Assembly and Functions of Photosynthetic Water Oxidases

描述(申请人提供)：生物产生的02是地球上大气氧气的主要可再生来源。在所有当代光自养生物中，水是光合作用产生氧气的来源，没有其他生物过程能够将水分解成氧气。包括人类在内的所有利用有氧代谢的有机体的生存和健康都依赖于光合作用产生的O2。这种化学的催化是如此复杂，以至于到目前为止所研究的所有含氧光自养生物中似乎只有一种类型的酶位点存在。这项提议的长期目标是揭示生物水分裂的化学基础。要使用的方法是分解包含酶活性部位的不稳定的无机核心(Mn4OxCal CI1-2)，并使用已知的完全恢复功能酶的受控的光和暗反应序列，从简单的无机辅因子逐个原子地重建它。这一体外过程被称为光活化，并作为体内生物发生反应的模型。利用替代辅因子组装活性部位的“无机突变体”将被用来探索天然辅因子的功能。关于组装的动力学、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。将使用的工具包括用于检测溶解的O2浓度、质子浓度的新方法，以及用于时间分辨和顺磁系统多维光谱的电子自旋共振技术的集合(EPR-PLUS)。研究将集中在产氧植物和原始蓝藻的酶上。拟议研究的生物医学益处应提供：1)有关水裂解酶的结构及其催化机理的基本信息；2)对金属组装成金属蛋白的机制的一般了解；3)基于光合作用酶所采用的原理设计水氧化催化剂的分子蓝图；4)关于复杂的多金属酶如何协同激活底物以催化键断裂化学的一般见解。

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.