EPR AND ENDOR OF PARAMAGNETIC BIOMOLECULES

我们的总体目标是了解生物系统如何包含 顺磁性金属中心和自由基起作用。 我们将探索 顺磁中心，以确定局部配位结构，共享 不成对电子分布和蛋白质诱导的证据 扰动 我们将调查这些中心主要通过 电子顺磁共振（EPR）和电子核共振（ESR）技术 双共振（ENDOR）。 在需要澄清机制的地方，EPR- 将采用相关的停流和冷冻骤冷方法。 我们的la 是世界上少数几个经常使用ENDOR来测量 生物分子中电子自旋的超精细耦合。 的 拟研究的系统包括： 1. 锰中心。 在丙酮酸激酶的Mn 2+中心，我们将 仔细研究目前不确定的约束力模式， 磷酸烯醇丙酮酸底物和丙酮酸产物。 在研究中， 在多锰光合放氧中心和模型 为此，我们将用ENDOR探测可交换氧结合的证据 到那个中心。 2. 核糖核苷酸还原酶。 我们将进行ENDOR测量， 揭示组装过程中早期自由基中间体的结构， 二铁/酪氨酰自由基辅因子， 还原 3. 激活博莱霉素。 我们将研究目前未知的 在活化的博来霉素中的氧-铁连接， 博来霉素诱导的DNA损伤的前体。 4. 电子输运的顺磁性中心。 目标是 了解血红素的配位环境，我们将探测血红素- 末端细胞色素c和醌醇氧化酶的铜催化核心，其中 氧气被还原成水。 在特定的泛醌结合位点， 电子传递链，我们将研究结构和 稳定半醌自由基动力学行为 5. 亚硝酸还原酶。 通过使用天然的和转基因的形式， 这种细菌酶，我们将阐明其活性位点结构 1型和2型铜，用于将亚硝酸盐转化为一氧化氮。 通过EPR监测，我们将测量这些金属的氧化还原行为 中心，并与冷冻淬火捕获，我们将探讨酶 机制

Our overall goal is to understand how biological systems that contain paramagnetic metal centers and free radicals work. We will probe the paramagnetic centers to determine local liganding structure, shared unpaired electron distribution, and evidence for protein-induced perturbation. We will investigate these centers primarily through the techniques of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR). Where mechanism needs to be clarified, EPR- related stopped-flow and freeze-quench methods will be employed. Our la is one of the few in the world which routinely uses ENDOR to measure hyperfine coupling of electron spins in biological molecules. The systems to be studied are: 1. Manganese Centers. At the Mn2+ center in pyruvate kinase we will closely examine the presently uncertain binding mode of phosphoenolpyruvate substrate and pyruvate product. Then in study aimed at the multimanganese photosynthetic oxygen-evolving center and models for it we will probe by ENDOR for evidence of exchangeable oxygen bound to that center. 2. Ribonucleotide Reductase. We will perform ENDOR measurements to uncover the structure of early radical intermediates in the assembly of the diiron/tyrosyl radical cofactor that is needed for nucleotide reduction. 3. Activated Bleomycin. We will examine the presently unknown mode of oxygen-iron ligation in the activated bleomycin that is a necessary precursor to bleomycin-induced DNA damage. 4. Paramagnetic Centers of Electron Transport. With the goal of understanding the heme's liganding environment, we will probe the heme- copper catalytic core of terminal cytochrome c and quinol oxidases where oxygen is reduced to water. At specialized ubiquinone binding sites of the electron transport chain we will investigate the structure and kinetic behavior of stabilized semiquinone radical. 5. Nitrite Reductase. By using native and genetically altered forms of this bacterial enzyme, we will clarify the active site structure of its Type-1 and Type-2 coppers that serve to convert nitrite to nitric oxide. With EPR monitoring we will measure redox behavior of these metal centers, and with freeze-quench trapping we will explore the enzymatic mechanism.