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 β‐carotene in two forms of the photosystem II reaction center (Dl‐D2‐cytochrome (Cyt) b559) complex containing one and two β‐carotene molecules per two pheophytin a. All the handling steps and HPLC analyses were done in darkness at room temperature and at 4°C. Two different pigment extracts were analyzed, a mixture of chlorophyll a, pheophytin a and 3‐carotene, and the isolated p‐carotene from that mixture. In both cases only the all‐frans‐β‐carotene was detected. The chromatographic profiles were similar at both temperatures only differing in the retention times that were longer at 4°C. This result was independent of the concentration of photosynthetic starting material. Furthermore, no differences were observed between Dl‐D2‐Cyt b559 complexes with one and two p‐carotene molecules per reaction center. The analysis of the β‐carotene chromatographic peak indicated no 15‐cis to all‐fraws isom‐erization occurred during the HPLC chromatography in our experimental conditions. Resonance Raman spectra were also recorded in the isolated Dl‐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 M‐trans isomer and confirm previous results. The presence in the reaction center suspension of artificial electron acceptors such as silicomolybdate or 2,5‐dibromo‐3‐methyl‐6‐iso‐propyl‐p‐benzoquinone that are able to quench 3P680 did not modify the resonance Raman spectra of the native Dl‐D2‐Cyt b559 complex. The results suggest that no isomerization takes place during the laser irradiation.