Characterization of a Purified Photosystem II-Phycobilisome Particle Preparation from Porphyridium cruentum

Abstract

Detergent preparations isolated from thylakoids of the red alga Por-phyridium cruentum, in a sucrose, phosphate, citrate, magnesium chloride medium consist of phycobilisomes and possess high rates of photo-system II activity. Characterization of these particles shows that the O2-evolving activity is stable for several hours and the pH optimum is about 6.5 to 7.2. Response of the system to light, electron donors and acceptors, and inhibitors verify that the observed activity, measured both as 02 evolution and 2,6-dichlorophenol-indophenol reduction, is due to photo-system II. Furthermore, photosystem II is functionally coupled to the phycobilisome in this preparation since green light, absorbed by phyco-bilisomes of P. cruentum, is effective in promoting both 02 evolution and 2,6-dichlorophenol-indophenol reduction. Photosystem II activity declines when light with wavelengths shorter than 665 nm is removed. Both 343,4-dichlorophenyl)-1,1-dimethylurea and atrazine inhibit photosys-tem II activity in this preparation, indicating that the herbicide binding site is a component of the photosystem II-phycobilisome particle. A PBS3 preparation with PSII activity has been isolated from a red alga, Porphyridium cruentum. Phycobilisomes are the pigment protein complexes, found only in red algae and cyanobac-teria, functionally analogous to Chl a/b complexes of higher plants. Energy absorbed by the PBS is primarily transferred to PSII. This leads to the assumption that the PBS is physically attached to PSII in the thylakoid membrane of red algae and cyanobacteria. Early attempts to fractionate the photosynthetic electron transport chain resulted in loss or significant decline in the 02 evolving activity of PSII. PSII preparations with high 02 evolving activity were first isolated by Stewart and Bendall (20) from a cyanobac-terium, Phormidium laminosum. Since then, similar preparations have been isolated from cyanobacteria and higher plants by others (2, 10, 11, 13, 19, 24). These workers did not attempt to retain the PBS light-harvesting complex. The specific interest 'Supported in part by Department of Energy Contract AS05-76ER-04310 and a Smithsonian Fellowship to B. M. C. 2 Present Address: Universite de Technologie de Compiegne, B.P. 233, 60206 Compiegne Cedex. 3Abbreviations: PBS, phycobilisome(s); PIIP, PSII-phycobilisome; SPCM, 0.5 M sucrose, 0.5 M potassium phosphate, 0.26 M potassium citrate, 10 or 15 mM MgCI2, pH 7; PBS-(Triton)-20C, phycobilisomes isolated at room temperature and solubilized with Triton X-100; PBS-(Triton)-5C, phycobilisomes isolated at 5°C and solubilized with Triton X-100; FeCN, ferricyanide; DMBQ, dimethylbenzoquinone; DCPIP, 2,6-dichlorophenol-indophenol; DPC, sym-diphenylcarbazide; I"o, concentration of inhibitor required for 50% inhibition. in our laboratory has been to ascertain if the PBS is closely associated with the PSII components as expected from energy transfer to PSII (14-16). We had already shown that in a high ionic medium (sucrose-phosphate-citrate) it was possible to retain intact PBS and PSII activity (7). Conditions normally used for PSII preparations (2, 4, 10,11, 13, 19, 24) result in the loss of PBS by dissociation. We recently reported the successful isolation in SPCM medium, of PIIP particles with high specific rates of PSII activity (1000-3000 ,umol 02* mg:' Chl * h-') from P. cruen-tum (5). Subsequently, this same medium has been used in the isolation of PSII particles and PBS from a cyanobacterium (17). The PIIP preparation from P. cruentum used in this study is deficient in PSI and has a low Chl content. By electron micros-copy the preparations were shown to be discreet particles, consisting primarily of phycobilisomes (6). Thylakoid membranes were extremely rare. In this presentation we show that green light, absorbed by phycoerythrin in the PBS of P. cruentum, is highly effective in driving PSII in PIIP preparations. This demonstrates that PSII does not merely co-isolate with the PBS but is functionally attached. Furthermore, we show that PIIP is a true PSII particle based upon its response to light, inhibitors, and electron donors and acceptors. Inhibitor studies indicate that the herbicide binding protein is a component of the red algal pho-tosynthetic apparatus and is also present in our PIIP preparations. MATERIALS AND METHODS Isolation of Phycobilisomes and PSII-Phycobilisome Particles. Porphyridium cruentum were grown as in Dilworth and Gantt (7) and havested after 7 d growth in the late exponential phase. The PIIP isolation was carried out in SPCM with particle solu-bilization using lauryl dimethylamine oxide as described in Clement-Metral and Gantt (5). PBS-(Triton)-20C and PBS-(Tri-ton)-5C were isolated basically as in Gantt et al. (12) by treatment with 1% Triton X-100, followed by isolation on sucrose step gradients. The final centrifugation for 2.5 h at 254,000g was omitted. PBS-(Triton)-5C were isolated at 4°C. PSII Assays. 02 evolution was measured at 25°C with a Clark-type electrode. Light from a slide projector was filtered through 10 cm water and a Schott KG 1 heat absorbing filter. The assay volume was 3.0 ml and typically contained 2 mM FeCN, 1 mM DMBQ, and 0.67 to 1% ethanol. The instrument was calibrated by the method of Robinson and Cooper (18). The solubility of 02 in air-saturated SPCM was found to be about 55% of the solubility of 02 in air-saturated water at 25°C. Therefore, values reported here are lower than those reported in Clement-Metral and Gantt (5) which were based on standardization of the electrode in air-saturated water. DCPIP measurements were done in an Aminco DW2 spectro-photometer with side illumination from a Dolan Jenner model 170-D illuminator. The absorbance change of 560 minus 520 nm was measured with a 3 nm slit width and an extinction 626