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06 FdSc Bioscience - Metabolism and Enzymology > Week 3 - TCA cycle and oxidative phosphorylation > Flashcards

Week 3 - TCA cycle and oxidative phosphorylation Flashcards

1
Q

Give a brief introduction / overview to the TCA cycle

A
  • Under aerobic conditions pyruvate is converted to acetyl-coenzyme A and is oxidised to CO2 in the TCA cycle (aka Krebs cycle and citric acid cycle)
  • Electrons stored in the form of NADH or FADH2 (reduced enzymes) are passed through the electron transport chain to molecular oxygen
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2
Q

What is oxidative decarboxylation and why is it required in the TCA cycle?

Overview oxidative decarboxylation

A
  • Pyruvate, producted from glycolysis, is a source of acetyl-CoA for the TCA cycle
  • For pyruvate to enter the TCA cycle it must enter the mitochondria and oxidative decarboxylation of pyruvate -> acetyl-CoA is the connecting link between glyoclysis and the TCA cycle
  • Reaction is catalysed by pyruvate dehydrogenase which is a multienzyme complex which is an assembly of three different enzymes
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3
Q

Describe the first reaction of the TCA cycle

A
  • The first reaction in the TCA cycle is the citrate synthase reaction
    • This involves the formation of citrate from oxaloacetate and acetyl-CoA
  • This involves nucleophillic attack by acetyl-CoA and thioester hydrolysis
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4
Q

Describe the second reaction of the TCA cycle

A
  • The 2nd reaction is the conversion of citrate to isocitrate
  • Reaction is catalysed by aconitase
  • This is an isomerisation reaction and is a two-step process
  1. Dehydration reaction occurs which involves the removal of a water molecule from citrate to produce aconitate
  2. Rehydration of a water molecule in a different position to produce isocitrate
  • Conversion of 3o alcohol to 2o alcohol making subsequent reaction in the TCA cycle an easier process
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5
Q

Why does the isomerisation of citrate to isocitrate make subsequent reaction in the TCA cycle easier?

A
  • Oxidation of isocitrate involves the cleavage of a C-H bond which requires less energy than the breakage of a C-C bond directly from oxidizing citrate
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6
Q

Describe the third reaction of the TCA cycle

A
  • Oxidative decarboxylation of isocitrate to α-ketoglutarate is catalysed by isocitrate dehydrogenase and is linked to the reduction of NAD+ to NADH
  • This is the 1st oxidation reaction of the TCA cycle, the production of NADH is the first link between the TCA cycle and oxidative phosphorylation and the electron transport pathway
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7
Q

Why and how is the third step of the TCA cycle regulated?

A
  • This reaction is a link between two metabolic pathways therefore isocitrate dehydrogenase is a regulated reaction
  • Both NADH and ATP are allosteric inhibitors of isocitrate dehydrogenase
  • ADP is an allosteric activator
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8
Q

Describe the fourth reaction of the TCA cycle

A
  • The α-ketoglutarate dehydrogenase reaction is the 2nd oxidative decarboxylation reaction of the TCA cycle
  • α-ketoglutarate dehydrogenase is a multi-enzyme complex (similarly to pyruvate dehydrogenase)
  • Reaction produces NADH and a thioester product called succinyl CoA and CO2
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9
Q

Describe the fifth reaction of the TCA cycle

A
  • Succinyl-CoA is converted to succinate catalysed by succinyl-CoA synthetase
  • This is an example of substrate-level phosphorylation which means that the substrate provides the energy for phosphorylation
  • This reaction is coupled to the production of GTP which in turn can yield ATP
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10
Q

Describe the sixth reaction of the TCA cycle

A
  • Succinate is oxidised to fumarate
  • Catalysed by succinate dehydrogenase which is a membrane-bound enzyme associated with the inner mitochondrial membrane
  • This reaction leads to the production of FADH2 from FAD
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11
Q

Describe the seventh reaction of the TCA cycle

A
  • The trans-hydration of fumarate with water yields L-malate
  • Catalysed by fumarase
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12
Q

Describe the eighth reaction of the TCA cycle

A
  • The conversion of L-malate to oxaloacetate, catalysed by malate dehydrogenase, is the last step of the TCA cycle
  • Oxidation of L-malate is coupled with the reduction of NAD+ to NADH
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13
Q

