Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Abstract

The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio-and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa QB protein of chloroplast membranes.The purple, nonsulfur bacterium Rhodopseudomonas sphaeroides contains an intracytoplasmic membrane system that is derived from invaginations of the cytoplasmic membrane (1, 2). These membranes are designated as chromatophores because they are the site of localization of bacteriochlorophyll (BChl) and other pigments that function to harvest the light energy that drives photosynthesis. The bacterial reaction center (RC) is the site at which the absorbed radiant energy is converted into chemical energy by a charge separation across the photosynthetic membrane (3). The RC of R. sphaeroides is mechanistically an analog of photosystem II (PSII) in higher plant chloroplasts (4,5). In both PSII and the bacterial RC, a photon of light causes the oxidation of a special RC chlorophyll, with the resultant transfer of an electron to a tightly bound quinone (QA), which is then oxidized by a secondary quinone (QB) to form a stable semiquinone (Q-). A second photoreduction results in a second electron transfer to QB, resulting in production of the fully reduced quinol, which diffuses away from the RC and is cycled through the quinone pool to donate electrons to the remaining components of the electron transport chain (2-5).The RC of R. sphaeroides contains three polypeptides with estimated molecular weights of 28, 24, and 22 kDa, which are designated the H, M, and L subunits, respectively. In addition, purified RCs contain four BChls, two bacteriopheophytins, two ubiquinones, and one iron (2). Studies with purified antibodies suggest that the H and M subunits transverse the membrane, while the L subunit is minimally exposed on the periplasmic surface (6). The M and L subunits are resistant to protease treatments, indicating that they are integral membrane polypeptides (7, 8). The function of the H subunit is unknown, since the M-L complex still catalyzes charge separations even after removal of the H subunit (9). A possible structural role for the coordination of RC and light-harvesting chlorophyll interactions has been proposed for the H subunit in the RC of Rhodopseudomonas capsulata (10,11). The M subunit binds azidoanthraquinone and is believed to be the location of the primary quinone (QA) binding site (12). The secondary quinone (QB) binding site has not been unequivocally established, although antibodies prepared against the M subunit prevent electron flow from QA to QB, sugges...

Characterization of the photoinduced electron transfer reaction from the photosynthetic system in Rhodobacter sphaeroides to an exogenous electron acceptor

“…We found that v A and v B with R. sphaeroides chromatophores are not affected by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the buffer. We also examined the effect of 2-tert-butylamino-4-ethylamino-6-methylthio-1,3,5-triazine (also known as terbutryn), which has been reported to interfere with electron transport from Q A to Q B in purple bacteria more effectively than other triazine class inhibitors such as 2-chloro-4-ethylamino-6-isopropylaminos-chlorotriazine (also known as atrazine) [29,30]. Terbutryn was added to pH 8.0 buffer containing 25 lM R. sphaeroides chromatophores and 0.1 mM DCBQ, and v A and v B were measured.…”

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

“…In the bacterial reaction centers, QA and QB are bound to the M and L reaction center polypeptides, respectively. The L subunit has also been shown to bind PSll herbicides (Brown et al, 1984;DeVitry and Diner, 1984;Okamura, 1984). The molecular structures of the reaction center of Rhodopseudomonas viridis (Deisenhofer et ai., 1985) and Rhodobacter sphaeroides (Allen et al, 1987) have recently been determined by x-ray crystallographic analysis.…”

THE 32000 DALTON QB PROTEIN OF PHOTOSYSTEM II

“…This was confirmed by the observation that the inhibitors binding at the PQ binding site of the Qs-protein of PS I1 also inhibit U Q binding (albeit with a decreased affinity) of the QB-protein in the purple sulfur bacterium Rhodopseudomonas sphaeroides (Stein and Wraight, 1984). Indeed, photoaffinity tagging of the bacterial reaction center or chromatophores with ['4C]azidoatrazine results in the incorporation of most of the label in a single band; the L-subunit of the reaction center (deVitry and Diner, 1984;Brown et al, 1984). This finding was somewhat surprising as antibodies to the M-subunit, but not to the L-subunit, inhibited electron transfer from Qa to QB , and an azidoanthroquinone (ubiquinone analogue) covalently binds to the M-subunit following photoactivation (Marinetti et al, 1979).…”