固氮红细菌(Rhodobacter azotoformans) 423 nm特征吸收峰成因与定位

Formation origination of characteristic absorption peaks at 423 nm in Rhodobacter azotoformans

摘要:

【背景】在不产氧光合细菌中，因420?425 nm特征峰位于类胡萝卜素(carotenoid，Car)吸收区域，通常被认为是由Car积累引起，但固氮红细菌R7菌株呈现的423 nm特征峰不具备Car三指峰特征。【目的】阐明R7菌株423 nm特征吸收峰形成的物质基础及胞内定位。【方法】采用吸收光谱、薄层层析、高效液相色谱、质谱、超速离心和离子交换层析等方法阐明423 nm吸收峰形成原因。【结果】谷氨酸钠明显促进R7菌株活细胞呈现423 nm特征峰，色素提取液中该峰蓝移至415 nm，但其生长、细菌叶绿素(bacteriochlorophyll，BChl)和Car含量大幅度降低，而添加酵母提取物则反之。色素组成分析表明，在检测到的色素成分中，只有镁卟啉单甲基酯IX (Magnesium Protoporphyrin IX Monomethylester，MPE)呈现415 nm特征吸收峰。MPE可定位于光合膜上并呈现出423 nm特征峰。对色素蛋白复合体(Pigment Protein Complex，PPC)的研究显示，添加谷氨酸和酵母提取物的菌体细胞虽然都检测到3种PPC组分[2个外周捕光复合体(Peripheral Light Harvesting Complex 2，LH2)和1个光反应中心(Reaction Center，RC)]，但源自谷氨酸菌体细胞的RC和1个LH2则呈现423 nm特征吸收峰，表明R7菌株可产生2种不同类型的LH2，且MPE可定位于一种LH2和RC。【结论】R7菌株所呈现的423 nm特征峰不是由Car积累所致，而是由MPE积累所形成，且能与LH2和RC结合定位于光合膜上。MPE是BChl合成的中间产物，其合成受严格调控，不容易获得。MPE代谢调控的深入研究可为光合作用光氧化损伤与保护机理增添新内容。

Abstract:

[Background] The characteristic peak at 420?425 nm was frequently observed in anoxygenic phototrophic bacteria and often assigned to carotenoid component(s). However, the absorption peak at 423 nm in Rhodobacter azotoformans R7 did not exhibit the characteristic absorption of carotenoid(s). [Objective] To elucidate the formation mechanism of the 423 nm absorption peak in strain R7. [Methods] Analyses by UV-VIS spectrophotometry, thin layer chromatography, high performance liquid chromatography, mass spectrometry, ultracentrifugation and ion exchange chromatography were conducted to investigate the formation origin of the 423 nm peak. [Results] A characteristic absorption maximum at approximately 423 nm was displayed in the in vivo absorption spectrum of strain R7 cultured in glutamate medium, while it was shifted to approximately 415 nm in the absorption spectrum of pigment extract. However, the cell growth and the amounts of bacteriochlorophyll (BChl) and carotenoid (Car) of strain R7 were significantly reduced by addition of glutamate, compared to those with the supplement of yeast. Pigment composition analysis showed that the 415 nm peak was only characterized by magnesium protoporphyrin IX monomethylester (MPE). MPE could locate on the intracytoplasmic membrane with an absorption peak at 423 nm. These results indicated that the 423 nm absorption peak was caused by MPE accumulation. The analyses of pigment protein complex (PPC) showed that three components of PPCs were detected in both yeast and glutamate cultures, however, only the absorption spectra of peripheral light harvesting complex 2 (LH2) and the reaction center (RC) of glutamate cultures gave the 423 nm characteristic peak. Collectively, strain R7 was capable of produce two different types of LH2; MPE locating on either LH2 or RC could form the characteristic peak of 423 nm. [Conclusion] The characteristic peak at 423 nm in strain R7 originated from MPE accumulation rather than carotenoid. The accumulated MPE inside cells could bind to LH2 and RC and locate inside the intracytoplasmic membrane. It is difficult to obtain a large amount of MPEs as it is a biosynthetic intermediate of BChl. Strain R7 was capable of accumulating MPE, therefore, the further study for biosynthesis regulation of MPE contributes to elucidate the novel mechanism of photooxidation and photoprotection in photosynthesis.