Draw a diagram of the entire TCA cycle

A
  • See image below
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14
Q

Give a word summary of the TCA cycle

A
  • TCA cycle results in the production of 2 molecules of CO2 , 1 ATP, 3 NADH and 1 FADH2 per cycle (x2 for a whole glucose molecule)
  • Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O -> 2 CO2 + 3 NADH + 2 H+ + FADH2 + GTP + CoASH
  • The γ phosphoryl group of GTP can be transferred to ADP to produce ATP catalysed by nucleoside diphosphokinase
  • Per glucose molecule (two turns of TCA cycle) the net reaction for both glycolysis and TCA is shown below:
    • Glucose + 2 H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi -> 6 CO2 + 10 NADH + 10 H+ + 2 FADH2 + 4 ATP
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15
Q

How is the TCA cycle regulated?

A
  • [Acetyl-CoA], [ATP], [NAD+] and [NADH] are the main regulatory signals of the TCA cycle which regulate pyruvate dehydrogenase, citrate synthase, isocitrate dehyrogenase & α-ketoglutarate dehydrogenase
  • All of the above enzymes are inhibited by NADH meaning when [NADH] high, which is subsequently oxidised to ATP, the TCA cycle halts
  • ATP inhibits pyruvate dehydrogenase and isocitrate dehydrogenase
  • TCA cycle stimulated when either ratio ADP/ATP or NAD+/NADH is high since this signals cell had low amount of ATP and NADH
    *
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16
Q

What is the difference in the production of ATP from glycolysis and the TCA cycle vs ATP produced as a results of NADH and FADH2?

A
  • ATP produces in glycolysis and the TCA cycle is a results of substrate-level phosphorylation
  • NADH-dependent ATP synthesis is a result of oxidative phosphorylation
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17
Q

What is the terminal electron acceptor in oxidative phosphorylation?

A
  • Terminal electron acceptor is molecular oxygen
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18
Q

Describe the different state of the components of the electron transport chain

A
  • Each constituent of the electron transport chain normally exist in two oxidation states and each consituent is successively reduced and re-oxidised as electrons pass through the chain from NADH FADH2 to the terminal electron acceptor
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19
Q

What is established as a result of electron transport?

What is the significance of this?

A
  • A proton gradient is established across the innre mitochondrial membrane
  • This proton gradient drives ATP synthesis
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20
Q

What happens to the co-enzymes as a result of the electron transport?

A
  • The e- transport chain reoxidises NADH and FADH2
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21
Q

What occurs as a result of the electron transport chain?

A
  • As the coenzymes are reoxidised, the electon transport chain channels the free energy obtained from the oxidation of food material into the synthesis of ATP
22
Q

What happens to the electrons from NADH and FADH2?

A
  • The electrons move from the coenzymes to oxygen which is reduced to water
23
Q

What are the different molecular species found in the electron transport chain?

A
  • Flavoproteins: contains bound FMV (flavin mononucleotide) and FAD prosthetic groups
  • Coenzyme Q : ubiquinone
  • Cytochromes such as: cytochrome C (a molile electron carrier)
  • Iron-sulfur proteins
  • Protein-bound copper
24
Q

How can the electron transport chain be broken down and why is it categorised in this way?

A
  • Broken down into four parts because there are four distinct proteins complexes in the electron transport chain, all of the complexes are membrane bound
  1. NADH-coenzyme Q reductase
  2. Succinate-coenzyme Q reductase
  3. Coenzyme Q-cytochrome C reductase
  4. Cytochrome C oxidase
25
Q

Describe complex I (NADH-coenzyme Q reductase)

A
  • Several polypeptide chains, one flavin mononucleotide (FMN) and several Fe-S clusters
26
Q

What is the function of complex I in the electron transport chain?

A
  • Complex I is a link between glycolysis, the TCA cycle and the rest of the electron transport chain by accepting electrons from NADH
27
Q

What is the first step of complex I in the electorn transport chain?

