Telfer A, Bishop S M, Phillips D, Barber J
Wolfson Laboratories, Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London, United Kingdom.
J Biol Chem. 1994 May 6;269(18):13244-53.
Singlet oxygen formation by photosystem II reaction centers isolated from Pisum sativum has been detected by two chemical trapping techniques: histidine-dependent oxygen uptake and bleaching of p-nitrosodimethyl-aniline by the intermediary endoperoxide of histidine. The quantum yield of singlet oxygen formation determined by these methods was estimated to be 0.16 by comparison with the known quantum yields of standard singlet oxygen sensitizers. Singlet oxygen was formed on illumination of reaction centers under conditions that lead to formation of the triplet state of the primary electron donor, P680. Experiments with deuterated buffer and active oxygen scavengers indicated that singlet oxygen was the only active oxygen species produced by this reaction. Neither azide nor histidine, which are scavengers of singlet oxygen, protected against photobleaching of the chlorophyll of reaction centers that occurs concomitantly with singlet oxygen formation, suggesting that bleaching involves singlet oxygen generated within the protein matrix of the complex. Singlet oxygen sensitized exogenously by rose bengal (when excited specifically at 550 nm) was also found to bleach reaction center chlorophyll in a manner similar to the intrinsic mechanism. We conclude that singlet oxygen formed within the hydrophobic interior of the reaction center attacks the chlorophylls of P680, and presumably also amino acids in the vicinity, and that only the singlet oxygen that escapes to the medium is affected by added scavengers or deuterated medium. These experiments extend our earlier report of the detection of singlet oxygen by its luminescence at 1270 nm when isolated photosystem II reaction centers are illuminated (Macpherson, A. N., Telfer, A., Barber, J., and Truscott, T. G. (1993) Biochim. Biophys. Acta 1143, 301-309). Moreover, our results support the hypothesis that production of singlet oxygen underlies the vulnerability of photosystem II to photodamage and hence necessitates the rapid turnover of the D1 protein of the reaction center.
通过两种化学捕获技术检测了从豌豆中分离出的光系统II反应中心产生单线态氧的情况：组氨酸依赖性氧摄取以及组氨酸中间内过氧化物对对亚硝基二甲基苯胺的漂白作用。与标准单线态氧敏化剂的已知量子产率相比，通过这些方法测定的单线态氧形成的量子产率估计为0.16。在导致初级电子供体P680形成三重态的条件下，照射反应中心时会形成单线态氧。用重水缓冲液和活性氧清除剂进行的实验表明，单线态氧是该反应产生的唯一活性氧物种。作为单线态氧清除剂的叠氮化物和组氨酸都不能防止与单线态氧形成同时发生的反应中心叶绿素的光漂白，这表明漂白涉及复合物蛋白质基质内产生的单线态氧。还发现孟加拉玫瑰红（在550 nm处特异性激发时）外源敏化的单线态氧以类似于内在机制的方式使反应中心叶绿素漂白。我们得出结论，反应中心疏水内部形成的单线态氧攻击P680的叶绿素，大概也攻击附近的氨基酸，并且只有逸出到介质中的单线态氧会受到添加的清除剂或重水介质的影响。这些实验扩展了我们早期的报告，即当分离的光系统II反应中心受到光照时，通过其在1270 nm处的发光检测到单线态氧（Macpherson，A. N.，Telfer，A.，Barber，J.，和Truscott，T. G.（1993年）Biochim. Biophys. Acta 1143，301 - 309）。此外，我们的结果支持这样的假设，即单线态氧的产生是光系统II易受光损伤的基础，因此需要反应中心的D1蛋白快速周转。
