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ABSTRACT2-Azido-4 -ethylamino-6 -isopropylamino-s-triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido ['4C] Many commercial herbicides inhibit photosynthetic electron transport by interrupting electron flow at the reducing side of photosystem II (PS II) (1, 2). There are several lines of evidence which indicate that this inhibition occurs at the level of a protein-bound plastoquinone called "B" (3). This electron carrier acts as the second stable electron acceptor of PS II (4, 5). It has been proposed that the mode of action of compounds such as atrazine or diuron is via high-affinity binding to the PS II complex (6). Herbicide binding induces a change in the redox potential of the quinone cofactor of B, thus making the transfer of electrons from the primary acceptor (Q) thermodynamically unfavorable (3,5). In addition, binding may decrease kinetic interactions of Q and B.Evidence that PS II inhibitors interact with a polypeptide of the PS II complex, possibly the apoprotein of B, comes from studies involving the enzyme trypsin (7). Proteolytic digestion of surface-exposed membrane polypeptides results in a destruction of inhibitor binding sites with a concomitant inactivation of the secondary acceptor, B (8). Attempts have.been made to correlate trypsin-mediated changes in polypeptides of the chloroplast membrane or detergent-solubilized PS II particles with changes in inhibitor binding properties (9). To date, however, the polypeptide(s) that determines the inhibitor binding site has not been identified.A variety of radiolabeled inhibitors of PS II have been used for the characterization of the properties of the herbicide-binding site; these studies have resulted in the demonstration of a single binding site per electron transport chain (3, 10). In addition, the affinity of various inhibitors for this site has been determined (6, 8). The association of PS II inhibitors with their binding site is noncovalent. For this reason, attempts at physical isolation of proteins labeled by a radioactive inhibitor have failed because detergent fractionation or electrophoretic separation rapidly leads to a new equilibrium and dissociation of the acceptor-inhibitor complex. The approach we have used to overcome this difficulty in identification of the herbicide receptor is to attach a radiolabeled photoaffinity azido derivative of atrazine to its high-affinity receptor polypeptide in a covalent manner. It is well established that activation of the azido function of photoaffinity-labeled compounds by UV irradiation produces a nitrene that is highly reactive (11). In preliminary investigations this was found to covalently link the azido-atrazine to chloroplast membranes (12, 13). MA...