A
  • 1st step of this complex involves binding NADH and the transfer of electrons from NADH to FMN
    • NADH + [FMN] + H+-> [FMNH2] + NAD+
28
Q

Describe the second and third step of complex I of the ETC

A
  • 2nd step is the transfer of electrons from FMNH2 to a series of Fe-S clusters
  • 3rd and final step is the transfer of 2 electrons from Fe-S proteins to coenzyme Q (UQ) which serves as a mobile electron carrier
29
Q

Outline the difference between NADH and Fe-S ?

A
  • NADH is carries and transfers two electrons whereas Fe-S proteins are one electron donors
30
Q

What is the critical characteristic of coenzyme Q and what is the significance of this?

A
  • Coenzyme Q is highly hydrophobic allowing it to freely permeate through the hydrophobic core of the inner mitochondrial membrane
31
Q

Besides the oxidation of NADH and the reduction of coenzyme Q, what else happens at this stage simultaneously?

A
  • Complex I simultaneously stimulates the transport of protons from the mitochrondrial matrix to the intermembrane space
  • The stoichiometry for this reaction is 4 H+ transported for every tow e- passed from NADH to Coenzyme Q
    • QH2 + 2 H+ + 2 e –> UQH2
32
Q

Describe the structure of complex II

A
  • Two Fe-S clusters and FAD bound to histidine residue, it is an integral protein of the inner mitochondrial membrane
33
Q

Descibe the function of complex II in the ETC and the associated reactions

A
  • When succinate is converted to fumarate it is coupled with the production of FADH2 (catalysed by succinate dehydrogenase)
  • FADH2 transfers its electrons to the Fe-S clusters in complex II which are then transferred to coenzyme Q
    • Succinate -> fumarate + 2H+ + 2e-
    • QH2​ + 2H+ + 2e- -> UQH2
  • No protons are transferred across the inner mit membrane membrane
  • Link between the TCA cycle and the ETC
34
Q

How many molecules of ATP are produce by the oxidation of FADH2 vs NADH?

A
  • 1 molecule of FADH2 generates 2 ATP whereas NADH produces 3 ATP
35
Q

Describe the transfer of electrons in complex III ( coenzyme Q- cytochrome C reductase)

A
  • reduced coenzyme Q transfers its electrons to cytochrome C via coenzyem Q-cytochrome c reductase through a mechanism known as the Q cycle
  • Complex II inolves three cytochromes, one Fe-S protein and a haem c
    • Fe atom at the centre of porphyrin ring cycles between Fe2+ and oxidises Fe3+
36
Q

In addition to the transfer of electron in complex III what else happens

A
  • when copmplex III transports electrons through the Q cycle it is accompanies by proton transport across the inner mitochrondiral membrane
  • 2 protons are taken up by Complex III on the matrix side and four protons are released into the intermembrane space for each pair of electrons that passes through the Q cycle
37
Q

What happens once electrons are transferred through Complex III?

A
  • Transfer of electrons through Complex III are passed from cytochrome c1 to cytochrome c
  • Cytochrome c is classed as an electron carrier because it is watre soluble and loosely associated with the inner mitochondrial membrane
  • Once cytochrome c receives electrons from cyctochrome c1 in complex III it migrates along the mitochondrial membrane in a reduced state carrying electrons to complex IV
38
Q

Describe complex IV (cytochrome c oxidase)

A
  • Complex IV transfers electrons from cytochrome c to oxygen which is reduced to form water
  • 4 cyt c (Fe2+) + 4 H+ + O2 - - - – - - - -> 4 cyt c (Fe3+) + 2 H2O
  • Cytochrome c binds on the cytosolic side of Complex IV and the electrons are transferred through copper (CuA / CuA& CuB) and haem centres to reduce oxygen on the matrix side of theinner mitochrondrial membrane
  • e- transfer is accompanied with transport of 2H+ into intermembrane space
39
Q

What is the significance of proton transfer by the ETC?

A
  • Transfer of electrons through the ETC drives H+ out of the mitochrondrial matrix and into the intermembrane space which creates and electrochemical gradient
40
Q

What is an alternative name for ATP synthase and what is its role?

