Lecture 12: Oxidative Phosphorylation: Shuttles and inhibtors

Question 1

what do mitochondrial shuttle systems do?

Answer 1

move metabolites across impermeable inner mitochondrial membrane

Question 2

why do we need shuttle systems?

Answer 2

NADH can’t cross into inner mitochondrial membrane on its own!

so instead we just transfer an e- pair (reducing equivalents) from cytosol

Question 3

Aerobic regeneration of NAD+

Answer 3

mitochondrial shuttle systems oxidize cytosolic NADH

sliiiiiiide 4

Question 4

malate-aspartate shuttle in liver and heart and kidney

Answer 4

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

Question 5

what cytosolic enzyme oxidizes NADH under ANEROBIC conditions?

Answer 5

lactate dehydrogenase

Question 6

what are the 2 e- from cytosolic NADH used for in mitochondrial matrix?

Answer 6

enter ETS and later make ATP!!!!!!!

Question 7

glycerol-3-phosphate shuttle system used in the brain and muscle cells

Answer 7

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

Question 8

what’s the “cost” of using the glycerol-3P shuttle (in terms of proton motive force and ATP synthesis)

Answer 8

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)

Question 9

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?

Answer 9

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

Question 10

What about the shuttle systems matter in bookkeeping?

Answer 10

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)

Question 11

BOOKKEEPING SLIDE 8!

Answer 11

BOOKKEEPING SLIDE 8

Question 12

see slide 9?

Answer 12

see slide 9?

Question 13

so… ETS and Ox phos?

Answer 13

FUNCTIONALLY COUPLED!

we can see this by experimenting with inhibitors

Question 14

what do you need to add to get O2 consumption and ATP synth to happen?

Answer 14

ADP and Pi
succinate

order doesn’t matter!

Question 15

why add succinate?

Answer 15

provide more e- for ETS!

Question 16

what if we add CN- (cyanide)?

Answer 16

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

Question 17

why succinate increases rates of Ox Phos and O2 consumption

why cyanide inhibits Ox Phos AND O2 consumption

Answer 17

we need succinate to inc production of O2 and ATP (for more e-)

cyanide blocks complex 4

Question 18

What if we add oligomycin

Answer 18

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

Question 19

what if we add DNP after oligomycin?

Answer 19

adding DNP creates a “hole” in the membrane, which makes the proton gradient dissappears
thus, O2 consumption can continue

SLIDE 11

Question 20

more on DNP

Answer 20

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

Question 21

why does DNP cause you to burn fat?

Answer 21

dissipate proton gradient leads to less ATP around, body creates nrg by burning fat

Question 22

UCP1 uncoupling protein

Answer 22

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

Question 23

what happens when we inhibit… complex 3?

Answer 23

No ETS activity, very low ATP synth

no e- flow, protons depleted so very little H+ avalible for ATP synth

Question 24

what happens when we inhibit…using DNP

most confusing one

Answer 24

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???

Question 25

what happens when we inhibit…ATP synthase

Answer 25

very low ETS activity
NO ATP synth
there’s no H+ flow through ATP synthase, so H+ builds up, causing ETS to slow down

Question 26

what happens when we inhibit…ATP synthase and add DNP

Answer 26

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

Question 27

analogy of pressureized sprayer

1. pump handle
2. nozzle
3. release valve
4. insecticide solvent

Answer 27

1. redox nrg
2. ATP synthase
3. UCP
4. protons