CATALASE AND PHOTOSYNTHETIC WATER OXIDASE

这项建议的长期目标是更好地了解 两个多核催化剂的分子结构和催化机理 具有结构和功能同源性的锰酶。 锰过氧化氢酶是新的（非血红素型）过氧化氢酶，其用于 保护免受过氧化氢引起的氧化损伤，一种有效的 生物毒素，通过转化为水和氧气。 光合 水氧化酶是一种复杂的多亚基蛋白， 一种光化学反应中心蛋白。 它含有四锰 簇沿着与钙离子和氯离子需要催化氧化 从水到氧气 这种化学反应的催化作用是如此复杂， 一种单一类型的酶位点似乎已经在自然界中进化了。 的 包括人类在内的所有需氧生物的健康都依赖于此 过程，因为它是大气中主要的可再生能源， 氧气 极端嗜热菌栖热菌锰过氧化氢酶 嗜热菌，将比较水氧化酶从菠菜和 蓝藻 提出的方法和具体目标是：1）两个新的 在水氧化酶从其apo- 蛋白质和无机试剂（Mn 2+、Ca 2+、Cl）已被 发现了 这些将使用以下方法进行动力学表征， 能够揭示结合的无机离子的数量， 氧化态、热力学结合常数和1H/2 H同位素 速率依赖性 这将使用两个新开发的 能够超灵敏检测O2和质子的仪器，以及 通过分光光度法; 2）这些辅因子的结合位点 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）将是 使用磁共振光谱（EPR、ESE-ENDOR和 ESEEM）同位素标记的蛋白质样品和辅因子; 3） 功能性“无机突变体”的组装，例如，通过置换 其中，Ca 2+与Sr 2+和Mn 2+与Ru 2+的结合将 在努力催化新的反应或积累 部分组装的中间体; 4）抑制机制， Mn过氧化氢酶的催化作用将在原子水平上通过 检测过氧化氢过程中形成的反应性中间体 快速混合分光光度法歧化，瞬时FT-EPR， 共振拉曼和核磁共振光谱，并使用X射线 我们的合作者V. Barynin博士的晶体学; 5）底物2 H/1H 18 O/16 O同位素效应将被用来表征 在过渡态中键的形成和断裂; 6）表征 锰过氧化氢酶定点突变体的研究开始将集中在 产生单体蛋白质亚基，用于NMR结构分析。

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.