Assembly and Function of the Cyanobacterial H2O-Oxidation Complex

氧气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严格需要的氧气。氧合光合作用依赖于与膜结合的光系统II(PSII)复合体的H2O氧化催化位置相关的四个锰原子簇(Mn)4。最近在分子结构测定方面的进展证实，(Mn)4)被隔离在PSII复合体中，该复合体是由内源和外源蛋白形成的，靠近类囊体膜的管腔表面。许多悬而未决的问题仍然存在，在某些情况下，它们的谜团只会因为新的可用结构而加深。本项目的总体目标是将蓝藻PSII H2O氧化复合体(WOC)的结构特征与MN簇的组装和功能的动力学方面联系起来。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中的光合作用机制基本相似，但同时也有更有效的方法在分子水平上探测光合作用过程，从而缩短了检验研究基本思想所需的时间。这项工作结合了分子遗传学、生化和生物物理的方法来阐明光激活的过程，这是形成WOC核心的锰原子催化四聚体的顺序光依赖组装。His-Tag纯化方法和菌株的发展促进了这项研究，以温和分离高活性和纯洗涤剂溶解的PSII复合体。产生大量突变型和野生型PSII颗粒和外源蛋白质的能力现在允许对WOC的离子和蛋白质进行系统操作，以更好地了解其组装和功能。虽然大多数突变体已经产生，但一些额外的突变正在进行位点特异性标记，以使用半胱氨酸扫描突变/化学修饰方法来探测结构重排。这些实验有助于并行生物物理分析(如FTIR、EPR)，因为它定义了特定突变的His标记的PSII颗粒的组成和动力学性质，并突出了分离和分析它们的最佳准备程序。总而言之，这些努力通过考虑关于光驱动组装和激活MN团簇的机制的动力学方面，补充了晶体结构确定方面的正在进行的进展。

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.