Isolation and characterization of oxygen-evolving Photosystem II membranes from the cyanobacterium Synechocystis 6803

Abstract

A simple and rapid procedure has been designed for isolation of a highly active Photosystem II (PS II) subfraction from the cyanobacterium Synechocystis 6803. The thylakoid membranes used as starting material were isolated from cells treated with red light to increase the relative PS II content. The cell walls were broken in a glass-bead cell disruptor. The isolated thylakoids were pretreated with 0.01% dodecyl β-maltoside, followed by solubilization in 2.5% Triton X-100 to selectively extract photosystem I. The membrane residue, still containing PS II, was collected by a rapid differential centrifugation. The isolation PS II membranes possess rates of oxygen evolution as high as 2800–3200 μmol O 2/mg Chl h, and a low proportion of contaminating photosystem I. The yield of PS II is 30–35% on a PS II activity basis. Typically, PS II membranes corresponding to 2 mg of chlorophyll could be obtained within 6 h of preparative work, including the cell breakage. The purity and high intactness of the material allowed EPR analysis of various PS II parameters, including signal II slow and the S 2 state multiline signal. Considering the simplicity of the subfractionation procedure, the high yield and functional intactness, this preparation should have the potential of playing the same important experimental role for studying PS II in Synechocystis 6803 as the so called BBY-preparation has played for studies on PS II from plants. In particular, the preparation should be essential for studies on structure-function relations after site-directed mutagenesis of various PS II subunits in Synechocystis 6803 cells.