Studies on the Herbicide Binding Site in Isolated Photosystem II Core Complexes from a Flat-Bed Isoelectrofocusing Method

Abstract

Isoelectrofocusing has been used to separate various chlorophyll-protein complexes of photosystem two (PS II). Light-harvesting complexes containing chlorophyll a and chlorophyll b (LHC II) were located in bands having p/s in the region of 4.5. At slightly higher pH other light-harvesting complexes containing little or no chlorophyll b were found. In the most basic region of the isoelectrofocusing gel, were located PS II core complexes characterized by con­taining the proteins of CP47, CP43, D 1, D 2 and α-subunit of cytochrome b559. The number of PS II core bands depended on the particular conditions employed for the separation procedure and in some cases were contaminated by CP 29. It is suggested that this heterogeneity resulting from different protonation states of the PS II. The least-acidic PS II core complex (pI 5.5) was found to bind the herbicides atrazine, diuron and dinoseb. In contrast, a PS II core complex with a p / of 4.9 bound only diuron. Its inability to bind atrazine was shown to be due to the low pH but no such explanation could be found for dinoseb. When atrazine-resistant mutant Senecio vulgaris was used, no binding of radioactive atra­ zine was observed with the PS II cores having a p i of 5.5. It is therefore suggested that the normal atrazine binding observed with PS II cores involves the high affinity site detected with intact membranes. With the PS II cores, however, this site has a reduced affinity probably due to structural modification in the D 1-polypeptide resulting from the isolation procedures.

Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach

“…Some effects that have been attributed to the phosphorylation of the PSII core polypeptides include an inhibition of light-saturated PSII electron transfer (18,19), a destabilization of the anionic semiquinone QB-, and an accumulation of QA- (17). Recently, the existence of PSII core populations that differ in the extent of phosphorylation of their polypeptides has been described (9,11). It was possible to convert the cores from one form to another in the dark; however, this conversion was never complete because at least two differently phosphorylated populations were always detected (see Table II Table IV.…”

“…Control thylakoids were illuminated in the absence of ATP and MgCl2, washed with the suspension buffer, dark-adapted for 2 h, and finally NaF was added to a concentration of 5 mm. PSII membranes were isolated (3), solubilized in 1% DM (0.5 mg of Chl mnL1), and then applied to the cathode region of a flat-bed of granulated gel (9). The bed (2.5 x 22 cm) of Ultrodex (LKB) was prepared in 0.1 M glycine, 0.…”

“…1% DM, and 2% ampholine carrier ampholytes (pH range 3.5-5.5) and left at 40C in the dark. Isoelectrofocusing was carried out at a constant power of 3 W/tray for 16 h in an LKB Multiphor apparatus (9,11). PSII core populations separated in the granulated gel were eluted in a buffer containing 50 mm Mes (pH 6.5), 15 mm NaCl, 5 mM MgCl2, and 0.1% DM, and then filtered.…”

“…These PSII cores have been shown to have different capacities to bind dinoseb (9), suggesting that protein phosphorylation may reduce the ability of the QB pocket to bind herbicides.…”

Phosphorylation and disassembly of the photosystem II core as an early stage of photoinhibition

“…We have recently shown that a heterogeneity, which is probably regulated by light conditions, exists in the phosphorylated PSII core populations in grana membranes (Giardi et al 1990. These PSII cores show different QB pocket functioning with respect to plastoquinone (PQ) binding (Giardi et al 1992a).…”