Bioenergetics 6: Oxidative phosphorylation

Question 1

What experiment did Jagendorf and Mitchell to show how ATP synthase

Answer 1

They made artificial vesicles from mitochondrial proteins, including one containing ATP synthase. This turned the membrane inside out so that when the vesicles were made in a higher pH solution inside and placed into a lower pH solution outside , it started pumping H+ out to the low pH and make ATP from the ADP + Pi

Question 2

What is an example of a possible first proton pump

Answer 2

Rhodopsin in bacteria pumps out H+ by energy from light and this makes the proton gradient required to drive ATP synthase

Question 3

What are the two types of poisons that stop ATP synthase

Answer 3

Uncouplers that disrupt the proton gradient as well as

poisons that stop the electron flow to ATP synthase

Question 4

What are the components of the proton motive force

Answer 4

Electro -> positive charge in intermembrane space, negative inside
Chemical -> concentration gradient

Question 5

What causes the cristae to fold and where does proton flow: How can mitochondria work with such a low pH difference

Answer 5

The V shape of dimerised ATP synthases fold the inner membrane to give cristae their shape, increasing the density of negative charge at the base of the ATP synthase creating locally lowered pH gradient. This directs proton flow to the ATP synthase.

Question 6

How do H+ flow through the ATP synthase: 5 steps

Answer 6

1. H+ ions enter the half channel in the stator which is anchored in the membrane
2. H+ ions enter binding sites within the rotor, changing the shape of each subunit so that the rotor spins within the membrane
3. Each H+ ion makes one complete turn before leaving the rotor and passing through a second half of the channel in the stator into the mitochondrial matrix
4. Spinning of the rotor causes an internal rod to spin as well. This rod extends into the knob below it which is held stationary by part of the stator
5. The turning of this rod activates the catalytic knob to produce ATP from ADP and Pi

Question 7

How many H+ needed to make a full rotation in ATP synthase in human mitochondria vs Chloroplast

Answer 7

8 H+ in humans, 14 H+ in chloroplast

Question 8

What is the structure of the internal rod that makes it trigger the catalytic knob

Answer 8

It doesn’t sit straight within the Cknob so when it moves around it exerts pressure on the subunits and changes their conformations

Question 9

What is the force that pushes ADP and Pi together in substrate level phosphorylation vs oxphos

Answer 9

Substrate level relies on the charge of the substrate that the phosphate is going to be added to but Ox phos uses brute force to push the two together

Question 10

For each turn of ATP synthase how many ATPs made and why

Answer 10

it makes 3 ATPs because there are 6 subunits paired together to make sites. (a and b)

Question 11

Where did the subunits for catalytic knob come from

Answer 11

helicases-> they hydrolyse ATP (backwards to mitochondria) and move strands of DNA

Question 12

How does the amount of ATP produced by NADH produced by the glycolysis differ

Answer 12

The electrons from NADH in cytosol are transferred by 2 shuttles

1. Malate aspartate shuttle (slow and complicated but efficient) makes 2.5 ATP
2. G3PDH shuttle (made in glycolysis) makes 1.5 ATP because it uses FADH2, meaning it loses one ATP but its fast.

Question 13

What is the net ATP per 1 glucose from NADH and FADH2 derived

Answer 13

Glycolysis :
2 ATP = 2
2 NADH= 5 or 3 ATP depending on transport

PDH= 2 NADH= 5 ATP

CAC
-2ATP =2
-3 NADH x 2.5 x 2(pyruvate) = 15
2FADH2 =3

Total 28-38 ATP

Question 14

Why is the ATP yield from respiration so vague

Answer 14

1. Temperature : as when membranes are hotter, they are more leaky which means that some of the gradient is lost as H+ can make it back so not making as much ATP
2. The two shuttles that take NADH from glycolysis have different amount of ATP
3. The protons are also used for other processes (ADP/ATP exchange, substrate uptake, ion exchange …) loss of proton gradient

Question 15

Egs of disease states that affect ATP synthase

Answer 15

1. Leighs disease Mutation in C ring of ATP synthase that results in proton slippage/ wastage
2. Mutation in complex 1 causes more reliance on C2 which means less ATP
3. Oxidative stress on the mitochondrial membranes (from free radicals) lose proton gradient

Question 16

UCP1 eg. Dinitrophenol causes what

Answer 16

Causes the proton gradient to be uncoupled from ATP synthase, generating heat as they shuttle H+ across the IMM. This means that respiration happens very quickly

Question 17

What happens to the membrane potential (caused by proton gradient) if you add a substrate to make ETC go. Vs making ETC and OXphos go

Answer 17

It increases with ETC but when u add ADP + Pi for OxsPhos to go then it decreases a little bit but not back to the bottom

Question 18

What happens to the membrane potential (caused by proton gradient) if you add an inhibitor of ATP synthase to ETC + Oxphos line vs an UCP1

Answer 18

for ATP synthase inhibitor It increases because the ETC keeps going. UCP1 it goes down because the proton gradient gets dissipated.

Question 19

What do uncouplers do to respiration vs ATP production

Answer 19

it makes it respiration go faster but ATP production goes down