Inhibition of plastoquinol oxidation at the cytochrome bf complex by dinitrophenyl ether of iodonitrothymol (DNP-INT) depends on irradiance and H uptake by thylakoid membranes.

Inhibitory analysis is a useful tool for studying reactions in the photosynthetic apparatus. After introducing by Aachim Trebst in 1978, dinitrophenylether of iodonitrothymol (DNP-INT), a competitive inhibitor of plastoquinol oxidation at the cytochrome (cyt.) bf complex, has been widely applied to study reactions occurring in the plastoquinone pool and the cyt. bf complex. Here we examine the inhibitory efficiency of DNP-INT by implementing three approaches to estimate the extent of blockage of electron flow from the plastoquinone pool to photosystem I in isolated thylakoids from spinach (Spinacia oleracea). We confirm that DNP-INT is a potent inhibitor of electron flow to photosystem I and demonstrate that inhibitory action of DNP-INT depends on irradiance and H uptake by thylakoid membranes. Based on these findings, we infer that affinity of the quinol-oxidizing site of the cyt. bf complex to DNP-INT is increased in the light due to hydrogen bonding between DNP-INT molecules and acidic amino acid residue(s), which is (are) protonated in the light.