Assembly and Function of the Cyanobacterial H2O-Oxidation Complex

含氧光合作用最终是支持地球上生命的绝大多数生物能量的来源，并负责产生包括人类在内的所有动物呼吸所需的氧气。含氧光合作用依赖于四个锰原子簇（Mn）4，与膜结合光系统II（PSII）复合物的H2O氧化的催化位点相关。分子结构测定的最新进展证实，（Mn）4）被隔离在PSII复合物的一个区域内，该区域由内源性和外源性蛋白质形成，靠近类囊体膜的内腔表面。许多悬而未决的问题仍然存在，在某些情况下，新的结构只会加深这些问题的神秘性。本项目的总体目标是连接的结构特点的蓝藻PSII水氧化复合物（WOC）的组装和功能的锰簇的动力学方面。蓝细菌被用作实验模型，因为这些普遍存在的生物体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基本相似，但与此同时，在分子水平上探测光合作用过程的更有效方法是可用的，从而缩短了测试研究基本思想所需的时间。这项工作结合了分子遗传学、生物化学和生物物理学的方法来阐明光活化的过程，这是形成WOC核心的Mn原子催化四聚体的顺序光依赖性组装。该研究是促进His标签的纯化方法和菌株的发展，温和的高活性和纯洗涤剂溶解PSII复合物的分离。产生大量突变体和野生型PSII颗粒和外源蛋白的能力现在允许系统操纵WOC的离子和蛋白质，以更好地了解其组装和功能。虽然大多数突变体已经产生，但正在进行一些额外的突变以使用半胱氨酸扫描诱变/化学修饰方法进行位点特异性标记以探测结构重排。实验是有益的平行生物物理分析（如FTIR，EPR）通过定义特定的突变体His标记的PSII颗粒的组成和动力学特性，并突出最好的制备程序分离和分析它们。总之，这些努力补充了正在进行的发展，在晶体结构的测定，考虑动力学方面的光驱动组装和激活的Mn集群的机制。

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.