The Interaction of Calcium and Chloride at the Oxygen Evolving Complex of Photosystem II

智力价值在地球上的能源利用中，高等植物的光合作用具有深远的影响。它是几乎所有生命形式的碳氢化合物能量的来源，通过捕获太阳的光能，它几乎是呼吸所需的氧气的唯一来源。对光合作用过程的透彻理解对于我们未来满足自身能源需求的能力至关重要。光系统II是高等植物和蓝藻两个主要的光吸收反应中心之一。它还包含放氧地点，一个不同寻常的Mn4Ca催化中心。水多步转化为氧气的机理尚不清楚。钙和氯化物都是这一过程中的重要参与者，它们似乎在制氧过程中相互作用。作为Mn4Ca团簇的一部分，钙可能在氧的形成中起着直接作用。虽然氯化物不直接配位到Mn4Ca原子簇，但它是向更高氧化态转变所必需的，似乎稳定了钙离子。本项目旨在帮助阐明钙在高等植物光系统II中的作用及其与氯激活的关系。所使用的方法将表征Mn4Ca催化中心的钙耗尽或破坏的影响。将使用两种生化干扰钙功能的方法：适度降低pH和中等浓度的氯化钠处理；以及氟化物处理，这是一种竞争性的氯激活抑制剂。这些处理的结果将使用电子顺磁共振(EPR)光谱进行检查，这是一种用于研究具有未配对电子的分子的技术。这个项目的一个主要目标是阐明钙和氯对放氧复合体EPR信号的影响。为了补充这些研究，第二个主要目标是使用酶动力学来表征钙和氯化物在氧气释放激活过程中的相互依赖。广泛影响这个项目将在一个主要侧重于让本科生和硕士研究生参与研究的系中进行。这所大学是一所中等规模的公立学校，学生人口具有种族多样性，传统上强调本科教育。该大学最近经历了研究生项目的增长，包括该项目所在系的一个新的博士项目。原则调查员以前已经培训了许多初级研究人员，他们继续深造或在工业界从事职业生涯，目前的项目旨在继续这种类型的培训。该项目涉及一系列适用于不同经验水平的生化和生物物理技术。初级研究人员可以相当独立地从事涉及酶动力学分析的实验，并参与用于EPR光谱分析的样品的准备和工作。那些对研究的物理方面更感兴趣的人可以直接参与光谱测量和分析。总体而言，学生可以接触到常用的生化方法和更专业的光谱技术。由于光合作用过程对支持生命具有重要意义，因此在校园内开展一个专注于光能转换和氧气生产的项目将有助于提高人们对能源利用问题的认识。

Intellectual MeritIn the utilization of energy on earth, photosynthesis by higher plants has a profound effect. It is the source of hydrocarbon energy for nearly all life forms through the capture of the sun's light energy and it is almost the sole source of dioxygen for respiration. A thorough understanding of the photosynthetic process is central to our ability to provide for our own energy needs in the future. Photosystem II is one of the two major light absorbing reaction centers of higher plants and cyanobacteria. It also contains the site of oxygen evolution, an unusual Mn4Ca catalytic center. The mechanism of the multiple step conversion of water into dioxygen is still not well understood. Both calcium and chloride are important participants in this process and they appear to interact with each other during oxygen production. As a part of the Mn4Ca cluster, calcium probably has a direct role in oxygen formation. Although chloride is not directly coordinated to the Mn4Ca cluster, it is required for transitions to higher oxidation states and appears to stabilize the calcium ion. This project is intended to help clarify the role of calcium in photosystem II of higher plants and its relationship to activation by chloride. The approach used will be to characterize the effects of calcium depletion or disruption at the Mn4Ca catalytic center. Two methods for biochemically disturbing the function of calcium will be used: treatment with mildly reduced pH and moderately high sodium chloride concentration; and treatment with fluoride, a competitive inhibitor of chloride activation. The results of these treatments will be examined using electron paramagnetic resonance (EPR) spectroscopy, a technique that is used to study molecules with unpaired electrons. A major objective of this project is to clarify the effects of calcium and chloride on the EPR signals from the oxygen evolving complex. To complement these studies, a second major objective is to use enzyme kinetics to characterize the interdependence of calcium and chloride in the activation of oxygen evolution.Broader ImpactThis project will be carried out in a department that has a major focus on involving undergraduate and Master's level graduate students in research. The university is a mid-sized public institution with a student population that is ethnically diverse and a traditional emphasis on undergraduate education. The university has recently experienced a growth in graduate programs, including a new PhD program in the department housing the project. The Principle Investigator has previously trained numerous beginning researchers, who have gone on to further graduate study or careers in industry, and the current project is designed to continue this type of training. The project involves a range of biochemical and biophysical techniques appropriate for various levels of experience. Beginning researchers can work fairly independently on experiments involving enzyme kinetics analyses and participate in the preparation and work-up of samples intended for EPR spectroscopy. Those more interested in the physical aspects of the research can participate directly in spectroscopic measurements and analysis. Overall, students gain exposure to both commonly used biochemical methods and more specialized spectroscopic techniques. Because of the great importance that photosynthetic processes have for supporting life in general, the presence on campus of a project that is focused on light energy conversion and oxygen production will help to raise awareness about energy utilization issues.