A
  • F1F0-Synthase is a molecular motor complex responsible for carrying out ATP synthesis
41
Q

Describe the strucutre of ATP synthase

A
  • F1 unit consists of 5 subunits denoted α, β, γ, δ and ε
  • F0 unit consists of three hydrophobic subunits denoted a, b and c
  • Catalytic sites for ATP synthesis are situated on the β subunits of the F1 complex
  • F0 forms the transmembrane pore that protons flow through and stimultes the rotor to turn
    • Flow of protons through this channel drives the conformational changes in α and β subunits of the F1 complex that synthesis ATP
42
Q

Describe the binding-change mechanism for ATP synthesis

A
  • Paul Boyer proposed a binding change mechanism for ATP synthesis, this model predicts that the F1 unit has three distinct active sites in the β subunits:
    1. Open (O) conformation is inactive with low affinity for ligands
    2. Loose (L) conformation is also inactive
    3. Tight (T) conformation is active with high affinity for ligands
  • It works as follows
  1. ATP synthesis is initiated by binding of ADP and Pi to the L site
  2. Conformational change convers the L site to T conformation and also convers T to O and O to L
  3. Final step is synthesis of ATP at the T site which is released from the O site
43
Q

Describe ATP-ADP translocase

A
  • ADP and ATP are both highly charged molecules meaning they do not readily cross biological membranes
  • ADP-ATP translocase is a transport system
  • Exit of ATP from mitochondria is strictly coupled to the entry of ADP by the ADP-ATP translocase
  • Coupling assures that ATP, celluar energy, can transport cellular energy throughout the cell and ADP enter the mitochondria for reporocessing
44
Q

Give a brief overview of shuttle systems and what they are for

A
  • The inner mitochondrial membrane is impermeable to NADH
  • Shuttle system harvests cytosolic NADH for delivery to the mitochrondrial matrix
    • This is because glycolysis generates NADH which occurs in the cytosol
  • Failure to reoxidise NADH to NAD+ would cause glycolysis to halt due to NADH limitation
45
Q

What is the function and composition of the glycerophosphate shuttle?

A
  • Made of two glycerophosphate dehydrogenases, one on the cytosolic face of the inner mitochondrial membrane and one in the cytosol
  • they work together to transport electrons into the mitochondrial matrix
46
Q

Describe how the glycerophosphate shuttle works

A
  1. NADH, produced in cytosol, transfers its electrons to dihydroxyacetone phosphate which reduces it to glycerol-3-phosphate
  2. Glycerol-3-phosphate is reoxidised by a mitochondrial membrane, which is FAD dependant, to regenerate dihydroxylacetone phosphate and enzyme bound FADH2
  3. The 2 e- of FADH2 are transferred to coenzyme Q
  • Essentially shuttle links the oxidation of cytosolic NADH with mitochrondrial reduction of FAD
  • As e- are transported to FAD the shuttle will only yield 1.5 ATP
  • This is an irreversible pathway ensuring that TCA cycle operates effectively even in depleting NADH in the cytosol
47
Q

Describe how the malate-aspartate shuttle works

A
  • Malate-aspartate shuttle works across the inner mitochondrial membrane
  1. Oxaloacetate is reduced in the cytosol to malate via malate dehydrogenase
  2. malate is transported across mitochondrial membrane through the α-ketoglutarate-malate carrier
  3. In matrix malate is reoxidised to oxaloacetate, by malate dehydrogenase, which is coupled to NADH synthesis
    1. Meaning NADH readily enters ETC
  4. Oxaloacetate cannot pass the membrane readily so it is transaminated to aspartate
  5. Aspartate travels across mitochondrial memrbae via aspartate-glutamate carrier
  6. Aspartate is converted to oxaloacetate in cytosol
  7. Shuttle generates 2.5 ATP per NADH
48
Q

What is the difference between the α-ketoglutarate-malate carrier and the aspartate-glutamate carrier?

A
  • Unlike the α-ketoglutarate-malate carrier, the aspartate-glutamate carrier is reversible
49
Q

What is the net production of ATP combined from glycolysis, the TCA cycle, the ETC and oxidative phosphorylation?

A
  • Net production of ATP from glycolysis, TCA cycle, ETC and oxidative phosphorylation is approx. 30-32 ATP per glucose molecule
50
Q

What is the equation for the oxidation of glucose using the glycerophosphate shuttle?

A
  • Glucose + 6 O2 + ~ 30 ADP + ~ 30 Pi ——-> 6 CO2 + ~ 30 ATP + ~ 36 H2O

06 FdSc Bioscience - Metabolism and Enzymology (8 decks)

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