CBI 8: Krebs Cycle and Oxidative Phosphorylation Flashcards
What is the second main stage of cellular aerobic respiration?
- the Krebs cycle
- also known as the tricarboxylic acid cycle or TCA cycle
Where do oxidative decarboxylation and the TCA cycle take place?
- in the mitochondrial matrix
- hence pyruvate needs to be transported via the mitochondrial membranes using a special carrier protein
Describe the outer membrane of the mitochondrion
- has a lipid bilayer
- similar lipid and protein content to other cell membranes
- has protein carriers in the outer membrane that allow small molecules in, such as:
- ions
- nucleotides
- metabolites
Describe the mitochondrial matrix
- contains most of the proteins and the mitochondrial DNA
- TCA cycle takes place here
Describe the inner membrane of the mitochondrion
- the membrane is extensively folded and compartmentalised
- contains the membrane proteins involved in the electron transport chain and oxidative phosphorylation
Briefly describe what oxidative decarboxylation is and give its overall reaction equation
- in order for TCA cycle to begin, the metabolite (e.g. pyruvate) needs to be converted to acetyl-CoA
- oxidative decarboxylation links the end of glycolysis and the start of the Krebs cycle
What enzyme catalyses oxidative decarboxylation?
- pyruvate dehydrogenase complex
- a very large and complex enzyme
- uses different co-factors to allow reactions to happen at multiple sites
Describe the steps of oxidative decarboxylation
Note: this is simplified and in reality, each step is much more complex!
- also the final decarboxylation steps that completely convert glucose to CO2 occurs in the TCA cycle
1. Decarboxylation: - pyruvate is decarboxylated, so CO2 is produced
2. Oxidation: - 2 electrons released, taken up by NAD+ and 2H+
3. Transfer to CoA: - acetyl-CoA formed as a thioester bond forms
How many molecules of pyruvate undergo oxidised decarboxylation per molecule of glucose?
- two pyruvate molecules
Briefly describe the hydrolysis of the thioester bond between the CoA and the acetyl group (in acetyl CoA)
- hydrolysing the acetyl group is associated with a large, negative change in the Gibbs free energy
What is the overall reaction of the TCA/Krebs cycle?
Give a brief overview of the Krebs cycle
- acetyl-CoA enters the Krebs cycle
- the C2 fragment gets converted to CO2
- in parallel, electrons and protons are transferred to NAD+ and FAD
- produces one molecule of ATP of GTP (depending on cell type)
Describe the Krebs Cycle
- Condensation reaction of C2-fragment, acetyl-CoA, with C4-fragment, oxaloacetate
- forms citrate
- reaction is driven by the breakage of high energy thioester bond in acetyl-CoA
- catalysed by enzyme citrate synthase - Citrate is isomerised to isocitrate
- catalysed by aconitase
- first, a dehydration reaction occurs
- then a hydration step
- this results in an interchange of hydrogen with hydroxide - First oxidative decarboxylation
- isocitrate is oxidised, transferring two electrons to NAD+ forming NADH and carbon dioxide is released
- first of the four oxidation-reduction reactions in the Krebs cycle
- catalysed by isocitrate dehydrogenase
- alpha-ketoglutarate is formed - Second oxidative decarboxylation forming succinate-CoA
- release of one molecule of CO2
- formation of one molecule of NADH
- catalysed by alpha-ketoglutarate dehydrogenase complex - Substrate-level phosphorylation
- the thioester bond in succinyl-CoA is broken to provide energy for substrate-level phosphorylation
- one molecule of ATP or GTP is formed
- there are two isoforms of the enzyme succinyl-CoA-synthetase, specific for ATP or GTP
- ADP is for higher energy, GDP lower (e.g. liver) - Oxidation of succinate to form fumarate
- produced FADH2
- FAD is covalently bound to the enzyme, succinate dehydrogenase
- it is the only enzyme in the Krebs cycle embedded in the inner mitochondrial membrane (and also called complex II) - Hydration of fumarate
- water is added
- generates a new stereocenter
- the enzyme, fumarase, is stereospecific and only produces L-malate - Oxidation to form oxaloacetate
- catalysed by malate dehydrogenase
- a third molecule of NADH is formed
- has a highly positive change in Gibbs energy and would normally be unfavourable
- but due to constant use of products, oxaloacetate and NADH, in the Krebs cycle and electron transport chain, the reaction equilibrium is driven towards the products
Video:
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Describe the chemical structure of NAD and NADH
- consists of:
- adenine
- two ribose sugars
- a diphosphate group
- a nicotinamide group
Describe the chemical structure of FAD and FADH
- an ADP unit that is connected via a sugar moiety to an isoalloxazine ring
- the ring is the active electron carrier part and can accept two electrons and protons
How many carbon dioxide molecules is yielded for each glucose molecule?
