Study on the photosynthetic mechanism by spectroscopic measurements of cyanobacterial mutants

利用光致傅里叶变换红外（FTIR）差分光谱分析了嗜热蓝藻热聚球菌（Thermosynechococcus elongatus）突变体，研究了光系统II （PSII）配合物的结构和反应。得到了以下结果：利用组氨酸标记CP43蛋白的高活性PSII核心复合物，研究了水氧化的反应机理。我们首次检测了底物水在反应周期中单个s态跃迁（S_1→S_2、S_2→S_3、S_3→S_0、S_0→S_1）的反应，水OH的FTIR波段变化幅度为3500 ~ 3650 cm^<-1>)。用^<13>C和^<15>N同位素取代法鉴定了氨基酸侧链和多肽主干的变化。此外，通过测量不同pH值下s态循环的闪光诱导FTIR光谱，研究了单个s态转变的pH依赖性。除S_1→S_2外，3个s态转变在酸性区均有明显的抑制作用，表明在这些转变中存在质子释放。我们制备了将D2-Tyr160 （YD）替换为Phe （D2-Y160F）的长叶藻突变体，并研究了外围电子给体Y_D对初级电子给体叶绿素P680的作用。D2-Y160F突变体光诱导形成P680时的FTIR差谱显示，WT中碳甲氧基C=O拉伸带在1751 cm^<-1>处明显下降。这表明Y_D通过氢键网络与P680相互作用，Y_D的氧化状态通过这种相互作用控制P680的氧化还原电位。

We have examined the structure and reactions of photosystem II (PSII) complexes by analyzing the mutants of the thermophilic cyanobacterium Thermosynechococcus elongatus by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy. The following results were obtained.1.Using the highly active PSII core complexes in which the CP43 protein is histidine tagged, the reaction mechanism of the water oxidation was studied. We have for the first time detected the reactions of substrate water in the individual S-state transitions (S_1→S_2,S_2→S_3,S_3→S_0,S_0→S_1) in the reaction cycle as the changes in FTIR bands of the water OH stretches (3500-3650 cm^<-1>). The changes in the amino acid side chains and polypeptide backbones were also identified using ^<13>C and ^<15>N isotope substitution. Furthermore, pH dependence of the individual S-state transitions was studied by measurements of flash-induced FTIR spectra of S-state cycle at various pHs. Remarkable inhibition in the acidic region was observed in the three S-state transitions other than the S_1→S_2, indicating the presence of proton release at these transitions.2.We have prepared the T.elongatus mutant in which D2-Tyr160 (YD) was replaced with Phe (D2-Y160F) and studied the role of Y_D, the peripheral electron donor to the primary electron donor chlorophyll P680. The FTIR difference spectrum upon light-induced P680 formation of the D2-Y160F mutant showed a clear downshift of carbomethoxy C=O stretching band at 1751 cm^<-1> observed in WT. This observation indicates that Y_D interacts with P680 by a hydrogen-bonding network and it is suggested that the oxidation status of Y_D controls the redox potential of P680 through this interaction.