MBC - Cell integrity Flashcards
What is the difference between substrate-level and oxidative phosphorylation?
Substrate-level: direct generation of ATP/GTP by kinases
Oxidative: indirect bulk generation of ATP in mitochondria
How is oxidative phosphorylation increased in the inner membrane of mitochondria?
The cristae are folds which increase surface area for ETC proteins
What is complex I?
NADH-Q oxidoreductase/NADH dehydrogenase
What are the 4 membrane proteins in ETC?
Complex I - NADH-Q oxidoreductase/NADH dehydrogenase
Complex II - Succinate-Q reductase/ succinate dehydrogenase
Complex III - Q-cytochrome C oxidoreductase
Complex IV - cytochrome C oxidase
What is complex II?
Succinate-Q reductase / succinate dehydrogenase
What is complex III?
Q-cytochrome c oxidoreductase
What is complex IV?
cytochrome C oxidase
What are the 2 electron carriers in ETC?
Co-enzyme Q (ubiquinone)
Cytochrome C
Why does electron only go one way in the ETC?
Because complex IV has higher affinity for e- than complex I
How does Complex I acquire e- from NADH?
Complex I has a higher affinity for e- than NADH
How does proton go from matrix to intermembrane space?
Complex I, III, IV accept e- and by doing so releases energy to pump protons across membrane
Where is complex II from?
An enzyme from the TCA cycle (It sits in the inner mitochondrial membrane)
What does complex II do?
It uses FAD as cofactor to directly communicate w/ Coenzyme Q.
As e- pass from FADH2 to Coenzyme Q, Complex II picks up a pair of protons, regenerating FAD and forming QH2
When does ETC end?
When complex IV has 4e- to from H2O
Why are fewer ATP molecules produced when FADH2 is reoxidised by ETC compared with NADH?
Complex I is bypassed and fewer protons are pumped to the innermembrane space
What does cytochrome c do?
transfers electrons from complex III to complex IV
How do redox reactions govern oxidative phosphorylation?
By substrates’ ability to accept/donate e-
How to define redox reactions?
Electron transfer reaction involving a reduced & oxidised substrate
What does a reduced substrate do?
It donates e- and gets oxidised
What does an oxidised substrate do?
It accepts e- and gets reduced
What is a redox couple?
A substrate that can exist in both reduced and oxidised form
What is the reduction/redox potential?
Ability for redox couple to accept/donate e-
What is the standard redox potential measured against?
The hydrogen electrode
What does a standard NEGATIVE redox potential indicate?
Tendency to donate e-, it has more reducing power than H
eg NAD+/NADH
What does a standard POSITIVE redox potential indicate?
Tendency to accept e-, it has more oxidising power than H
eg Fe3+/2+ , 0.5O2/H2O
How does e- energy progress along the chain?
Transfer of electron is energetically favourable, e- loses energy as it progresses along chain
What is ATP synthase?
A multimeric enzyme that consist of a membrane bound part and a part that projects into the matrix
How does ATP synthase work to synthesise ATP?
Rotation of central shaft influences conformation and arrangements of subunit, energy from conformation promote formation of ATP from bound ADP + Pi
What are the steps to rotate the shaft of ATP synthase?
- H+ goes down proton gradient (IMS to matrix)
- Chemical energy stored in proton gradient is converted to mechanical/rotational energy
- H+ drive rotor by binding to rotor subunits (only protonated subunits can rotate into membrane)
How does hydrolysis happen with ATP synthase?
When H+ gradient is higher in the matrix than the intermembrane space, ATP breaks down into ADP + Pi to provide energy to move H+ to IMS
What is the oxygen electrode?
A device to measure O2 conc. in solution
What is the base of the oxygen electrode chamber formed by?
Teflon membrane (permeable to O2)
What is under the Teflon membrane?
Compartment with Pt cathode (+) & Ag anode (-)
What happens when voltage is applied between electrodes?
O2 diffuses through membrane - reduced at Pt cathode
At Ag anode, Ag oxidised to AgCl by KCl electrolyte
How can we use the oxygen electrode to test components of ETC?
- Prepare suspension of mitochondria + place it in
2. Observe oxygen consumption of suspension for a set time period to determine various substrates and inhibitors
How do we conduct the test w/ oxygen electrode?
- Measure baseline respiration
- Add known amount of ADP (rapid consumption of O2 -steeper gradient)
- Calculate ratio of amount of ADP phosphorylated by mitochondria to amount of O2 consumed
What is the ADP:oxygen index?
Measure of efficiency of mitochondrial phosphorylation system
What happens when all the ADP has been consumed? (Oxygen electrode test)
Level of respiration returns to baseline gradient
How long does each ATP molecule live for?
1-5 min(s)
Around how many times is ATP recycled a day?
300 times
What happens when oxidative phosphorylation/ATP synthesis is interrupted?
Cell death
What is the most common failure to oxidative phosphorylation/ATP synthesis?
Lack of O2
(Hypoxia - diminished O2)
(Anoxia - total lack of O2)
What is respiratory control?
Uptake of O2 by mitochondria which is controlled by ADP+Pi
Allows body to adapt O2 consumption to body energy requirements
What are metabolic poisons?
Molecules that interfere w/ ATP synthesis
- flow of e- along ETC
- flow of H+ through ATP synthase
What are some examples of metabolic poisons?
Cyanide (CN-) & Azide (N3-)
Malonate
Rotenone
Oligomycin
Dinitrophenol (DNP)
What does Cyanide & Azide do?
Bind w/ high affinity to Fe3+ of haem group in complex IV
What does malonate do?
competitively inhibit succinate dehydrogenase
(resembles succinate)
Slows down flow of e- from succinate to ubiquinone
What does rotenone do?
Inhibits transfer of e- from complex I to ubiquinone
what does oligomycin do?
Bind to stalks of ATP synthase, block flow of proton
Inhibiting oxidative phosphorylation
What does dinitrophenol (DNP) do?
Shuttles protons across inner mitochondrial membrane
Why is dinitrophenol dangerous?
It uncouples OxPhos from ATP production - increases metabolic rate & body temp
