PULSED EPR STUDIES OF BIOLOGICAL MANGANESE CLUSTERS

生物锰簇的脉冲 EPR 研究

• 批准号: 2185734
• 负责人: R David Britt
• 金额: $ 9.07万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185734
• 关键词: PULSED; EPR; STUDIES; BIOLOGICAL; MANGANESE

The molecular oxygen in our atmosphere is a product of a "water-splitting" reaction that occurs in the oxygen-evolving complex (OEC) of the Photosystem II component of plant photosynthesis. The catalytic core of the OEC is an ensemble of four manganese atoms arranged in a cluster of undetermined structure. Photosynthetic oxygen evolution occurs via a cycle that results in the removal of four electrons from two water molecules that bind to the Mn cluster. After this four-electron oxidation is complete, molecular oxygen is released, and the OEC returns to its initial state. This proposal concerns the use of novel pulsed electron paramagnetic resonance (EPR) methods designed to investigate this important enzyme system to a degree not possible with conventional continuous-wave EPR spectroscopy. The general goal is to utilize the pulsed EPR techniques of electron spin echo envelope modulation (ESEEM) and electron spin echo electron-nuclear double resonance (ESE-ENDOR) to measure the nuclear spin transitions of paramagnetic nuclei in protein ligands, cofactors, water molecules, and inhibitors bound to the Mn cluster, as well as spin transitions of the 55Mn nuclei of the cluster. The results will reveal new details concerning the structure and enzymatic function of this important biological metal center. More specifically, ESE-ENDOR experiments will be performed to measure 55Mn nuclear spin transitions of MN(III) and MN(IV) ions in a number of different synthetic exchange-coupled Mn clusters. Parallel experiments will be performed on the g=2 and g=4 Mn signals from the OEC in order to determine the degree to which each of the four Mn ions contributes to the EPR lineshapes. Other ESEEM and ESE-ENDOR experiments will measure spin transitions of the paramagnetic 17O isotope incorporated into oxygen bridges in the synthetic and biological clusters. Hyperfine and electric quadrupolar couplings will be determined for the 55Mn and 17O nuclei, and the results interpreted to provide new insights into the actual structure of the biological Mn complex. Corresponding experiments will be performed on the dinuclear Mn cluster of a manganese catalase enzyme. In addition, ESEEM and ESE-ENDOR experiments will be performed on Photosystem II membranes prepared with 1H20, 2H20, and H2 170-enriched buffers to determine at which stages of the oxygen-evolving cycle the two substrate water molecules bind to the cluster, and in what specific chemical form. ESEEM and ESE-ENDOR experiments will be performed to further characterize the protein ligation to the Mn cluster and to determine the proximity of the necessary cofactors Cl- and Ca2+ to the cluster. Additional pulsed EPR studies will determine if inhibitors of oxygen evolution that stabilize the g=4 Mn signal do so by binding directly to the Mn cluster.

我们大气中的氧分子是一种“水分解”的产物 反应发生在氧气释放复合物（OEC）的 光系统II是植物光合作用的组成部分。 的催化核心 OEC是四个锰原子排列成簇的系综， 不确定的结构 光合放氧通过一个循环发生 这导致从两个水分子中移除四个电子， 与Mn簇结合。 在这个四电子氧化完成后， 分子氧被释放，并且OEC返回到其初始状态。 该建议涉及使用新的脉冲电子顺磁 共振（EPR）方法，旨在研究这种重要的酶 在一定程度上不可能与传统的连续波EPR系统 谱 总的目标是利用脉冲EPR技术， 电子自旋回波包络调制（ESEEM）和电子自旋回波 电子-核双共振（ESE-ENDOR）测量核自旋 蛋白质配体、辅因子、水中顺磁核的跃迁 分子，和抑制剂结合到锰簇，以及自旋 团簇的55 Mn核的跃迁。 结果将揭示新的 关于这个重要的结构和酶功能的细节 生物金属中心 更具体地，将进行ESE-ENDOR实验以测量55 Mn 核自旋跃迁的MN（III）和MN（IV）离子在一些 不同的合成交换耦合锰簇。 平行实验 将对来自OEC的g=2和g=4 Mn信号进行分析， 确定四种Mn离子中的每一种对所述化合物的贡献的程度。 EPR线形 其他ESEEM和ESE-ENDOR实验将测量自旋 顺磁性17 O同位素掺入氧的跃迁 在合成和生物集群中的桥梁。 超精细和电动 四极耦合将被确定为55锰和17 O核，和 对结果的解释为实际结构提供了新的见解 生物锰络合物的一部分。 将进行相应的实验 在锰过氧化氢酶的双核锰簇上。 此外，本发明还提供了一种方法， 将在光系统II上进行ESEEM和ESE-ENDOR实验 用1H 20、2 H20和H2170富集的缓冲液制备的膜， 确定在氧气释放循环的哪个阶段， 水分子以何种特定的化学形式与团簇结合。 将进行ESEEM和ESE-ENDOR实验，以进一步表征 蛋白质与Mn簇的连接，并确定 必要的辅助因子Cl-和Ca 2+的集群。 附加脉冲EPR 研究将确定是否抑制氧气释放，稳定 g=4的Mn信号通过直接结合到Mn簇来这样做。