M2: ETC & OXPHOS 2 L13 Flashcards
What were the 2 competing theories on how ATP was synthesized and which one was right? Describe them.
- Chemi-osmotic theory: Movement of ions across a semi permeable membrane down their electrochemical gradient. This was driving the synthesis of ATP. CORRECT
- Chemical coupling hypothesis: ATP is synthesized from a high energy intermediate of the respiratory chain during oxidation. INCORRECT
What is the evidence proving that chemi-osmotic theory is correct?
- The respiratory chain can function in the absence of phosphate (Can uncouple oxygen consumption from the synthesis of ATP).
- The number of moles of ATP (2.5) generated through NADH oxidation was not an integer. You would expect a round number if a high energy intermediate made ATP, this suggests the high energy intermediate hypothesis was not correct.
- An intact IMM is required for OXPHOS. If you permeabilize the mitochondrial membrane you cant make ATP because you don’t get the proton motive force anymore because they can just leak back in.
- Key electron transport proteins span the IMM
- Uncouplers such as 2,4-dinitrophenol (DNP) inhibits ATP synthesis. (but you still get O2 consumption)
- Generating artificial proton gradient permit ATP synthesis without electron transport.
What did we learn from the chemical coupling hypothesis even though it was WRONG?
They thought that the energy changes from the reduction potentials through each complex of the ETC were enough to make ATP. But they were wrong. (Before, these complexes were called coupling sites). This energy doesn’t directly make ATP, but it partially contributed to the proton motive force which is responsible for generating ATP through ATP synthase
Explain how the different complexes of the ETC can be blocked (by poisons) to alter the oxygen consumption.
If you add mitochondria and you give a substrate (ex: B-hydroxybutyrate) this generates NAD linked respiration. B-hydroxybutyrate is a substrate of the CAC that will generate NADH so electrons will come into complex 1. When that happens respiration starts and oxygen consumption occurs.
Rotenone is a poison that inhibits complex 1, because electrons are coming into complex 1 electron flow stops and respiration stops.
Succinate bypasses complex 1 and goes to complex 2 (can bypass the poison). So when you add succinate, respiration restarts.
Then you can block complex 3 with antimycin. This blocks any electrons coming in ahead of complex 3 so respiration stops.
Then you can add TMPD and ascorbate which give electrons straight to cytochrome c and you get consumption of oxygen.
Explain the experiment done on complex 1 to determine what its P/O ratio is.
They wanted to know how complex 1 contributed to the proton motive force. They inhibited complex 3 with Antimycin A. Then ferricyanide was added as an artificial electron acceptor so electrons could continue to flow if they were donated upstream. If you block complex 3, and give electrons into complex 1, nothing happens because electrons would stop. But if you have a release valve through ferricyanide, electrons can come in through complex 1 and come out through ferricyanide. This electron path allows complex 1 to only pump protons. So we isolated the function of complex 1. They found a P/O ration of 1. (Makes sense bc 2.5-1.5 = 1).
Explain the experiment done on complex 3 to determine what its P/O ratio is.
Isolating complex 3: they added exogenous cytochrome c which allows electrons to flow (release valve) and cyanide to block complex 4 activity. This forces electrons to exit through the exogenous cytochrome c. They bypass complex 1 by giving succinate (enters through complex 2). P/O ratio is 0.5 ATP per oxygen.
To test whether the protons were being pumped by complex 2 or 3, they blocked complex 3 with antimycin A. When they did this no protons were pumped. This confirmed that complex 3 pumps protons but complex 2 does not.
Explain the experiment done on complex 4 to determine what its P/O ratio is.
Isolating complex 4: they used artificial electron donors (ascorbate and TMPD) to reduce cytochrome c. You’re bypassing everything upstream which isolates complex 4. Complex 4 oxidizes cytochrome c and makes water. They got a P/O ratio of 1.
What is DNP and how does it work?
DNP is a weak acid found in the inter membrane space of the mitochondria. In the inter membrane space there is a higher proton concentration relative to the matrix. DNP carries protons through the inner mitochondrial membrane and drops them off in the matrix. It can do this because DNP is lipophilic.
What is the consequence of DNP in the mitochondria?
Decreases the proton motive force because it is a proton leak pathway. This causes the mitochondria to oxidize more NADH, consume more oxygen, and pump more protons to try and maintain the proton motive force. Conclusion: respiration increases in the presence of chemical uncouplers.
Why can DNP be used for weight loss?
DNP causes massive amounts of weight loss because you are dissipating the energy in your body to generate heat rather than work or energy (not making ATP).
What is UPC1? What is significant about it?
UPC1 is an endogenous uncoupler in brown adipose tissue. UPC1 allows protons to re-enter the matrix and increases energy expenditure. They think that by understanding how brown adipose tissue increases energy expenditure they could combat obesity accelerated diseases.
What is the most believed origin of the mitochondria?
The endosymbiosis hypothesis: Aerobic bacteria was engulfed by an archaea. This provided the host with oxidizable substrates and the endosymbiont was provided with energy. Most of the genes from the endosymbiont transferred to the nuclear genome= the relationship was sealed. These bacterium are mitochondria.
How many proteins are encoded for by the mitochondrial genome?
13
Where are most mitochondrial proteins encoded?
In the nucleus of the cell. They need to be translated in the cytosol and imported into the mitochondria.
How can mitochondrial diseases be obtained?
Mitochondrial diseases arise from genes mutated in mitochondrial DNA but can also arise when you have mutations in nuclear encoded genes (including OXPHOS assembly factors that help put together complexes 1-4 of ETC) that affect mitochondrial function.
