Molecular basis of the heat denaturation of photosystem II.
Molecular basis of the heat denaturation of photosystem II.
复制标题
光系统热变性的分子基础II。
DOI：
10.1021/bi00442a023
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Brudvig,GW
中科院分区：
文献类型：
--
作者：
Thompson,LK;Blaylock,R;Sturtevant,JM;Brudvig,GW
耶鲁大学化学系，纽黑文，康涅狄格州，06511收到1988年12月19日；修订稿收到1989年4月27日摘要：通过将差示扫描量热法观察到的吸热转变指定为光系统II复合体中特定蛋白质的变性来研究光系统II膜蛋白复合体的热变性。在先前的PSII膜的DSC研究中[Thompson，L.K.，Sturtevant，J.M.，&Brudvig，G.W.(1986)生物化学25,6161]，在30-70℃的温度范围内观察到五个DSC峰(AbA2，B，C和D)。A2峰被指定为对水氧化必不可少的成分的变性，而B峰被指定为对电子传递链的其余部分至关重要的成分的变性。我们现在通过热凝胶分析和电子顺磁共振(EPR)测量来扩展这些研究。热凝胶分析是一种依赖于膜蛋白变性时溶解性变化的技术，已被用于确定PSII复合体的所有主要膜蛋白的变性温度。EPR实验被用来监测叶绿素光氧化和酪氨酸的稳定性。DSC变性图谱中的B、C和D峰分别被指定为几种蛋白质的变性，这提供了有关PSII复合体组织成结构和功能单位的信息。峰B对应于外围核心蛋白和紧密结合的天线蛋白的变性，峰C对应于PSII核心的变性，峰D对应于松散结合的天线蛋白。在A2峰期间，没有观察到膜蛋白变性。A2峰被过氧化氢酶、超氧化物歧化酶、低氯和高pH改变。这些结果表明，当高氧化、隔离的锰络合物(水氧化中的活性中心)在高温下可进入水相时，会出现异常尖锐的A2峰。我们提出了一种锰配合物与氢氧化物离子反应的机理，它涉及到过氧化氢或超氧化物，并导致锰的还原和释放。提出的模型提供了众所周知的锰络合物的不稳定性和氯化物在稳定络合物中的作用的洞察。这可能有助于未来纯化过程的发展，并可能解释PSII的水氧化器对热变性的敏感性。iotossystem II(PSII)1是一种多组分膜蛋白复合体，它利用光能驱动上坡的电子传递反应和氧化水。水氧化反应发生在由四个锰离子组成的活性中心，是整个光合作用电子传递链中最热敏感的组成部分(Katoh&San Pietro，1967)。我们用差示扫描量热法(DSC)结合其他技术研究了PSII的热变性，以便深入了解复合体的结构组成和组成蛋白的单独作用，并确定水氧化器热敏性的分子基础。
Department of Chemistry, Yale University, New Haven, Connecticut 06511 Received December 19, 1988; Revised Manuscript Received April 27, 1989 abstract: The thermal denaturation of the photosystem II (PSII) membrane protein complex is investigated by assigning the endothermic transitions observed by differential scanning calorimetry (DSC) to the de-naturation of particular proteins of the PSII complex. In a prior DSC study of PSII membranes [Thompson, L. K., Sturtevant, J. M., & Brudvig, G. W.(1986) Biochemistry 25, 6161], five DSC peaks were observed in the 30-70 C temperature range (Ab A2, B, C, and D). The A2 peak was assigned to denaturation of a component essential for water oxidation and the B peak to denaturation of a component critical to the remainder of the electron-transport chain. We have now extended these studies with thermal gel analysis and electron paramagnetic resonance (EPR) measurements. Thermal gel analysis, a technique which relies on a change in the solubility properties of a membrane protein upon denaturation, has been used to determine the temperatures of denaturation of all of the major membrane proteins of the PSII complex. EPR experiments have beenused to monitor chlorophyll photooxidation and the stability of TyrD+. Peaks B, C, and D in the DSCdenaturation profile are each assigned to the denaturaation of several proteins, which provides information on the organization of the PSII complex into structural and functional units. Peak B corresponds tothe denaturation of peripheral core proteins and closely associated antenna proteins, peak C to the PSII core, and peak D to the loosely associated antenna proteins. No membrane protein is observed to denature during the A2 peak. The A2 peak is altered by the presence of catalase, superoxide dismutase, low chloride, and high pH. These results suggest that the abnormally sharp A2 peak occurs when the highly oxidizing, sequestered Mn complex (the active site in water oxidation) becomes accessible to the aqueous phase, at elevated temperatures. We propose a mechanism for the reaction of the Mn complex with hydroxide ions, which involves peroxide or superoxide and results in the reduction and release of Mn. The proposed model provides insight into the well-known instability of the Mn complex and the role of chloride in stabilizing the complex. This may enable the future development of purification procedures and may explain the sensitivity of the water-oxidizing apparatus of PSII to heat denaturation.I^ iotosystem II (PSII) 1 is a multicomponent membrane protein complex which utilizes light energy to drive uphill electron-transport reactionsand oxidize water. The water oxidation reaction, which occurs at an active site composed of four Mn ions, is the most heat-sensitive component of the entire photosynthetic electron-transport chain (Katoh & San Pietro, 1967). We haveused differential scanning calorimetry (DSC) in combination with othertechniques to investigate the heat denaturation of PSII, in order to provide insight into the structural organization of the complex and the individual roles of the component proteins, as well as to determine the mo-lecular basis of the heat sensitivity of the water-oxidizing apparatus.