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

高等植物的光合作用对地球上的能量利用有着深远的影响。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，它几乎是呼吸用的二氧的唯一来源。对光合作用过程的透彻理解对于我们将来满足自身能源需求的能力至关重要。光系统II是高等植物和蓝藻的两大吸光反应中心之一。它还包含了一个不寻常的Mn4Ca催化中心——氧释放位点。水多步转化为二氧的机理尚不清楚。钙和氯在这个过程中都是重要的参与者，它们在氧气生产过程中似乎相互作用。作为Mn4Ca簇的一部分，钙可能在氧的形成中起直接作用。虽然氯化物不直接配位到Mn4Ca簇，但它是过渡到更高氧化态所必需的，并且似乎稳定了钙离子。本项目旨在帮助阐明钙在高等植物光系统II中的作用及其与氯化物活化的关系。所使用的方法将是表征钙耗尽或破坏在Mn4Ca催化中心的影响。两种生物化学干扰钙功能的方法将被采用：轻度降低pH和适度高氯化钠浓度的处理；氟是氯离子活化的竞争性抑制剂。这些处理的结果将使用电子顺磁共振（EPR）光谱进行检查，这是一种用于研究未配对电子的分子的技术。本项目的一个主要目的是阐明钙和氯对析氧复合体的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在氧释放激活中的相互依赖性。更广泛的影响该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部门进行。这所大学是一所中等规模的公共机构，学生群体种族多样，传统上强调本科教育。该大学最近经历了研究生课程的增长，包括该项目所在部门的一个新的博士课程。首席研究员之前已经培训了许多初级研究人员，他们已经继续深造或在工业界工作，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合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进行涉及酶动力学分析的实验，并参与EPR光谱样品的制备和工作。那些对物理方面的研究更感兴趣的人可以直接参与光谱测量和分析。总的来说，学生将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生命的支持具有重要意义，因此在校园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.