Structure And Function Of α-carotene In Cytochrome B₆f From B. Corticulans

In oxygenic photosynthesis, the integral membrane cytochrome b₆f complex (Cyt b₆f) connects the reaction centers of photosystems I and II electronically by oxidizing lipophilic plastoquinol and reducing plastocyanin or cytochrome c6. This electron transfer process is coupled to proton translocation across the membrane that generates a transmembrane electrochemical proton gradient used to synthesize adenosine triphosphate (ATP). Therefore, Cyt b₆f plays a very important role in the light energy transducing process of photosynthesis. Although High-resolution X-ray crystallographic structures of the Cyt b₆f unambiguously show chlorophyll a (Chl a) and β-carotene are intrinsic components of the Cyt b₆f complex, their physiological functions are not yet available. It plays a key role to further investigate the functions of these pigments in Cyt b₆f complex for elucidating the mechanism of photosynthetic energy transduction. In this work, Cyt b₆f complex from a kind of marine green alga, Bryopsis corticulans (B.corticulans) was used for experimental materials, due to no available data of Cyt b₆f complex from marine green alga in these days. In order to purify the Cyt b₆f complex from a kind of marine green alga, B.corticulans, the purification procedure of spinach Cyt b₆f was modified: Firstly, the 2 M NaBr membrane washing step was repeated to completely remove the extrinsic protein; Secondly, the second ammonium sulfate fractionations was changed and the precipitates formed from 38-45% were determined to be the Cyt b₆f preparation. Using this modified method, the pure and active Cyt b₆f was purified from B.corticulans chloroplast at the first time. SDS-PAGE shows that the molecular masses of four major polypeptides( Cytf ,Cyt b6 ,Rieske[Fe-s] and subunitIV ) in the Cyt b6f are 34.8, 24, 18.7and 16.7 kD, respectively. The ratio of Cyt b6 to Cyt f is 2.01. The purity is 9.9 nmol cyt f/mg. The electron transfer activity is 73 e/s. Through the analysis of HPLC and resonance Roman spectroscopy, the carotenoid in the B.corticulans Cyt b6f was determined to be α-carotene, a carotenoid different form β-carotene in Cyt b6f complex. The molecular ratios of all-trans, 9-cis -α-carotene and Chl a to Cyt f were determined to be 0.2, 0.7 and 1.2, respectively. It was shown that the α-carotene existed in an asymmetry protein environment by the measurement of CD spectroscopy. No lipid was detected in this complex by TLC. To study the function of 9-cis-α-carotene in B.corticulans Cyt b6f, fluorescence excitation spectroscopy, time-resolving absorption spectroscopy and the light bleaching experiment on Chl a in this complex were employed. The presence of efficient singlet excitation transfer from α-carotene to Chl a was found with an overall efficiency as high as 62%, and this energy transfer was considered to be occurred through F?ster mechanism. The photoprotective function of α-carotene to Chl a was proved, and this photoprotective function was performed through scavenging the singlet oxygen molecules. Moreover, the interactions between Chl a molecule and some amino acid residues existing in its protein mircoenvironment were proved. It also was thought to be one way for Chl a to protect itself. In addition, after alternate treatments with light induction and dark adaptation, the configuration changes of α-carotene in B.corticulans Cyt b6f were studied by HPLC. Base on these results, the reason why 9-cis-α-carotene was selected to bind in B.corticulans Cyt b6f was primarily discussed .