The configuration of β-carotene in the photosystem II reaction center

Different pigment extraction procedures and HPLC methods were tested to investigate the geometric configuration of the beta-carotene in two forms of the photosystem II reaction center (D1-D2-cytochrome (Cyt) b559) complex containing one and two beta-carotene molecules per two pheophytin a. All the handling steps and HPLC analyses were done in darkness at room temperature and at 4 degrees C, Two different pigment extracts were analyzed, a mixture of chlorophyll a, pheophytin a and beta-carotene, and the isolated beta-carotene from that mixture. In both cases only the all-trans-beta-carotene was detected. The chromatographic profiles were similar at both temperatures only differing in the retention times that were longer at 4 degrees C. This result was independent of the concentration of photosynthetic starting material. Furthermore, no differences were observed between D1-D2-Cyt b559 complexes with one and two beta-carotene molecules per reaction center. The analysis of the beta-carotene chromatographic peak indicated no 15-cis to all-trails isomerization occurred during the HPLC chromatography in our experimental conditions. Resonance Raman spectra were also recorded in the isolated D1-D2-Cyt b559 complex at room and liquid nitrogen temperature with excitation at 514.5 nm from an Ar+ laser. Spectra of control preparations showed main bands at 1532, 1264, 1213, 1185, 1154 and 1003 cm(-1) corresponding to the all-trans isomer and confirm previous results. The presence in the reaction center suspension of artificial electron accepters such as silicomolybdate or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone that are able to quench (3)P680 did not modify the resonance Raman spectra of the native D1-D2-Cyt b559 complex. The results suggest that no isomerization takes place during the laser irradiation.