What does treatment of the mitochondrial membrane with urea do?
uncouples ATP synthase by causing dissociation of the F1 portion
What part of ATP synthase contains the catalytic subunit?
F1
What is the Fo subunit of ATP synthase?
- membrane bound portion
- contains the proton channel
What inhibits the Fo subunit?
Oligomycin
Can O2 still be reduced to H2O when F1 is removed from ATPase? Why or why not?
Yes, Removing F1 does not inhibit the reducing abilities of the ETC complexes
note: ATP is NOT being synthesized if not F1 is present so respiration is UNCOUPLED
How do uncouplers work?
- They get protonated in the relatively acidic intermembrane space where protons are being pumped
- Pronation neutralizes their charge allowing them to cross the inner membrane
- once in the matrix they loose the proton in the relatively basic environment
- The net affect is that protons are moving backwards
T or F: the general leakyness of the mitochondrial membrane causes uncoupling.
True
Give two examples of common uncouplers of the mitochondrial membrane.
- CCCP
- DNP
- FCCP
T or F: if no electrons flow, then no ATP can be made.
True
T or F: if no ATP is made then electrons cannot flow.
True
Why can electrons not flow in a normal cell if no ATP is being made?
- The proton gradient builds up to be so large that even the energy released by oxidation of NADH will not be enough to pump protons against such a large gradient
- During ATP synthesis by ATPase protons are pumped back to the matrix
In the presence of an uncoupling agent no ATP is made yet electrons continue to flow. Explain this.
- The proton gradient is reduced by the uncoupling agents that bring the protons back to the matrix side.
What is the respiratory control ratio, and what should it be in good preparations of isolated mitrochondria?
- Ratio of State 3 repiration to state 4 respiration
- Should be 5-6
T or F: uncouplers allow the mitochondria to resume to state 3 respiration even in the absence of ADP and Pi.
True, state 3 respiration will only stop at this point when the cell runs out of O2
What structural changes would you expect to see in a mitochondria if a uncoupling agent had been added.
- State 3 respiration will happen
- Large periplasmic space, Condensed Matrix
Why would a bear want to uncouple its membrane in the winter, and what protein would be used to do this?
- Uncoupling generates heat because the cell is working to build enough to shut of TCA and ETC but never does
- The end result of this continued cycle is heat production
- The bear would use UCP1
How does UCP1 work?
- Transports Fatty acid anions across the INNER mitochondrial membrane FROM MATRIX TO INTERMEMBRANE SPACE in exchange for Cl- or OH-
- The Fatty acids get protonated as soon as they cross the membrane
- Since they are neutral and lipophilic they can cross back over to the MATRIX without a transporter carrying the H+ with them
What did the experiement of Racker and Walther with light activated proton pumps show.
Exp. Recap.
- They made a vesicle that with bacteriohodopsin (light driven pump) that pumped in protons in when light was on, the only other thing on the vesicle was ATP synthase
- ATP was made when the light shined, since ATP synthase and Bacteriohodopsin were the only things present it proved NOTHING BESIDES ATP SYNTHASE AND A PROTON GRADIENT was need to make ATP.
What does the c subunit of ATP synthase consist of?
- 2 helices with an aspartate in the middle, which is REQUIRED for the proton channel
Would a substitution of glutamate for aspartate at the c subunit allow for continued function of ATP syntase?
Yes, what’s needed in that position is a carboxylate group that can easily grab and release a proton
How does DCCD (dicyclohexyl carboiimide) work?
- Reacts with c subunit blocking proton flow via the channel
Does ATP synthase rely on the mitochondrial genome to function?
Yes, Fo subunits are coded for by the mitochondrial genome
T or F: the F1 particle contributes to the long stalk.
True
T or F: oligomycin binds to the oligomycin sensitivity conferring protein.
FALSE