- four molecules of carbon dioxide
- two from one Kreb cycle and there are two Kreb cycles per glucose molecule
Briefly summarise what happens to the electrons from the Krebs cycle
- electrons released from the Krebs cycle formed NADH and FADH2
- these electrons are used to reduced oxygen to water
- a highly exergonic reaction, with deltaG at -220.1kJ/mol
- NADH + 1/2 O2 + H+ → H2O + NAD+
- the energy of this reaction is released stepwise as the electrons flow through four protein complexes that are embedded in the inner mitochondrial membrane
- the inner mitochondrial membrane is not permeable to ions and other small molecules
- the energy released by transferring electrons through those membrane proteins powers the transport of protons across the inner mitochondrial membrane
- thus, a proton gradient is generated, which powers the formation of ATP from ADP and inorganic phosphate
- this whole process can be called oxidative phosphorylation
What are the enzyme complexes involved in the electron transport chain?
- Complex I: NADH-Q oxidoreductase
- Complex II: Succinate-Q reductase
- Complex III: Q-cytochrome c oxidoreductase
- Complex IV: Cytochrome c oxidase
What are the two electron carriers that faciliate flow between enzyme complexes in the electron transport chain?
- ubiquinone (coenzyme Q)
- cytochrome c
Describe ubiquinone (coenzyme Q)
- electron carrier
- a hydrophobic molecule that is located in the inner mitochondrial membrane
- shuttles electrons between complex I/II and III
- can be reduced by two electrons to QH2
Describe cytochrome c
- an electron carrier
- can only carry one electron at the time
- a small water-soluble protein with a heme centre atom, carrying an iron
- transfers electrons from complex III to IV
Describe the electron transport chain
1.
- NADH binds to complex I and is oxidised to NAD+ and H+
- the two electrons are then transported via internal metal clusters to ubiquinone
- it takes up two protons from the matric to get reduced to QH2
- this electron transfer is the driving force for complex I to transfer 4H+ from the matrix to the intermembrane space
2.
- Complex II catalyses the reaction of succinate to fumarate
- this leads to the reduction of FAD
- it then transfers its two electrons and protons to the other part of this enzyme complex
- from there, electrons and protons are taken up by ubiquinone to form QH2
3.
- Now the electrons from the Kreb cycle have been transferred to ubiquinone
- ubiquinone gives its 2 electrons to complex III
- this electron transfer leads to 4 protons released into the intermembrane space, while two protons are taken up from the matrix (this is the Q cycle)
- the electrons then get transferred to cytochrome c
- since cytochrome c can only carry one electron at a time, two cytochrome c molecules are needed
4.
- Cytochrome c shuttles the electrons to complex IV
- this needs to happen four times
- complex IV catalyses the reduction of molecular oxygen to water by taking up four protons from the matrix side
- four protons are also pumped across the inner mitochondrial membrane
What are the two ways a proton gradient is generated during the electron transport chain?
- by directly pumping protons from the matrix to the intermembrane space
- by using protons from the matrix side for reactions that take place inside the complexes
What is the proton-motive force?
