Assembly and Functions of Photosynthetic Water Oxidases

描述（由申请人提供）：生物O2生产是地球上大气氧的主要可再生来源。水是在所有当代光合自养生物中通过光合作用产生的O2的来源，并且没有其他生物过程能够将水分解成O2。使用有氧代谢的所有生物体（包括人类）的存在和健康取决于光合作用衍生的O2。这种化学的催化作用是如此复杂，以至于迄今为止所研究的所有产氧光合自养生物中似乎只存在一种类型的酶位点。这项提案的长期目标是揭示生物水分解的化学基础。要使用的方法是分解不稳定的无机核心（Mn 4 OxCal Cl 1 -2），包括酶的活性位点，并从简单的无机辅因子，原子一个原子地重建它，使用受控序列的光-和暗-反应，这是已知的完全恢复功能酶。这种体外过程被称为光活化，并作为体内生物发生反应的模型。通过使用替代辅因子组装活性位点的“无机突变体”将用于探测天然辅因子的功能。关于组装动力学、原子组成/结构和原子力的信息将在组装中间体和完整的全酶上获得。要使用的工具包括新的方法，用于检测溶解的02浓度，质子浓度和收集的电子？用于顺磁系统的时间分辨和多维光谱学的自旋共振技术（EPR-plus）。研究将集中在产氧植物和原始蓝藻的酶。该研究的生物医学效益应包括：1）关于水分解酶的结构及其催化机制的基本信息; 2）对金属组装成金属蛋白的机制的一般理解; 3）基于光合酶所采用的原理设计水氧化催化剂的分子蓝图; 4）对复杂的多金属酶如何在用于催化键断裂化学的底物的活化中协作的一般见解。

DESCRIPTION (provided by applicant): Biological 02 production is the major renewable source of atmospheric oxygen on Earth. Water is the source of 02 created by photosynthesis in all contemporary photoautotrophic organisms and no other biological process is capable of splitting water into 02. The existence and health of all organisms that use aerobic metabolism, including humans, depends on photosynthetically derived 02. Catalysis of this chemistry is so complex that only a single type of enzymatic site appears to exist within all oxygenic photoautotrophs examined to date. The long term goal of this proposal is to reveal the chemical basis for biological water splitting. The approach to be used is to disassemble the labile inorganic core (Mn4OxCal CI1-2) comprising the active site of the enzyme and to reconstruct it from simple inorganic cofactors, atom-by-atom, using a controlled sequence of photo- and dark- reactions that is known already to fully restore the functional enzyme. This in vitro process is called photoactivation, and serves as a model for the in vivo biogenesis reactions. The assembly of "inorganic mutants" of the active site by use of surrogate cofactors will be used to probe the function of the native cofactors. Information on the kinetics of assembly, the atomic composition/structure and the atomic forces will be acquired on the assembly intermediates and the intact holo-enzyme. Tools to be used include novel methods for detection of dissolved 02 concentration, proton concentration and a collection of electron??spin resonance techniques for time-resolved and multi-dimensional spectroscopy on paramagnetic systems (EPR-plus). Studies will focus on the enzyme from O2-producing plants and primitive cyanobacteria. The biomedical benefits of the proposed research should provide: 1) fundamental information about the structure of the water splitting enzyme and its mechanism of catalysis; 2) general understanding of the mechanism of assembly of metals into metalloproteins; 3) a molecular blueprint for design of a water oxidation catalyst based on the principles adopted by the photosynthetic enzymes; 4) general insights into how complex multi-metal enzymes cooperate in activation of substrates for catalysis of bond cleavage chemistry.