Assembly and Function of the Cyanobacterial Photosystem II Complex

智力价值：氧气光合作用是生物圈中主要的太阳能转换过程，因此它是地球上生命的基础。光系统II(PSII)可以被认为是这一过程的关键酶，因为它利用光能分解水(水氧化)，从水中的氧气中释放出紧密结合的氢，同时使这些氢以一种形式被用来从无机碳(二氧化碳)中生成能量丰富的有机碳化合物。该项目研究了PSII的水氧化酶、受损的PSII蛋白的替换以及将Mn和Ca组装到活性部位的过程。PSII是一种动态结构，由于其在正常运行过程中不断遭受光损伤，因此处于持续的修复状态。修复是通过一个复杂的组装途径进行的，包括去除受损的D1蛋白，插入和蛋白质分解处理新生的D1蛋白前体，以及组装作为水氧化反应核心的锰团簇(4Mn-Ca)的金属原子。D1蛋白是上述大多数光损伤的基因位点。一种专门的和保守的D1损伤检测和替换机制已经进化到修复损伤，但仍有待了解。1.4Mn-Ca组装机制。水氧化的活性部位含有四个锰和一个钙离子(4Mn-Ca)，其中大部分被不稳定的D1蛋白结合。4MN-Ca的组装需要光来驱动组装成活性金属簇。由于尚不清楚的原因，这种组装过程的量子效率非常低。之前由国家科学基金会支持的分析揭示了这一过程的新的动力学特征。关于这些新的动力学特征的起源的假设将使用定点突变以及跟随组装过程的敏感的生化和生物物理技术来评估。金属蛋白质组装过程正在一个动力学框架中考虑，如果有效，将代表金属簇组装的一个新的、随机的概念。在PSII修复周期中，检测在移除和替换受损的D1蛋白过程中的结构变化。D1蛋白被深埋在大的PSII复合体中，因此，在替换受损的D1以及插入和激活新的蛋白质副本的过程中，PSII复合体的主要重排必须发生。如何检测PSII复合体的内部损坏？启动修复的信号是什么？在替换过程中，d1蛋白发生了什么变化？与金属簇的组装相关的结构变化是什么？实验上使用了两种互补的方法来解决这些问题：靶向结构反应性方法，使用包含非天然半胱氨酸的突变菌株，这些突变菌株将与硫醇反应试剂反应，以定量探测表面暴露中特定部位的变化。这种定向的方法将使用强大的质谱学方法进行补充，这些方法有可能发现蛋白质中其他位置暴露的组装相关变化。广泛影响：教育影响包括学生接触到对有趣的生物学问题的多学科攻击，以及培训使用、操作和理解先进的质谱仪。计划与美洲原住民生物科学(NABS)计划开展教育合作。该项目包括一个教学模块，暂定名称为：“光与生命”。它将包含“生物困境”(一个多方面的现实世界问题)的练习和材料，供中学教师在家庭科学课上使用。这些项目将从研究项目中借用材料，并仿照早期项目中最成功的活动，其中包括与暑期学院期间访问校园的高中教师密切合作。

Intellectual Merit: Oxygenic photosynthesis is the major solar energy conversion process in the biosphere consequently it is at the very foundation of life on planet Earth. Photosystem II (PSII) may be considered to be the key enzyme of this process since it uses light energy to split water (water oxidation) liberating the tightly bound hydrogens from the oxygen of water and, at the same time, renders these hydrogens in a form used for producing energy-rich organic carbon compounds from inorganic carbon (carbon dioxide). This project investigates the water-oxidation enzyme of PSII, the replacement of damaged PSII proteins, and assembly of Mn and Ca into the active site.PSII is a dynamic structure that is under a constant state of repair owing to the incessant photodamage it incurs during its normal operation. The repair occurs via a complex assembly pathway involving the removal of damaged D1 protein, insertion and proteolytic processing of the nascent D1 protein precursor and the assembly of the metal atoms of the manganese cluster (4Mn-Ca), which is the heart of the water oxidation reaction. The D1 protein is the locus of most of the above mentioned photodamage. A dedicated and conserved D1 damage detection and replacement mechanism has evolved to repair the damage, but remains to be understood. 1. Mechanism of 4Mn-Ca assembly. The active site of water-oxidation contains a cluster of four manganese and one calcium ions (4Mn-Ca) most of which are bound by the labile D1 protein. The assembly of the 4Mn-Ca requires light to drive the assembly into an active metal cluster. For reasons that remain unclear, this assembly process occurs with very low quantum efficiency. Previous NSF-supported analysis revealed new kinetic features of this process. Hypotheses regarding the origin of these new kinetic features will be evaluated using site-directed mutagenesis together with sensitive biochemical and biophysical techniques that follow the assembly process. The metalloprotein assembly process is being considered in a kinetic framework, that if valid, would represent a new, stochastic concept of metal cluster assembly.2. Detection of structural changes during the removal and replacement of damaged D1 protein during the PSII repair cycle. The D1 protein is buried deeply within the large PSII complex and, consequently, major rearrangements of the PSII complex must occur during the replacement of damaged D1 and the insertion and activation of a new copy of the protein. How is internal damage within the PSII complex detected and what are the signals to initiate repair? What is happening to the D1 protein during replacement? What are the structural changes associated with the assembly of the metal cluster? Two complementary approaches to experimentally address these questions are used: targeted structure-reactivity approaches using mutant strains containing non-native cysteines that will be reacted with thiol-reactive reagents to quantitatively probe site-specific changes in surface exposure. This directed approach will be complemented using powerful mass spectroscopic methods which have the potential to discover assembly associated changes in exposure at other locations in the protein.Broader Impacts: Educational impacts include student exposure to a multidisciplinary attack on an interesting biological problem and training to utilize, operate, and understand advanced mass spectrometers. An education collaboration with the Native Americans in Biological Sciences (NABS) program is planned. The project incorporates a teaching module tentatively entitled: "Light and Life". It will contain exercises and materials for the "Biological Dilemma" (a multi-faceted real-world issue) and for secondary school teachers to utilize in their home science classes. These will borrow materials from the research project and be modeled on the most successful activities of earlier programs, which involved close collaboration with the high school teachers visiting the campus during the summer academies.