CATALASE AND PHOTOSYNTHETIC WATER OXIDASE

过氧化氢酶和光合水氧化酶

• 批准号: 2180114
• 负责人: G CHARLES DISMUKES
• 金额: $ 17.66万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180114
• 关键词: Raman spectrometry; active sites; catalase; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme mechanism; enzyme structure; manganese; metalloenzyme; oxidation; photosynthesis; water

The long term objective of this proposal is to better understand enzyme catalysis by metalloenzymes which utilize metal clusters. Specifically, the molecular structure and mechanism of catalysis by two multinuclear manganese enzymes which appear to share structural and functional homologies will be compared: manganese catalase and the photosynthetic water oxidizing enzyme. The photosynthetic water oxidizing enzyme is a complex multisubunit protein which utilizes a tetramanganese cluster and calcium and chloride ions to catalyze the photochemical oxidation of water to oxygen by an unknown mechanism. Catalysis of this chemistry is so complex that only a single type of enzymatic site has been identified in nature, from the most primitive cyanobacteria to present day plants. The health of all aerobic organisms, including man, is critically dependent upon this process since it is the only renewable source of atmospheric oxygen in the biosphere. Manganese catalases are rare in comparison to heme catalases and possess a spin-coupled dimanganese center as the catalytic site in place of the simpler heme cofactor. Like the heme catalases they function to remove the cellular by-product hydrogen peroxide which is a common and potent toxin to all cells. The mechanism of this process is not fully understood. The enzyme from the high temperature thermophile, Thermus thermophilus, will be studied. The proposed methods and their specific goals are: 1) High resolution multinuclear (1H, 15N, 17O, 19F, 35C1) electron nuclear double resonance (ENDOR) spectroscopy will be used to identify the magnetic nuclei located within magnetic contact (< 5A) to the manganese ions in several oxidation states: The ligand hyperfine data will be used to determine the atomic positions using a newly developed model. 2) High frequency 55Mn ENDOR and 35 GHz electron paramagnetic resonance (EPR) spectroscopies will be used to measure the nuclear electric quadrupole interaction and the magnetic hyperfine interaction of the individual Mn ions which monitor the internal electric and magnetic fields surrounding the cluster. This will be used to determine how this changes upon binding of inhibitors, inorganic cofactors, and substrate and upon redox changes of the Mn ions; 3) The identity of the protein derived radical produced in the calcium depleted water oxidase will be investigated by ENDOR spectroscopy; 4) Synthesis of catalytically active structural models of manganese catalase and their characterization by kinetic and spectroscopic methods; collaborative studies of synthetic dimanganese complexes with Dr. N. Kitajima which are structural models for multinuclear Mn enzymes; 5) Collaborative resonance Raman studies with Dr. R. Czernuszewicz on manganese catalase and synthetic mimics to determine the active site structure of magnetically silent oxidation states.

该建议的长期目标是更好地了解酶 利用金属簇的金属酶催化。 具体来说， 两个多核催化的分子结构和机制 锰酶似乎共享结构和功能 将比较同源物：锰过氧化氢酶和光合作用 水氧化酶。 光合水氧化酶是一种 使用四曼加尼亚群集的复杂多亚基蛋白 钙和氯离子以催化光化学的氧化 水通过未知机制到氧气。 该化学的催化是 如此复杂，仅确定了单一类型的酶位点 从自然界中，从最原始的蓝细菌到现在的植物。 包括人在内的所有有氧生物的健康状况都至关重要 取决于此过程，因为它是唯一可再生的来源 生物圈中的大气氧。 锰催化酶很少 与血红素催化酶进行比较，并具有自旋耦合的Dimanganese 中心为催化部位，代替简单的血红素辅因子。 喜欢 它们发挥作用的血红素过氧化氢酶可以去除细胞副产品 过氧化氢是所有细胞的常见且有效的毒素。 这 这个过程的机制尚未完全理解。 来自 将研究高温嗜热，热嗜热。 这 建议的方法及其具体目标是：1）高分辨率 多核（1H，15N，17O，19F，35C1）电子核双共振 （endor）光谱法将用于识别位于位置的磁核 在磁性接触（<5a）中与锰离子几种氧化中的 状态：配体超细数据将用于确定原子 使用新开发的模型的位置。 2）高频55mn endor 和35 GHz电子顺磁共振（EPR）光谱镜将是 用于测量核电四极相互作用和 监测的单个MN离子的磁性超细相互作用 集群周围的内部电场和磁场。 这 将用于确定抑制剂结合后如何变化， 无机辅因子，底物以及Mn离子的氧化还原变化； 3）钙在钙中产生的蛋白质的身份 耗尽的水氧化酶将通过endor光谱研究； 4） 锰过氧化氢酶的催化活性结构模型的合成 以及它们通过动力学和光谱方法的表征； 合成Dimanganese综合体与N. Kitajima是多核Mn酶的结构模型； 5） 与R. Czernuszewicz博士进行的合作共鸣拉曼研究 锰过氧化氢酶和合成模拟物以确定活性位点 磁性无声氧化状态的结构。