Characterization of a D1-D2-cyt b-559 complex containing 4 chlorophyll a/2 pheophytin a isolated with the use of MgSO4

Abstract

A D1-D2-cyt b-559 complex containing 4 chlorophyll a, 1 β-carotene and 1 cytochrome b-559 per 2 pheophytin a has been isolated from spinach with 30% yield using a Q-Sepharose Fast-Flow anion-exchange column equilibrated with 0.1% Triton X-100, 10 mM MgSO4 and 50 mM Tris-HCl (pH 7.2). The preparation was then stabilized with 0.1% dodecyl-β-d-maltoside. This method gave a yield 10 times higher than that using a Fractogel TSK-DEAE 650(S) column equilibrated with 0.1% Triton X-100, 30 mM NaCl and 50 mM Tris-HCl (pH 7.2). The PS II RC complex was characterized using absorption and fluorescence spectroscopy at 277 and 77 K. A selective reversible bleaching under reducing conditions with maximum at 682 nm, associated with pheophytin a reduction, and light-induced absorption differences with a lifetime of 1.0 ms, ascribed to the triplet state of P680 were measured and indicated that the isolated Dl-D2-cyt b-559 complex is active in charge separation. The results are compared with the data obtained for a PS II RC preparation containing 6 chlorophyll a, 2 β-carotene and 1 cyt b-559 per 2 pheophytin a.