Cytochrome b559 content in isolated photosystem II reaction center preparations

The cytochrome b559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized-minus-reduced difference extinction coefficient of cytochrome b559 at 559 nm. The relative amount of D1 and cytochrome b559alpha-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b559alpha-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their alpha-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b559alpha-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes.