Photo-assembly and Efficiency of Photosynthetic Water Oxidases: Probing the Catalytic Core Atom by Atom

在这个由化学部生命过程化学项目颁发的奖项中，G。Charles Dismukes和Gennady M.来自新泽西罗格斯州立大学的Ananyev试图理解控制无机辅因子组装以形成光合生物光系统II（PSII）内的水氧化复合物（WOC）的物理化学原理。这种复合物是自然界唯一的酶溶液，可以利用阳光将水分解为分子氧、质子和电子。 尽管明显的保护WOC的催化核心，包括一个Mn 4CaO 5簇，两个氯离子，和近端氨基酸残基，催化周转率发生在PSII物种在体内，指示功能差异，尚未理解的大的差异。 该项目的一般实验方法使用有机，生物化学和无机化学策略。预期的结果将是确定可能的非天然无机辅因子的允许范围，这些辅因子可以支持PSII-WOC的催化功能，确定它们的相对结合亲和力和位点，并测量它们在不同物种的催化中的动力学性能。一个长期的目标是了解这类酶进化保守的化学基础。 这些研究将使用光合生物模型和使用天然细胞（体内）和分离的PSII复合物（体外）进行。将使用一些互补技术，允许在宽范围的闪烁频率的动力学性能进行比较，包括：超灵敏的电化学O2浓度和速率测量，叶绿素检测的快速重复率荧光法，和染料检测的动力学pH值测量。由于WOC中没有单一的无机成分可以独立发挥作用，我们将进行系统的“原子对原子”的分析，揭示它们的协同作用。我们对光合水氧化机制的理解的进展将为两个领域的替代能源研究提供信息。 首先，从这里提出的实验的见解可能会指导转基因光合生物的设计，例如，预计将有改善的生物量积累。 第二，阐明光合作用水氧化的原理将指导人工光合作用系统的水氧化催化剂的设计，能够生产可再生燃料作为化石燃料的替代品。 拟议的研究将使罗格斯大学的本科生和研究生，包括妇女和那些代表性不足的背景，为未来的职业生涯和可再生能源领域的高级培训的研究培训。这些拟议的研究将利用罗格斯大学两个NSF IGERT赠款的教育培训项目。 本提案中描述的新仪器的建造和现有仪器的升级将改善我们实验室的基础设施，并适应罗格斯大学的其他用户，同时支持多个合作者及其资助机构。

In this award from the Chemistry of Life Processes Program in the Chemistry Division, Drs. G. Charles Dismukes and Gennady M. Ananyev, from Rutgers, The State University of New Jersey, seek to understand the physico-chemical principles that govern the assembly of the inorganic cofactors to form the water-oxidation complex (WOC) within Photosystem II (PSII) of photosynthetic organisms. This complex is nature's sole enzymatic solution for splitting water into molecular oxygen, protons, and electrons using sunlight. Despite apparent conservation of the WOC catalytic core, comprised of a Mn4CaO5 cluster, two chloride ions, and proximal amino acid residues, large differences in catalytic turnover rates occur between PSII species in vivo, indicative of functional differences that are not yet understood. The general experimental approach of this project uses organismal, biochemical and inorganic chemical strategies. The expected outcome will be to determine the allowed range of possible non-native inorganic cofactors that can support catalytic functioning of PSII-WOCs, determine their relative binding affinities and sites, and measure their kinetic performance in catalysis across different species. A long term goal is to understand the chemical basis for the evolutionary conservation of this enzyme class. These studies will be conducted using model photosynthetic organisms and using both native cells (in vivo) and isolated PSII complexes (in vitro). A number of complementary techniques will be used that allow comparison of kinetic performance over a wide range of flashing frequencies including: ultra-sensitive electrochemical O2 concentration and rate measurements, chlorophyll-detected Fast Repetition Rate fluorometry, and dye-detected kinetic pH measurements. Because no single inorganic component of the WOC works independently, we will conduct a systematic 'atom-by-atom' analysis that reveals their synergistic contributions.Advances in our understanding of the mechanism of photosynthetic water oxidation will inform alternative energy research in two areas. First, insights from the experiments proposed here may guide the design of genetically modified photosynthetic organisms that, for example, are expected to have improved biomass accumulation. Second, elucidation of the principles of photosynthetic water oxidation will guide the design of water oxidation catalysts for artificial photosynthesis systems capable of producing renewable fuels as alternative to fossil fuels. The proposed studies would allow research training of Rutgers undergraduate and postgraduate students, including women and those of underrepresented backgrounds, for future careers and advanced training in the renewable energy field. These proposed studies will leverage educational training programs sponsored by two NSF IGERT grants at Rutgers. The new instruments built and upgrades to existing instruments described in this proposal will improve the infrastructure available in our laboratory and accommodate other users at Rutgers, while supporting multiple collaborators and their funding agencies.