Johnson - Metabolism (Oxidative Phosphorylation) Flashcards
what is the first step in oxidative phosphorylation? which enzyme(s) (and cofactors) catalyse this?
1) NADH —> NAD{+} +2e{-} +H{+} (catalysed by complex 1, NADH dehydrogenase and cofactor FMN)
2) 2e{-} passed between 7 (out of 9) Fe-S clusters, reduces UQ –> UQ{2-} which picks up 2H{+} from matrix
3) free energy causes conformational change in membrane domain and pumps 4H+ across MIM
what is the second step in oxidative phosphorylation? which enzyme(s) (and cofactors) catalyse this?
1) succinate —> fumarate + 2e{-} (catalysed by succinate dehydrogenase, uses FAD cofactor) THIS STEP IS PART OF KREBS
2) 2e{-} passed from FAD(H2) to 3 Fe-S clusters to bound haem molecule and eventually reduces UQ—>UQ{2-} in the ubiquinone pool which picks up 2H{+} from the matrix
what is the third step in oxidative phosphorylation? which enzyme(s) (and cofactors) catalyse this?
1) 2 molecules of UQH2 are oxidised to UQ releasing 4e{-}
2) 1st of 2 UQH2 releases 2e- :
• 1e- goes to a Fe-S cluster and then a haem group, and then finally reduces Cyt c (Cyt C carries 1e-)
• 1e- goes to 2 haems to reduce UQ to UQH2 (technically this 1e- reduces UQ to UQ-)
3) 2nd of 2 UQH2 releases 2e-
• 1e- goes to a Fe-S cluster and then a haem group, and then finally reduces Cyt c
• 1e- goes to 2 haems to reduce UQ to UQH2 (this e- reduces UQ- to UQ2- which can then pick up 2H+ from the matrix, forming UQH2)
4) overall 4H{+} are pumped across the MIM per UQH2 (2 from matrix, 2 from UQH2)
what is the fourth step in oxidative phosphorylation? which enzyme(s) (and cofactors) catalyse this?
1) 2e- from 2CytC(red) molecules are passed via a pair of Cu2+ ions, to a haem group, then to a haem/Cu2+ pair
2)2CytC(red) +4H{+} + ½O2 —> 2CytC(ox) + H2O + 2H{+}
2H{+} from matrix are pumped across the MIM
what is the fifth step in oxidative phosphorylation? which enzyme(s) (and cofactors) catalyse this?
1) one full turn of the F0 rotor ring carries 12H+ across the membrane causing one full turn of the F1 ATPase head forming 3x ATP (catalysed by ATP synthase)
describe Mitchell’s chemiosmotic hypothesis and the theory it replaced
Common belief that the ETC generated some form of high-energy intermediate which donated a high energy phosphate group directly to ADP (ie substrate level phosphorylation mechanism)
Peter Mitchell’s theory was the electrochemical proton gradient generated by ETC was used to generate ATP (won Nobel Prize in ‘78)
give evidence that supports the theory of chemiosmosis
When actively respiring the appearance of mitochondria changed in EM:
Ratio of matrix : intermembrane space volume had changed dramatically
This suggested that electron transport was coupled to changes in osmotic potential
The use of an uncoupler (molecule that facilitates diffusion of H+ across MIM) led to ATP synthesis being abolished. Showed H+ gradient needed to make ATP.
Further compelling evidence provided by Walter Stoeckenius:
Added isolated bacteriorhodopsin to reconstituted lipid vesicles with ATP synthase from cow heart mitochondria (this allowed ATP to be made)
Proved no ‘high energy intermediates’ were needed
describe the structure of F1F0 ATP synthase
ATP synthase has 2 domains:
F1 domain protrudes out of the MIM into the matrix and is comprised of 9 major subunits α3β3γδε
F0 domain embedded in the MIM comprised of 8-15c subunits, 1a subunit and the b2 subunit which acts as a stator (stationary part of a motor in which another part rotates)
describe how protons enter the c-subunit ring of ATP synthase and how they are released
The c-subunits have an aspartate residue which lies at the centre of the MIM This accepts protons which enter via the subunit half-channel When exposed to the proton rich inter membrane space the aspartate takes up a proton When eventually (after a full rotation of the c-ring) aspartate is exposed to the proton poor matrix it releases the proton
describe how c-subunit rotation is coupled to γ-subunit rotation. explain how this relates to the binding change model.
The rotation of the c-subunit causes the γ-subunit at their centre to rotate, this couples the movement of protons down their concentration gradient to the rotation of the F1 domain. PMF drives c-ring rotation.
The 3β subunits of the F1 domain can exist in 3 conformations, which bind ATP tightly (T) or loosely (L) or release it (open, O). The interaction with the γ-subunit determines the conformation of each β-subunit.
The binding change model:
The rotation of the γ-subunit interconverts the conformation of the 3β subunits between 3 states:
1) In the O-state ATP is released
2) In the L-state ADP + Pi is bound
3) In the T-state ADP + Pi is converted to ATP
what two components make up the proton motive force, Δp?
The Δp is formed form the combination of the membrane potential (Δψ), i.e. the difference in charge between the 2 sides of the membrane, and the proton concentration gradient (ΔpH)
give the equation to work out proton motive force Δp
Δp = Δψ – 2.3 RT/F ΔpH R= the gas constant; 8.31x10-3 kJ⋅mol−1⋅K−1 T = temp; 298K F = Faraday constant; 96.5 kJ
give the equation to work out the free energy change associated with 1 mole of protons moving from the IMS to the matrix
ΔG = -nfΔp n = number of moles f = faraday constant: 96.5 kJ Δp = proton motive force
how many moles of H+ are moved from the IMS to matrix to make 1 mole of ATP by using ATP synthase? Use ΔG = +57kJmol-1 (ADP + Pi –> ATP) and ΔG = -14.3 kJmol-1 (moving 1 mole of H+ across the MIM)
57/14.3 = 4 moles H+ are moved from intermembrane space to matrix per mole ATP synthesised
why does the size of the c-ring in ATP synthase vary across species?
The higher the proton motive force the greater the free energy change associated with moving 1 mole of H+ across the MIM. This means that it will require fewer H+ to be moved across the MIM per ATP synthesised. The ratio of H+/ATP varies between species depending on the size of the proton motive force, thus the size of the c-ring varies from 8 in beef heart mitochondria to 15 in cyanobacteria
