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的来源，并负责产生包括人类在内的所有动物呼吸所严格需要的氧气。氧光合作用依赖于一个由四个锰原子组成的簇（Mn）4，它与膜结合光系统II （PSII）复合物的h2o氧化催化位点有关。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，该区域由类囊体膜腔面附近的内源性和外源性蛋白质形成。许多悬而未决的问题仍然存在，在某些情况下，新的结构只会加深它们的神秘感。该项目的总体目标是将蓝藻PSII h2o -氧化络合物（WOC）的结构特征与Mn簇的组装和功能的动态方面联系起来。蓝藻被用作实验模型，因为这些无处不在的生物的光合作用机制与高等植物和藻类的光合作用机制基本相似，但同时，更有效的方法可以在分子水平上探测光合作用过程，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。该研究得益于his标签纯化方法和菌株的发展，这些方法和菌株可以温和地分离高活性和纯洗涤剂溶解的PSII配合物。产生大量突变型和野生型PSII颗粒和外源性蛋白质的能力现在允许系统地操纵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，但一些额外的突变正在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。这些实验有助于平行生物物理分析（如FTIR， EPR），通过定义特定突变his标记的PSII颗粒的组成和动力学特性，并突出分离和分析它们的最佳制备程序。总之，这些努力补充了晶体结构确定的持续发展，通过考虑有关光驱动组装和Mn团簇激活机制的动力学方面。

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.