Confusion on Inhibitors and Effects on ETC and ATP synthesis

[betterfuture]

Can someone explain the effects of the drugs on the ETC/proton gradient and their effects on ATP production and O2 consumption. I am honestly trying to get these down because whenever I encounter a question on anything related to inhibitors regarding ETC or ATP synthesis, I almost always get all the questions wrong.

-Oligomycin

-DNP

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[theonlytycrane]

Sample question:

And also check out a slight variant at this post:

http://forums.studentdoctor.net/threads/mitochondia-confusion.1196700/#post-17692342

 

[aldol16]

Okay, so let's start with oligomycin. Oligomycin is an inhibitor of ATP synthase. So if you know how ATP synthase works, it's driven by a proton "channel" (more like a proton wheel) that spins and that twisting force is conveyed up a rod that also turns and forces ATP molecules out at the active site. Remember that ATP synthesis is not the RDS for ATP synthase - rather, it's the release of ATP that requires the energy. So oligomycin is an inhibitor that blocks the proton channel, which then stops oxidative phosphorylation and respiration because there's no way to dissipate the proton gradient that builds up so at some point, Complexes I, III, and IV can no longer pump against the huge proton gradient. And since ATP synthase isn't turning, there's no ATP and the cell starves for energy.

DNP, or dinitrophenol, is an uncoupler. What it does is it uncouples proton transport from oxidative phosphorylation. That's a fancy way for saying it shuttles protons across the membrane so that the proton gradient can be dissipated. Think of it as punching holes in the inner membrane if it makes it easier for you. It's called an uncoupler because it uncouples proton transport from ATP synthesis. So complexes I, III, and IV can resume pumping protons across the membrane and the DNP will keep shuttling it back across, helping to dissipate the proton gradient so the complexes can stay active. Therefore, O2 will be consumed to do this. However, no ATP will be produced.

 

[theonlytycrane]

@aldol16 would you recommend knowing about oligomycin and DNP as outside knowledge?

 

[aldol16]

@aldol16 would you recommend knowing about oligomycin and DNP as outside knowledge?

No, I would recommend knowing what an uncoupler and inhibitor does, though. They might throw something new at you like "Compound A was found to block ATP synthesis but not affect O2 consumption. What is the likely mechanism of action?"

 

[betterfuture]

So for Oligomycin
(1) it inhibits ATP synthase by blocking the H+ from going down the Fo portion
(2) Since H+ can't go down the channel, that energy can't be used to release ATP molecules
(3) The H+ are basically left behind in the inner mitochondrial membrane space
(4) Complex I, III, and IV "notice" that there are still H+ present that they pumped previously and therefore won't pass down anymore electrons which will subsequently stop them from pumping anymore H+
(5) O2, the final acceptor, stops receiving electrons. So O2 builds up??? Or what happens to respiration. Still confused here

For DNP
(1) It basically pumps the H+ back into the matrix
(2) When it does this, the electrochemical gradient is destroyed and ATP can't be released from ATP synthase if there is no energy
(3) Complex I, III, IV don't "notice" this so they continue passing electrons and pumping H+
(4) O2 "notices" that ATP is not being generated and the cells need ATP, so it begins to increase respiration rate??? Confused here too.

 

[aldol16]

So for Oligomycin
(1) it inhibits ATP synthase by blocking the H+ from going down the Fo portion
(2) Since H+ can't go down the channel, that energy can't be used to release ATP molecules
(3) The H+ are basically left behind in the inner mitochondrial membrane space
(4) Complex I, III, and IV "notice" that there are still H+ present that they pumped previously and therefore won't pass down anymore electrons which will subsequently stop them from pumping anymore H+
(5) O2, the final acceptor, stops receiving electrons. So O2 builds up??? Or what happens to respiration. Still confused here

Correct on all counts. For (4), it's crucial to note that it's not them "noticing" that there are too many protons present in the intermembrane space but rather that the reactions fueling the pumps only generate some value of delta G that is coupled to the delta G of pumping against a gradient. Once that proton gradient starts building up even more, the delta G supplied by the reaction no longer is sufficient to maintain the pump.

For (5), the O2 doesn't build up because remember that [O2] is constantly in flux due to gas exchange. So the organism just breathes less and consumes less oxygen.

For DNP
(1) It basically pumps the H+ back into the matrix
(2) When it does this, the electrochemical gradient is destroyed and ATP can't be released from ATP synthase if there is no energy
(3) Complex I, III, IV don't "notice" this so they continue passing electrons and pumping H+
(4) O2 "notices" that ATP is not being generated and the cells need ATP, so it begins to increase respiration rate??? Confused here too.

For (4), remember that O2 doesn't "notice" anything. It's a molecule and so it's not a regulator in your body. Remember what complex IV does?

 

[betterfuture]

So basically, for Oligomycin, your saying the reaction of the complex pumping becomes nonspontaneous since it is not coupled, leaving it unfavorable to proceed, hence why they stop.

And for the rate of respiration, that's just Le Chatlier's principle in action?


And for DNP, your saying since complex I, III, and IV continue pumping, and I believe in complex IV, oxygen accepts the electrons and pumps H+ across, then respiration is undisturbed? Respiration rate is normal, but ATP decreases?

 

[aldol16]

So basically, for Oligomycin, your saying the reaction of the complex pumping becomes nonspontaneous since it is not coupled, leaving it unfavorable to proceed, hence why they stop.

No, with oligomycin, it's still coupled. That's why the pumping stops. In other words, think of a pump in a basement that pumps water outside, to ground level. Say your basement floods. The pump has a certain energy input that allows it to do work against gravity and pump water to ground level. Usually, once the water reaches ground level, it just rolls away. But say now you've walled off your backyard so that the water that's pumped there can no longer escape. This is equivalent to treating a cell with oligomycin. Now, that water you pump out of your basement is going to start building up in your backyard. Say it builds up so that your backyard is covered with 5 cm of water. Now, the pump is not only pumping against gravity but also against the additional pressure exerted by the 5 cm of water. Eventually, the water level of the backyard will get so high that it will exert a downward pressure that completely counteracts the energy input of the pump so that the pump no longer has enough energy to pump water out of your basement against its gradient.

And for DNP, your saying since complex I, III, and IV continue pumping, and I believe in complex IV, oxygen accepts the electrons and pumps H+ across, then respiration is undisturbed? Respiration rate is normal, but ATP decreases?

Yes, basically. You will continue consuming O2 but you won't be making any ATP.