Lecture 21: Oxidative Phosphorylation Part II

Question 1

Review Slide 3

Answer 1

Not a great way to start off this deck, but whatever.

Question 2

Describe the chemiosmotic hypothesis

Answer 2

• ETC accompanied by transport of protons from matrix to cytoplasmic side of inner membrane
• Generates a pH gradient and membrane potential
• Constitutes a proton motive force
• Used to drive ATP synthesis
• See Slide 5

Question 3

What evidence exists for the chemiosmotic hypothesis?

Answer 3

• Prepared synthetic phospholipid vesicles
• Purified Bacteriorhodopsin and ATP synthase
• Reconstituted the 2 purified proteins into the vesicles
• Added ADP + Pi into the system
• Exposed to light
• Resulted in the generation of ATP
• See Slide 7

Question 4

Describe the structure of ATP synthase

Answer 4

• Aka Complex V
• Embedded in inner membrane
• Ball and stick structure
• F0 subunit is stick – embedded in membrane
• Has a proton channel
• F1 subunit is ball, protrudes into matrix side
• Contains catalytic domains
• F1 subunit made of 5 types of polypeptide chains with different stoichiometrie
• Alpha3, Beta3, gamma, delta and epsilon
• Alpha and beta arranged alternately in a hexameric ring
• Both bind nucleotides but only beta are catalytically active
• Above the alpha and beta is a stalk made of gamma and epsilon proteins
• Gamma subunit has long helical coil that extends into the center of the alpha3 and beta3 hexamer
• The gamma subunit breaks the symmetry of the 3 beta subunits making each one distinct
• F0 has a proton channel made of 8-14 c subunits embedded in membrane
• F0 and F1 connected in 2 ways:
  – 1. Central gamma and epsilon stalk
  – 2. Exterior column – 1 a su, 2 b su, and delta su

Question 5

Describe the function of ATP Synthase

Answer 5

• ATP synthase molecules associate with each other to form dimers
• Dimers come together to form oligomers
• Stabilize the individual molecules to rotational forces required for catalysis
• Maintains curvature in inner membrane
• Cristae allow the proton gradient to be in close proximity to the ATP synthase
• See Slide 12

Question 6

Describe the role of the proton gradient

Answer 6

• ATP synthase when incubated with ADP and Pi formed ATP in absence of a proton gradient
• Role of proton gradient??
• Release the ATP from the synthase
• See Slide 14

Question 7

Describe the active site of ATP synthase

Answer 7

• The 3 beta su comprise active site of ATP synthase
• Not equal
• The gamma su passes through center, creating asymmetry
• The proton motive force causes the 3 su to sequentially change conformation
• Conformational change alters function

Question 8

What are the three steps in ATP synthesis

Answer 8

• Three steps in ATP synthesis
  – 1. Binding of ADP and Pi (L conformation)
  – 2. ATP synthesis (T conformation)
  – 3. Release of ATP (O conformation)
• Rotation of the Gamma su switches these forms
• See Slide 17 - 19

Question 9

Describe the proton conducting unit of ATP synthase

Answer 9

• The c subunit made of 2 alpha helices that span membrane
• An aspartic acid residue lies in the center of the membrane
• The a subunit has 2 half channels
• Allows proton to enter and pass partway but not completely
• Each 360º rotation of gamma su powered by 3 protons generates 3 ATP
• See Slides 20-22

Question 10

Describe the mechanism of action of ATP-ADP translocase

Answer 10

• ATP and ADP not permeable across mitochondrial membrane
• Need a carrier
• ATP-ADP translocase
• Flow of ATP and ADP coupled, i.e., ADP enters matrix only if ATP leaves
• See Slide 24

Question 11

Describe the regulation of cellular respiration

Answer 11

• Levels of ATP regulate respiration
• Electrons flow through ETC only when ADP phosphorylated to ATP
• Regulation by ADP levels called respiratory control
• See Slides 26-27

Question 12

Describe the regulation of ATP synthase

Answer 12

• Inhibitory factor I – inhibits hydrolytic activity of ATP synthase
• Prevents the reverse reaction, i.e., ATP breakdown
• In ischemia or oxygen deprivation
• In cancers – upregulated - facilitates the switch from aerobic to anaerobic respiration (Warburg effect)

Question 13

Describe the process of uncoupling and heat generation

Answer 13

• Some organisms can uncouple oxphos from ATP synthesis
• Used to generate heat and maintain body temperature (hibernating animals)
• Happens in brown adipose tissue
• Rich in mitochondria
• Reddish brown due to cytochromes and hemoglobin
• Inner mitochondrial membrane contains uncoupling protein (UCP 1) aka thermogenin.
• Transfers protons from cytoplasm to matrix side
• Energy converted to heat instead of ATP
• UCP 2 and UCP-3 also uncouple oxphos from ATP synthesis
• Play role in energy homeostasis
• See Slides 30-31

Question 14

Describe the inhibition of oxidative phosphorylation

Answer 14

• Inhibition of ETC – next slide
• Inhibition of ATP synthase – oligomycin (antibiotic and antifungal agent) inhibits influx of proteins into ATP synthase by binding to c su
• Uncoupling electron transport from ATP synthesis – by 2, 4 – dinitrophenol. Dissipates the proton gradient
• Inhibition of ATP export – atractyloside and bongkrekic acid inhibit ATP-ADP translocase
• See Slide 33