In pea thylakoids, [C-14]atrazine is found to have two binding sites per active oxygen-evolving complex; one with high-binding affinity and one with low-binding affinity. The high affinity site has a K(d) = 80 nM, is present at a concentration of 120 nM (1 site / 470 Chl); binds in less than 500 ms; and blocks the electron flow reaction, Q(a)-Q(b) --> Q(a)Q(b)-. The low affinity site has a K(d) = 420 nM; is present at a concentration of 120 nM (1 site / 470 Chl); binds with a half-time of 4 to 5 s; and partially inhibits the charge recombination reaction in Photosystem II, Sn+1Q(a)- --> SnQ(a). In the same thylakoids, the concentration of Photosystem II centers active in oxygen evolution is 100 nM (1 / 540 Chl) and the concentration of those active in charge separation is 120 nM (1 / 450 Chl). Therefore, there are approximately two atrazine-binding sites per Photosystem II reaction center; one with high affinity and one with low affinity. High-affinity labelling with [C-14]azidoatrazine is associated with a 34.5 kDa protein, which is identified as D1, using polyclonal antisera. Low-affinity labelling with [C-14]azidoatrazine is associated with a 30-32 kDa protein, which is identified as D2 with the monoclonal antibody FQC3. Our findings indicate that both high- and low affinity sites are specific for Photosystem II function.
