Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes

Abstract

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...

A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus

“…The inhibitors ophenanthroline and terbutryn were found by X-ray studies to bind within Q~ site in R. viridis RC [5,6]. These structural studies confirmed and extended the previous proposals based (1) on photoaffinity labelling with azido-atrazine which binds to the L and D1 subunits [7,8] and (2) on the localization of almost all mutations conferring herbicide resistances which map to the part of genes coding for L and D1 residues located in the QB site [9 11]. …”

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the ‘H’ subunit of bacterial reaction centres?

“…The secondary structures of the L-and M-subunits have been confirmed by the elucidation of the crystal structure of the Rhodopseudomonas viridis reaction centre by Michel, Deisenhofer and colleagues [32,33]. Moreover, a close functional similarity between the bacterial and eukaryotic reaction centres has long been assumed, based on spectrophotometric analyses of the electron acceptor reactions (see [34]) and on their similar sensitivities to triazine herbicides [35,36].…”

Problemática da ocorrência de diferentes espécies de capim-colchão (Digitaria spp.) na cultura da cana-de-açúcar

“…Embora ametrina, metribuzin, diuron e tebuthiuron tenham o mesmo mecanismo de ação, o sítio específico de ação desses herbicidas junto à quinona Qb é diferenciado (Christoffoleti et al, 2001). Portanto, essa pode ser a razão de ametrina e metribuzin com o mesmo mecanismo de ação (Pfister et al,1981;Oettmeier et al, 1984) Existem pelo menos quatro mecanismos gerais que podem explicar a resistência e/ou tolerância de plantas daninhas a herbicidas e influenciar o modo de ação desses compostos: baixa concentração do herbicida no local de ação; menor absorção e/ou translocação do herbicida; maior metabolização e/ou detoxificação do herbicida a substâncias menos tóxi-cas (De Prado et al, 1990;Sherman et al, 1996); e perda de afinidade do herbicida pelo sítio de ação específico (Sherman et al, 1996). Nesse sentido, Dias et al (2003) desenvolveram trabalho sobre tolerância de espécies de capim-colchão aos herbicidas aplicados na cultura da cana-de-açúcar, avaliando aspectos de eficácia de herbicidas e estudo dos mecanismos de tolerância.…”