CATALASE AND PHOTOSYNTHETIC WATER OXIDASE

这项建议的长期目标是更好地理解 两个多核催化剂的分子结构及其催化机理 具有相同结构和功能同源性的锰酶。 锰过氧化氢酶是一种新型(非血红素型)过氧化氢酶，可用于 防止过氧化氢引起的氧化损伤，过氧化氢是一种有效的 生物毒素，通过转化为水和氧气。光合作用 水氧化酶是一种复杂的多亚基蛋白质，与 一种光化学反应中心蛋白。它含有一种四锰 与催化氧化所需的钙离子和氯离子聚集在一起 从水到氧。这种化学的催化作用是如此复杂，只有 自然界中似乎进化出了一种单一类型的酶切位点。这个 包括人类在内的所有需氧生物的健康都有赖于此 过程，因为它是大气的主要可再生来源 氧气。极端嗜热菌Thermus的锰过氧化氢酶 嗜热菌，将与菠菜和一种 蓝藻。提出的方法和具体目标是：1)两个新的 水氧化酶在组装过程中形成的中间产物来自于它的脱脂酶. 蛋白质和无机试剂(Mn2+，Ca2+，Cl)已被 被发现了。这些将使用以下方法进行运动学表征： 能够揭示结合的无机离子的数量，他们的 氧化态、热力学结合常数和1H/2H同位素 费率依赖关系。这将使用两个新开发的 能够超灵敏地检测O2和质子的仪器，以及 分光光度法；2)这些辅因子在 蛋白质和辅因子间的相互作用(电场 Mn处的梯度以及磁偶极和标量耦合)将 使用磁共振波谱仪(EPR、ESE-Endor和 ESEEM)同位素标记的蛋白质样品和辅因子；3) 功能“无机突变体”的组装，例如通过替换 由RU2 Plus等公司将Ca2 Plus与Sr2 Plus和Mn2 Plus进行合并 被检查以努力催化新的反应或积累 部分组装的中间体；4)缓蚀机理和 锰过氧化氢酶的催化作用将在原子水平上通过 过氧化氢过程中形成的活性中间体的检测 利用快速混合分光光度、瞬时FT-EPR、 普林斯顿大学的共振拉曼和核磁共振光谱及其在X射线中的应用 我们的合作者V.Barynin博士的结晶学；5)底物2H/1H 并将测量18O/16O同位素效应来表征其程度 过渡态的成键和断键；6)表征 对锰过氧化氢酶定点突变体的研究将首先集中在 创建用于核磁共振结构分析的单体蛋白质亚基。

The long term objective of this proposal is to better understand the molecular structure and mechanism of catalysis by two multinuclear manganese enzymes which share structural and functional homologies. Manganese catalases are novel (non-heme type) catalases that serve to protect against oxidative damage caused by hydrogen peroxide, a potent biological toxin, by conversion to water and oxygen. The photosynthetic water oxidizing enzyme is a complex multisubunit protein associated with a photochemical reaction center protein. It contains a tetramanganese cluster along with Ca2 plus and Cl ions needed to catalyze the oxidation of water to oxygen. Catalysis of this chemistry is so complex that only a single type of enzymatic site appears to have evolved0 in Nature. The health of all aerobic organisms, including man, depends upon this process, since it is the dominant renewable source of atmospheric oxygen. Manganese catalase from the extreme thermophile, Thermus thermophilus, will be compared to the water oxidase from spinach and a cyanobacterium. The proposed methods and specific goals are: 1) two new intermediates formed during assembly of the water oxidase from its apo- protein and inorganic reagents (Mn2 plus, Ca2 plus, Cl) have been discovered. These will be kinetically characterized using methods that are capable of revealing the number of inorganic ions bound, their oxidation states, thermodynamic binding constants and 1H/2H isotopic rate dependences. This will be accomplished using two newly developed instruments capable of ultrasensitive detection of O2 and protons, and by spectrophotometry; 2) the sites of binding of these cofactors within the protein and the intercofactor interactions (electrical field gradient at Mn and magnetic dipolar and scalar couplings) will be determined using magnetic resonance spectroscopies (EPR, ESE-ENDOR and ESEEM) of isotopically labelled protein samples and cofactors; 3) assembly of functional "inorganic mutants", for example, by replacement of Ca2 plus with Sr2 plus and Mn2 plus by Ru2 plus, among others, will be examined in an effort to catalyze new reactions or to accumulate partially assembled intermediates; 4) the mechanism of inhibition and catalysis by Mn catalase will be examined at the atomic level by detection of the reactive intermediates formed during peroxide dismutation using rapid mixing spectrophotometry, transient FT-EPR, resonance Raman and NMR spectroscopies in Princeton and using X-ray crystallography by our collaborator Dr. V. Barynin; 5) substrate 2H/1H and 18O/16O isotope effects will be measured to characterize the degree of bond making and breaking in the transition state; 6) characterization of site-directed mutants of Mn catalase will begin with a focus on creation of monomeric protein subunits for structural analysis by NMR.