CATALASE AND PHOTOSYNTHETIC WATER OXIDASE

这项提案的长期目标是更好地了解酶 利用金属簇的金属酶催化。 具体地说， 两个多核催化剂分子结构和催化机理 锰酶似乎共享结构和功能 同源性进行比较：锰过氧化氢酶和光合作用 水氧化酶 光合水氧化酶是一种 利用四锰簇的复合多亚基蛋白， 钙离子和氯离子催化光化学氧化 水通过一种未知的机制转化为氧气 这种化学催化剂 如此复杂以致于仅鉴定出单一类型酶位点 从最原始的蓝细菌到现在的植物。 包括人类在内的所有需氧生物的健康， 因为它是唯一的可再生能源， 生物圈中的氧气。 锰过氧化氢酶在 与血红素过氧化氢酶相比， 中心作为催化位点代替更简单的血红素辅因子。 像 血红素过氧化氢酶的功能是去除细胞副产物 过氧化氢是一种对所有细胞都很常见的强效毒素。 的 这一过程的机制尚未完全了解。 来自 将研究高温嗜热菌Thermus thermophilus。 的 提出的方法及其具体目标是：1）高分辨率 多核（1H，15 N，17 O，19 F，35 C1）电子核双共振 （ENDOR）光谱将用于识别位于 在磁性接触（< 5A）的锰离子在几个氧化 状态：配体超精细数据将用于确定原子 使用新开发的模型。 2)高频55 Mn ENDOR 和35 GHz电子顺磁共振（EPR）光谱将被 用于测量核电四极相互作用和 单个Mn离子的磁超精细相互作用， 星系团周围的内部电场和磁场 这 将用于确定在抑制剂结合时这是如何变化的， 无机辅因子和底物以及Mn离子的氧化还原变化; 3)钙离子通道中产生的蛋白质衍生自由基的身份 耗尽的水氧化酶将通过ENDOR光谱法进行研究; 4） 锰过氧化氢酶催化活性结构模型的合成 并通过动力学和光谱方法对其进行表征; 与N. Kitajima的结构模型; 5） 与R.采努谢维奇 锰过氧化氢酶和合成模拟物以确定活性位点 磁性沉默氧化态的结构。

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.