H+ to p-side and uses 2 H+ on n-side per 2e-
–Also pumps 2H+ from n-side to p-side
4H+/2e- = 2H+/e-
–Lower affinity for oxygen – requires high aeration
the Cyd oxidase,
-Cytochrome bd oxidase - contains 3 haems (two haems b and one heme d)
–Redox loop – releases two H+ to p-side and uses 2 H+ on n-side per 2e-
–Not a proton pump
–2H+/2e- = 1H+/e-
–Higher affinity for oxygen – works at low oxygen tensions
How much PMF is generated when coupling NADH DH to Cyo?
The midpoint potential of the NAD/NADH couple being -320 mV, and oxygen is an excellent electron acceptor, with a plus 820 mV midpoint potential for the water/oxygen couple. Thus this electron transport chain is energetically very favorable as the electrons flow from NADH to oxygen, releasing plenty of energy that is coupled to proton translocation across the membrane, with the overall process allowing 8 protons to be moved into the periplasm for every 2 electrons that enter from NADH. So it costs roughly 3 protons to make an ATP by the ATP synthase so for each NADH we oxidise this way we can make about 2.7 ATPs (8 protons per NADH divided by 3)
How much PMF is generated when coupling NADH DH to Cyd?
in low oxygen conditions where the Cyd oxidase comes into play this value is lowered to three protons per electron because Cyd is not a proton pump, so less than for Cyo but we can still make a PMF for ATP synthesis by oxidative phosphorylation albeit with a lower yield of only 2 ATPs per NADH oxidised.
