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

该奖项由化学部门的生命过程化学项目颁发。来自新泽西州立大学罗格斯分校的G. Charles Dismukes和Gennady M. Ananyev试图了解控制光合生物光系统II （PSII）中无机辅助因子组装形成水氧化复合物（WOC）的物理化学原理。这种复合物是自然界唯一的利用阳光将水分解成氧分子、质子和电子的酶解溶液。尽管WOC催化核心由Mn4CaO5簇、两个氯离子和近端氨基酸残基组成，但PSII物种体内的催化转化率存在很大差异，这表明功能差异尚不清楚。本项目的一般实验方法采用有机、生化和无机化学策略。预期的结果将是确定可能支持PSII-WOCs催化功能的非天然无机辅因子的允许范围，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间催化的动力学性能。长期目标是了解这类酶的进化保护的化学基础。这些研究将使用模式光合生物和原生细胞（体内）和分离的PSII复合物（体外）进行。将使用许多互补技术，以便在广泛的闪烁频率范围内比较动力学性能，包括：超灵敏电化学O2浓度和速率测量，叶绿素检测快速重复率荧光法和染料检测动力学pH测量。由于WOC中没有单一的无机组分独立工作，我们将进行系统的“原子-原子”分析，以揭示它们的协同作用。我们对光合作用水氧化机制的理解的进步将为两个领域的替代能源研究提供信息。首先，这里提出的实验的见解可能指导转基因光合生物的设计，例如，有望改善生物量积累。其次，阐明光合作用水氧化原理将指导设计用于能够生产可再生燃料作为化石燃料替代品的人工光合作用系统的水氧化催化剂。拟议的研究将允许罗格斯大学的本科生和研究生，包括女性和那些代表性不足的背景，为未来的职业和可再生能源领域的高级培训进行研究培训。这些拟议的研究将利用由罗格斯大学两项NSF IGERT资助的教育培训项目。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可用的基础设施，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.