Module 12 - ATP synthase, Shuttle systems, and OxPhos Regulation

Question 1

What are the energy exchange ratios for NADH and FADH2?

Answer 1

1 ATP requires 4 H+

1 NADH = 10 H+ = 2.5 ATP
2 NADH = 20 H+ = 5 ATP

1 FADH2 = 6 H+ = 1.5 ATP
2 FADH2 = 12 H+ = 3 ATP

Question 2

The ATP synthase complex can be divided into two parts:

Answer 2

F1 - catalytic activity
F0 - proton channel that crosses the inner mitochondrial membrane

Question 3

What are the three functional units of the ATP synthase complex?

Answer 3

rotor: gamma and c10 that rotates around
headpiece: hexametric a3B3 ring
stator: includes the half-channel for H+

Question 4

Describe the binding change mechanism

Answer 4

the affinity of the B subunit for ATP is altered by proton flow through the Fo component
a Mg 2+ ion is required for the enzyme reaction

the dissociation constant increases by a million fold in the present of proton motive force

Question 5

What are the four steps of the binding change mechanism

Answer 5

1. the gamma subunit contacts all three B subunits, but leads to three different conformations
2. ATP binding affinities are defines as T, L, and O
3. as protons flow through Fo, the gamma subunit rotates. with each 120 degree rotation, the B subunits change from O–>L–>T–>O–>L–>etc.
4. in 1 full rotation of the gamma subunit, 3 ATP are generated

Question 6

What is the difference between ATP synthase and ATP hydrolase?

Answer 6

ATP synthase: rotates counterclockwise from the bottom view, H+ move into the mitochondria and ATP are synthesized

ATP hydrolase: rotates clockwise from the bottom view, H+ move out of the mitochondria and ATP is hydrolyzed to ADP and Pi

Question 7

How was it determined that ATP synthase is also ATP hydrolase?

Answer 7

the headpiece was attached to a solid surface and an actin filament was attached to the gamma subunit

rotation changed direction during synthase vs. hydrolase activity

Question 8

How can ATP synthase be used as a nanomotor?

Answer 8

the headpiece is attached to a solid surface and a magnetic bead is attached to the gamma subunit

six electromagnets in a circular arrangement around the chamber force rotation of the bead to drive ATP synthesis in the absence of a proton gradient

Question 9

How does H+ movement cause rotation of the ATP synthase rotor?

Answer 9

two channel model: protons drive rotation of the c ring in response to an electrochemical proton gradient through alternating protonation and deprotonation of a charges Asp residue in each C subunit

• H+ bind to carboxyl group of negatively charged Asp (becomes neutral)
• area jiggles to move the neutral area in to the membrane
• process continues and rotates the C ring around
• when it reaches the half channel again, Asp is deprotonated and the H+ moves toward the lower concentration inside the mitochondria

Question 10

What are atractyloside and Bongkrekic acid?

Answer 10

atractyloside: from Atractylis gummifera (common thistle plant), binds to the C state of ATP translocase

Bongkrekic acid: from bacteria that contaminate fermented soybeans, bind to the M state (prevent ATP from releasing)

Question 11

Describe the structure of ATP/ADP translocase. Is it a symporter or anti porter?

Answer 11

two conformations to exchange ADP and ATP, rocking bananas

antiporter

Question 12

What is phosphate translocase?

Answer 12

symporter or anti porter for transport of ATP, ADP, and Pi

symporter: moves H2PO4- and H+across the membrane in the same direction
antiporter: moves H2PO4- and OH- across the membrane in opposite directions

Question 13

How is an ATP moved out of the mitochondria?

Answer 13

• 1 H+ and 1 Pi moves in via the phosphate translocase
• 1 ADP moves in through the ATP/ADP translocase
• 3 H+ move in through the ATP synthase half channel, which turns ADP+Pi into ATP
• ATP exits the mitochondria via another ATP/ADP translocase

(total of 4 H+ used for every ATP)

Question 14

What does Bongkrekic acid do?

Answer 14

produced by bacteria that contaminate fermented soybeans used to make tempeh cakes

targets ATP translocase (prevents ATP from exiting the mitochondrial matrix)

Question 15

Why are membrane shuttle systems necessary for the mitochondria?

Answer 15

many cytosolic reactions generate NADH, but it cannot cross the inner mitochondrial membrane

instead, the cell uses an indirect mechanism to transfer only the electron pair from the cytosol to the matrix using a shuttle system

Question 16

Describe the malate-aspartate shuttle

Answer 16

• used in liver cells
• ## moves 2 e- from cytosolic NADH to mitochondrial NAD+ using malate

Question 17

Describe the glycerol-3P shuttle

Answer 17

• used in muscle cells
• cytosolic FADH2 to DHAP
• lose 1 ATP (2.5 from NADH but only 1.5 from FADH2)

Question 18

Why are there two shuttles? What are their differences?

Answer 18

malate-aspartate shuttle:
- liver cells
- 32 ATP
- more complicated and takes more time (more efficient)

glycerol-3P shuttle:
- muscle cells
- 30 ATP
- quick, few parts

Question 19

What are allosteric inhibitors and activators of OxPhos?

Answer 19

activators: ADP, AMP, Pi
inhibitors: ATP, NADH

Question 20

What does succinate (with ADP and Pi) do?

Answer 20

stimulates O2 consumption and ATP synthesis by donating 2 e- to FAD in complex 2

Question 21

How does cyanide effect the ETS/OxPhos?

Answer 21

blocks O2 consumption and ATP synthesis (electron transport disabled and no protons pumped)

Question 22

How does 2,4 dinitrophenol (DNP) effect the ETS/OxPhos?

Answer 22

protons are pumped, but they do not flow through ATP synthase to make ATP
remains in the mitochondrial membrane for a long time (builds up)

Question 23

How does oligomycin effect the ETS/OxPhos with DNP present?

Answer 23

electron transport happens but no ATP are generated (blocks ATP synthase but O2 consumption continues)

Question 24

What is UCP1?

Answer 24

uncoupling protein 1, located in brown adipose tissue, alternate channel away from ATP synthase that also produces heat

Question 25

Why do mitochondrial diseases have varying levels of severity?

Answer 25

when the mitochondria are put in egg cells, the amount of “bad mitochondria” in each one is different