Lecture 12: Oxidative Phosphorylation: Shuttles and inhibtors Flashcards
what do mitochondrial shuttle systems do?
move metabolites across impermeable inner mitochondrial membrane
why do we need shuttle systems?
NADH can’t cross into inner mitochondrial membrane on its own!
so instead we just transfer an e- pair (reducing equivalents) from cytosol
Aerobic regeneration of NAD+
mitochondrial shuttle systems oxidize cytosolic NADH
sliiiiiiide 4
malate-aspartate shuttle in liver and heart and kidney
molecs move in diff directions
oxidizes NADH to make NAD+
aspartate and malate are the molecs moving across the membrane
ONLY happens under aerobic conditions
what cytosolic enzyme oxidizes NADH under ANEROBIC conditions?
lactate dehydrogenase
what are the 2 e- from cytosolic NADH used for in mitochondrial matrix?
enter ETS and later make ATP!!!!!!!
glycerol-3-phosphate shuttle system used in the brain and muscle cells
NADH to NAD+ 2 molecs (DHAP and Glycerol-3P) move through outer and inner mitochondrial membranes
when in inner membrane space, we put electrons onto FADH2,
after that, FADH2 stuff goes into ETS at complex 2
what’s the “cost” of using the glycerol-3P shuttle (in terms of proton motive force and ATP synthesis)
NADH vs. FADH2. Glycerol-3P less effective at generating ATP b/c it move FADH2.
FADH2 bypasses complex 1, so it makes 4 less ATP (6 instead of 10)
what’s the ADVANTAGE of using the glycerol-3P shuttle in the brain and muscle cells as compared to using malate-aspartate shuttle cells in liver cells?
1) FASTER! brain and muscles cant be without energy! and this one only takes one enzyme
2) we may already have NADH in matrix, so if we can bypass that system, we don’t have to worry about concs of NADH. i.e. if concs of NADH high, we can bypass complex 1 with the shuttle system
What about the shuttle systems matter in bookkeeping?
SHUTTLE SYSTEM USED CHANGES FINAL ATP COUNT!
so with glycolysis if we use 2 NADH we get 5 ATP, if we use 2 FADH2 we get 3ATP
So final is 30 or 32 respectively
(recall, not 36 or 38 because we lose 4 protons per ATP in the ETS/ATP synthase/ATP movement)
BOOKKEEPING SLIDE 8!
BOOKKEEPING SLIDE 8
see slide 9?
see slide 9?
so… ETS and Ox phos?
FUNCTIONALLY COUPLED!
we can see this by experimenting with inhibitors
what do you need to add to get O2 consumption and ATP synth to happen?
ADP and Pi
succinate
order doesn’t matter!
why add succinate?
provide more e- for ETS!
what if we add CN- (cyanide)?
CN blocks complex 4
so O2 consumption stops (O2 consuption=electron flow through ETS)
ATP synth stops too b/c there is no more proton pumping
why succinate increases rates of Ox Phos and O2 consumption
why cyanide inhibits Ox Phos AND O2 consumption
we need succinate to inc production of O2 and ATP (for more e-)
cyanide blocks complex 4
What if we add oligomycin
we inhibited ATP synthase
so ATP production obvi stops
O2 consumption stops because we built up a proton gradient, so moving more protons up the gradient takes NRG!
we don’t have enough nrg avalible, so we stop pumping protons
what if we add DNP after oligomycin?
adding DNP creates a “hole” in the membrane, which makes the proton gradient dissappears
thus, O2 consumption can continue
SLIDE 11
more on DNP
it acts as a proton carrier and carries H+ across the inner membrane
thus we dissipate the proton gradient
a way for e- to move other than ATP synthase
why does DNP cause you to burn fat?
dissipate proton gradient leads to less ATP around, body creates nrg by burning fat
UCP1 uncoupling protein
controls thermoregulation in brown fat mammals
short circuts ETS by bypassing ATP synthase
proton gradient dissipated as heat when it flows through.
causes organism to warm up
what happens when we inhibit… complex 3?
No ETS activity, very low ATP synth
no e- flow, protons depleted so very little H+ avalible for ATP synth
what happens when we inhibit…using DNP
most confusing one
we WILL have ETS activity, but very LOW ATP synth
b/c high e- flow, but H+ crosses the membrane by ATP synth largely, so we would have poor atp synth? WHAT???
what happens when we inhibit…ATP synthase
very low ETS activity
NO ATP synth
there’s no H+ flow through ATP synthase, so H+ builds up, causing ETS to slow down
what happens when we inhibit…ATP synthase and add DNP
NO ATP activity
YES ETS activity
we have a high e- flow, as H+ cross the membrane by the DNP shuttle
ATP synthase is dead though
analogy of pressureized sprayer
- pump handle
- nozzle
- release valve
- insecticide solvent
- redox nrg
- ATP synthase
- UCP
- protons