Assembly and Function of the Cyanobacterial H2O-Oxidation Complex

蓝藻H2O氧化复合物的组装和功能

• 批准号: 0132356
• 负责人: Robert Burnap
• 金额: $ 31.5万
• 依托单位: Oklahoma State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0132356&HistoricalAwards=false
• 关键词: Assembly; Function; Cyanobacterial; H2O; Oxidation

Oxygenic photosynthesis is ultimately the source of the vast majority of biological energy that supports life on earth and is responsible for the generation of oxygen strictly required for respiration in all animals, including humans. Oxygenic photosynthesis depends upon a cluster of four manganese atoms, (Mn)4, associated with the catalytic site of H 2 O-oxidation of the membrane-bound photosystem II (PSII) complex. Recent advances in the determination of the molecular structure confirms that the (Mn)4) is sequestered within a region of the PSII complex formed by intrinsic and extrinsic proteins near the lumenal surface of the thylakoid membrane. Many outstanding questions remain and their mystery, in some cases, is only deepened by the newly available structure. The overall objective of this project is to connect the structure features of the cyanobacterial PSII H2O-oxidation complex (WOC) to the dynamical aspects of the assembly and function of the Mn cluster. Cyanobacteria are being used as the experimental model since the photosynthetic mechanism in these ubiquitous organisms is fundamentally similar to that found in higher plants and algae, but at the same time, more efficient methods for probing the photosynthetic process at the molecular level are available thereby shortening the time necessary to test the basic ideas of the research. The work combines molecular genetic, biochemical and biophysical approaches to clarify the process of photoactivation, which is the sequential light- dependent assembly of the catalytic tetramer of Mn atoms that forms the core of the WOC. The research is facilitated by the development of His-tag purification methods and strains for the gentle isolation of highly active and pure detergent-solublized PSII complexes. The ability to produce large amounts of mutant and wild-type PSII particles and extrinsic proteins now permits systematic manipulations of the ions and proteins of the WOC to better understand its assembly and function. While most mutants are already produced, a few additional mutations are being made to perform site-specific labeling to probe structural rearrangements using a cysteine-scanning mutagenesis/chemical modification approach. The experiments are of benefit to parallel biophysical analyses (e.g. FTIR, EPR) by defining the compositional and kinetic properties of specific mutant His-tagged PSII particles and highlighting the best preparative procedures for isolating and analyzing them. Together, these efforts complement on-going developments in the crystal structure determination by considering the dynamical aspects concerning the mechanism of light-driven assembly and activation of the Mn cluster.

氧光合物最终是支持地球生命的绝大多数生物能的来源，并负责在包括人类在内的所有动物中严格呼吸所需的氧气产生。充氧光合作用取决于四个锰原子（MN）4，与膜结合光系统II（PSII）复合物的H 2 O氧化的催化位点相关。分子结构测定的最新进展证实，（Mn）4）在由囊体膜的腔表面附近的固有和外在蛋白形成的PSII络合物区域内隔离。仍然存在许多杰出的问题，在某些情况下，它们的奥秘只有新近可用的结构才加深。该项目的总体目的是将蓝细菌PSII H2O氧化复合物（WOC）的结构特征与MN簇的组装和功能的动态方面联系起来。蓝细菌被用作实验模型，因为这些无处不在的生物中的光合作用机制与高等植物和藻类中发现的光合作用机制在根本上相似，但与此同时，同一时间，更有效的方法可以在分子水平上探索光合作用过程，从而可以缩短研究基本研究的必要时间。该作品结合了分子遗传，生化和生物物理方法来阐明光活化的过程，这是Mn原子的催化四聚体的顺序依赖性组装，形成了WOC的核心。通过开发HIS-标签纯化方法和菌株，可以轻轻分离高度活跃和纯净的清洁剂的PSII复合物，从而促进了这项研究。现在，产生大量突变体和野生型PSII颗粒和外部蛋白质的能力允许对WOC的离子和蛋白质进行系统的操作，以更好地了解其组装和功能。虽然大多数突变体已经产生，但还需要使用半胱氨酸扫描诱变/化学修饰方法进行一些其他突变来执行特定地点的标记以探测结构重排。实验可以通过定义特定突变体His标记的PSII颗粒的组成和动力学特性，并突出隔离和分析它们的最佳准备程序，从而对并联生物物理分析（例如FTIR，EPR）有益。这些努力共同考虑了通过考虑光驱动组装机理和MN簇的激活的机理的动力学方面来补充晶体结构确定中的持续发展。