LECTURE 23 - MIDTERM 3

Question 1

What is oxidative phosphorylation?

Answer 1

– it is the coupling of energy release during electron transport to ATP synthesis

– mechanism of oxidative phosphorylation is converting free energy of proton gradient for ATP synthesis

Question 2

Describe the Chemiosmotic Theory and it’s components.

Answer 2

– now widely accepted model for mechanism of oxidative phosphorylation

– states that free energy from electron transport drives an active transport system which pumps protons out of mitochondrial matrix into intermembrane spcae

– this action generates an electrochemical gradient for protons

– protons on the outside have thermodynamic tendency to flow back in down electrochemical gradient and this flow provides driving force for ATP synthesis

Question 3

What is ATP synthase?

Answer 3

– a reversible coupling device that can convert the energy of the electrochemical proton gradient into chemical bond energy or vice versa

• the direction depends on the magnitude of the electrochemical proton gradient

Question 4

What is the binding-change mechanism for ATP synthesis by ATP synthase?

Answer 4

– the beta subunit exists in three conformation states:

L- losse - binds ADP and Pi “loosely”

T - tight - catalyzes formation of ATP from bound ADP and Pi – the newly made ATP binds with very high affinity and is not released until the Beta subunit assumes the O conformation

O - open - ATP can now be released and ADP and Pi can enter the enzyme

– the conformation state of the beta subunit (L, T, or O) is determined by contact with the gamma subunit

Question 5

What is the conformation state of the beta subunit based on?

Answer 5

– dtermined by contact with the gamma subunit

– passage of protons through channels in F0 causes gamma to rotate counter-clockwise

– rotation of the gamma subunit allows a given beta subunit to change conformational states

– rotation is counter-clockwise

Question 6

T or F, rotation of the gamma subunit allows cycles of ATP synthesis and release through changes to the Beta subunit conformation

Answer 6

True;
subunit 1: L –> T –> O –> L –> T –> O

subunit 2: O–> L–> T –> O –> L –> T

subunit 3: T –> O –> L –> T –> O –> L

Question 7

What is rotating in F1?

Answer 7

– gamma is rotating in F1, everything else stays the same

Question 8

How does the proton-motive force drive rotation of the F0 assembly?

Answer 8

– an aspartic acid residue in the c subunit and two half-channels in the “a” subunit are key

Question 9

What is a key component of half-channels on the ATP synthase?

Answer 9

– channels do not span the membrane

– channels have an aqueous hydrophilic character (thus protons can flow into/out of the channels)

– aspartic acid positioned so that it comes into contact with two different environments –> interacts w/ two different environments

1: the hydrophilic half-channels in subunit a
2: hydrophobic environment of the membrane

Question 10

T or F, proton is 1 to 1 for C subunits w/ aspartic acid

Answer 10

– True, 1 aspartic acid; 1 proton

Question 11

What is a key feature of the aspartic acid in the half channel?

Answer 11

– the charge on the aspartic acid changes depending on the environment

– in a hydrophilic environment, the residue can be in the aspartate form (deprotonated) –> base form

– in the membrane, the residue is protonated and can exist in a hydrophobic environment –> acid form

Question 12

Describe the rotation of the C subunit.

Answer 12

– the intermembrane space half-channel is proton-rich, and thus the aspartic acid becomes protonated

– protonation of the C subunit causes the entire ring to turn to moving Asp to the hydrophobic membrane

– this positions a different c subunit next to the matrix half-channel, which is proton-poor and thus the aspartic acid is deprotonated

– then the cycle repeats

Question 13

What is the key point of the rotation of the C subunit?

Answer 13

– key point is that the proton gradient ensures protonation of the aspartic acid in the intermembrane half-channel

– causing movement

– and deprotonation in the matrix half-channel

– which resets the system

Question 14

T or F, the C subunit rotation clockwise means it’s hydrolyzing ATP and counterclockwise means it’s synthesizing ATP

Answer 14

False;

– clockwise = synthesizing ATP

– counter clockwise = hydrolyzing ATP

Question 15

T or F, there’s one matrix half-channel and one cytoplasmic half channel per ATP synthase

Answer 15

True

Question 16

How do protons enter the ATP synthase?

Answer 16

– protons enter from the intermembrane side, then ride around, and are released on the matrix side

– proton binding causes rotation, proton release resets the system to allow additional binding and additional rotation

– protons that are released in the matrix get pumped out again

Question 17

What allows the release of ATP from the ATP synthase?

Answer 17

– rotation of the c ring turns the gamma subunit, which causes conformation changes to the beta subunits, which allows release of ATP

Question 18

What is the point of oxidative phosphorylation?

Answer 18

– to generate ATP

Question 19

How is ATP transported out of the matrix? And how is ADP brought in?

Answer 19

– ATP-ADP translocase (specific transport protein) allows a 1:1 exchange of ADP for ATP

– ATP-ADP translocase is in high abundance and it makes up about 15% of the protein content in the inner mitochondrial membrane

Question 20

T or F, ATP transport is energetically expensive due to its negative charge

Answer 20

True; ATP has one more negative charge than ADP – this dampens the membrane potential

– difference in charges needs to be maintained for proton gradient

Question 21

Where does 25% of the energy yield from respiration go?

Answer 21

– goes back into regenerating the membrane potential

– part of the proton pumping we did during electron transport is used to maintain the electrical gradient

– therefore, some protons aren’t used to make ATP directly

Question 22

What is the importance of mitochondrial shuttles?

Answer 22

– mitochondrial shuttles allow transport across inner membrane

– inner membrane is extremely impermeable yet exchange w/ the cytoplasm is important for respiration

– solution = mitochondrial shuttles

Question 23

T or F, functionally, transport of OH- out is the same as transport of H+ in

Answer 23

True

Question 24

T or F, NAD+ in the cytoplasm must be regenerated for use during glycolysis (cytoplasm)

Answer 24

True

Question 25

T or F, ETC is happening in the inner membrane of the mitochondria

Answer 25

True

Question 26

T or F, the glycerol 3-phosphate shuttle transfers electrons (but not NADH itself) across the inner membrane

Answer 26

True; electrons are transferred to FAD and NADH is oxidized back to NAD+ without leaving the cytoplasm

Question 27

What is the whole point of the glycerol 3- phosphate shuttle? And what are the steps that occur?

Answer 27

– whole point of shuttle is to take NADH from cytoplasm for it to be used in ETC

glycerol 3-phosphate is located on inner mito membrane (is associated with FAD in a similar way to complex 2)

1. Reduction of DHAP by NADH in cytosol
2. Reoxidation of G3P and reduction of FAD
3. Transfer of an electron pair from FADH2 to coenzyme Q (in complex III)
4. DHAP returns to the cytosol

Question 28

What is the malate-aspartate shuttle?

Answer 28

– a more complicated shuttle that regenerates cytoplasmic NAD+ in heart, kidney and liver tissue

Question 29

What are the steps that occur in the Malate-Aspartate shuttle?

Answer 29

– Malate Dehydrogenase reduces Oxaloacetate to Malate and accepts e- from NADH

– Malate shuttle brings Malate across inner membrane

– Malate is reoxidized by Malate dehydrogenase and transfers e- back to NAD+ on Matrix side

– Oxaloacetate is transaminated to make Aspartate, which can transverse back across the membrane using the Aspartate shuttle

– Aspartate is converted back to Oxaloacetate