I am a undergrad in a cell physiology class and just did a learning exercise involving testing rates of the electron transport chain via the reduction of DCPIP, analyzed by spectrophotometry. ( I think... D: )

So from what I understand, uncouplers stop the "connection" of the ETC and ATP synthase indirectly. And in our lab period we used a hydrogen ionophore(ammonia) as our uncoupler( I think... D: ).

So for our results I obtained this graph**(The Y axis should be absorbance(600nm)) sorry**...:

enter image description here

Where tube 1 through 3 was a collection of thykloid membranes isolated from spinach leaves; tube 1 being kept in foil until the 10th min mark to then measure absorbance as a control. Where tube 2 and 3 were exposed to light for the 10mins, with absorbance readings being taking every 1 min. And with tube 3 having the added ammonia(from what I understand, the uncoupler).

Therefore how does the ammonia uncoupler speed up the electron transport chain?

My thoughts after googling are that since PQ( plastoquinone) is partially responsible for establishing the proton gradient for the thylakoid mebrane, since it transports H+ ions from the stroma to the lumen upon reduction, that if there was this ammonia uncoupler, that could travel through the membrane due to its lack of charge, it could then bind with the H+ ions forming $\ce{NH4+}$ in the lumen.

So then if the lumen now has less H+ ions then the high(lumen) to low(stroma) H+ gradient would be ruined/weakened.

So this is the part I guess confused with; if there is less of a H+ gradient across the thylakoid membrane, then how does that have any bearing on the reduction of the added DCPIP or the speed at which its reduced?

Since tube 3 went the fastest to clear( ~0 absorbance), this is only when it is reduced yes? This means then that in tube 3, the reduction of DCPIP happened the fastest.

So then does this mean that DCPIP, the artificial electron acceptor, takes over for PQ somehow? Why does PQ have trouble "doing its job" in the presence of the weakened H+ gradient?

P.S: Sorry for the rambling, but I felt including my train of thought was important for others to see where I misunderstood.


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