CBI 8: Krebs Cycle and Oxidative Phosphorylation Flashcards
1
Q
What is the second main stage of cellular aerobic respiration?
A
- the Krebs cycle
- also known as the tricarboxylic acid cycle or TCA cycle
2
Q
Where do oxidative decarboxylation and the TCA cycle take place?
A
- in the mitochondrial matrix
- hence pyruvate needs to be transported via the mitochondrial membranes using a special carrier protein
3
Q
Describe the outer membrane of the mitochondrion
A
- 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
4
Q
Describe the mitochondrial matrix
A
- contains most of the proteins and the mitochondrial DNA
- TCA cycle takes place here
5
Q
Describe the inner membrane of the mitochondrion
A
- the membrane is extensively folded and compartmentalised
- contains the membrane proteins involved in the electron transport chain and oxidative phosphorylation
6
Q
Briefly describe what oxidative decarboxylation is and give its overall reaction equation
A
- 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
7
Q
What enzyme catalyses oxidative decarboxylation?
A
- pyruvate dehydrogenase complex
- a very large and complex enzyme
- uses different co-factors to allow reactions to happen at multiple sites
8
Q
Describe the steps of oxidative decarboxylation
A
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
9
Q
How many molecules of pyruvate undergo oxidised decarboxylation per molecule of glucose?
A
- two pyruvate molecules
10
Q
Briefly describe the hydrolysis of the thioester bond between the CoA and the acetyl group (in acetyl CoA)
A
- hydrolysing the acetyl group is associated with a large, negative change in the Gibbs free energy
11
Q
What is the overall reaction of the TCA/Krebs cycle?
A
12
Q
Give a brief overview of the Krebs cycle
A
- 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)
13
Q
Describe the Krebs Cycle
A
- 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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14
Q
Describe the chemical structure of NAD and NADH
A
- consists of:
- adenine
- two ribose sugars
- a diphosphate group
- a nicotinamide group
15
Q
Describe the chemical structure of FAD and FADH
A
- 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
20
Q
Describe ubiquinone (coenzyme Q)
A
- 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
21
Q
Describe cytochrome c
A
- 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
22
Q
Describe the electron transport chain
A
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
24
Q
What is the proton-motive force?
A
- 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
25
Q
Describe ATP synthase structure
A
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
26
Q
Describe how the ATP synthase F0 subunit works and why this happens
A
- 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
27
Q
Describe how the ATP synthase F1 subunit works
A
- 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
33
Q
What is the total ATP produced from one glucose?
A
