Chlorophyll fluorescence measurements to assess the competition of substituted anthraquinones for the QB binding site

As analogs of the Photosystem II plastoquinone electron acceptor, QB, substituted quinones compete with QB for a common binding domain and thereby inhibit QB function. Substituted quinones interact with the QB binding niche via hydrogen bonds, and the extent of hydrogen bond formation is determined by quinone structure. We have previously shown that the quinone inhibitory activity can be quantitated using measurements of chlorophyll fluorescence quenching. To assess competition for the QB binding site, we report here measurements of the action of various pairs of substituted anthraquinones on the chlorophyll fluorescence emission of barley chloroplasts. The degree of competition between quinones for the QB binding site is classified as competition, partial competition, or no competition. Two quinones were classified as undergoing competition, i.e., interacting for the same or overlapping sites, if the chlorophyll fluorescence level in the presence of the two quinones was not as low as that achieved in the presence of either one of the quinones individually. Non-competitive quinones with different binding sites quenched chlorophyll fluorescence to the level expected if the quenching effects of the individual quinones were additive. Partial competition, or some interaction for the same or overlapping sites, was characterized by an extent of fluorescence quenching in the presence of two quinones that was more effective than either quinone alone but not as sizable as that expected when the two quinones act independently. These results reflect an interesting situation whereby substitution patterns can alter the binding characteristics within a single class of inhibitors. In an accompanying manuscript we report the results of CNDO molecular orbital calculations to demonstrate that the pi charge distribution in substituted quinones governs their binding properties.