- generated by protons that are pumped across the mitochondrial membrane
- it is energy created by a chemical gradient due to the difference in the concentration of protons on each side and a charge gradient due to the positive charge of these protons
Describe ATP synthase structure
ATP synthase is a complex structure
- consists of two regions:
- F0: the membrane-spanning region
- has a c ring that rotates in the membrane
- has the subunit a that contains two proton channels (one is open to intermembrane space, the other the matrix)
- F1: the catalytic unit
- has a hexameric ring built from three alpha and three beta subunits
- the two parts are connected by a central stalk (gamma ) and by an exterior column
Describe how the ATP synthase F0 subunit works and why this happens
- there is a glutamate residue right in the middle of the column-like c subunits
- the glutamate contains a carboxylic acid group and can either be protonated with no charge or deprotonated, carrying a negative charge
- glutamic acid will only be protonated at low pH, as we find in the matrix due to the proton gradient generated by the electron transport chain
- also, a negative charge within a hydrophobic environment like inside the lipid membrane will be very unfavourable
- thus for the c unit to enter the inner membrane, the charge will need to be neutralised
- the half channel of the a-unit that is open to the intermembrane space will easily transport protons inside the membrane
- due to the low pH, they can protonate glutamic acid on the c unit
- the gradient drives the c unit to rotate counter-clockwise inside the membrane, until the c unit reaches the other half channel of the a unit which is open to the matrix side
- due to the high pH on the matrix side, the glutamic acid will release the proton and can be loaded with another proton from the intermembrane side again
- this leads to the rotation of the c unit of ATP synthase, acting like a motor
Describe how the ATP synthase F1 subunit works
- the gamma unit directly connects to the c ring
- the unit is not a straight stalk but has a distinct shape which leads to conformational changes within the beta subunit (this is the catalytic subunit)
- there are three conformational stages in the beta subunit: loose, tight and open
- the loose state (L), allows ADP and phosphate to bind to the catalytic side
- the tight state (T), has a conformation that supports the formation of ATP from the substrate by tightly binding the product ATP
- the open state (O), amino acids on the active site are rearranged so that ATP can leave the enzyme
- all three conformation exist at the same time due to the structure of the gamma unit
- when the c-ring rotates, the gamma unit rotates 120 degrees in a counter-clockwise direction (from the top)
- this changes the conformational stages of all three beta subunits
- loose becomes tight, catalysing the formation of ATP
- tight becomes open: releasing ATP
- open becomes loose: ready to bind ADP and inorganic phosphate
- another rotation again causing the conformational change, closing the catalytic cycle of the subunits
- thus the role of the proton gradient is not to drive the reaction but to release ATP from the synthase
What are the net products of glycolysis?
- 2 ATP
- 2NADH
What are the net products of oxidative decarboxylation?
- 2 NADH
What are the net products of the Krebs cycle?
- 2 ATP (via GTP)
- 6 NADH
- 2 FADH2
Explain how much ATP is made from FADH2 and NADH
- it is estimated for the number of protons pumped per electron pair donated to the ETC (i.e. per NADH or FADH2 oxidised) are that 10 protons are pumped per NADH and 6 protons are pumped per FADH2
- evidence suggests that 4 protons are required for ATP synthesis and transport
- this includes the transit of 3 protons per ATP synthesised by ATP synthase and 1 proton used in the antiport of ATP and ADP across the mitochondrial membrane
- therefore, we can calculate that each NADH in the mitochondrial matric will yield a net of 2.5 ATP and each FADH2 will yield a net of 1.5 ATP
How do NAD+ and NADH cross the inner mitochondrial membrane if it is impermeable to them
Through one of two ‘shuttle’ systems
- the malate-aspartate shuttle: couples cytosolic NADH oxidation to mitochondrial NAD+ reduction (to form NADH in the mitochondrial matrix)
- the glycerol phosphate shuttle: couples cytosolic NADH oxidation to mitochondrial FAD reduction (to form FADH2 in the matrix)
What is the total ATP produced from one glucose?
Define oxidative decarboxylation
Reaction that simultaneously reduces a compound and removes carbon dioxide. An example is the conversion of pyruvate to acetyl-CoA that reduces NAD+ to NADH while releasing one molecule of CO2
Define Krebs/TCA cycle
Series of reactions that take place in the mitochondrial matrix that convert acetyl-CoA to two molecules of CO2, one molecules of ATP and releases eight electrons that are used to generate three molecules of NADH and one molecules of FADH2
Define electron transport chain (ETC)
A series of enzyme complexes that perform redox reactions, coupling the transfer of electrons - from electron donors (NADH and FADH2) to the terminal electron acceptor (oxygen) - to the movement of hydrogen ions across the mitochondrial inner membrane into the mitochondrial intermembrane space
Define oxidative phosphorylation
Reaction of the ETC and ATP synthase. Electrons are used to reduce oxygen to water while a proton gradient is established across the inner mitochondrial membrane. This proton gradient is the driving force of the ATP synthase to catalyse the reaction of ADP and Pi to ATP
