EPR and ENDOR of Paramagnetic Biomolecules

描述（由申请人提供）：我们的总体目标是了解含有顺磁性中心的生物系统如何工作。 我们探测这些顺磁中心，以确定当地的配位结构，未成对电子分布，蛋白质诱导的扰动的证据，顺磁物质的形成和相互转化的动力学变化。 我们的研究是通过电子核双共振（ENDOR）和电子顺磁共振（EPR）技术，包括快速混合流和停流EPR最近发展起来的。 除了EPR-ENDOR，我们还根据需要提供补充的光谱，动力学和生物化学方法。 要研究的系统是：1。 末端细胞色素c和醌醇氧化酶中偶联氧消耗和电子传递的生物能量重要性金属中心。 目的将是了解在血红素铜中心的氧化学中间体和对双核CUA中心的扰动影响。 2. 具有生物能量重要性的稳定自由基位点，其将电子转移与离子梯度的产生耦合。 目的是探测蛋白质-自由基氢键，并了解蛋白质如何扰动自由基自旋密度和电子结构。 3. 反硝化蛋白质，从亚硝酸盐酶促产生一氧化氮，或感知并结合一氧化氮以防止一氧化氮中毒。 利用低温还原技术对亚硝酸盐还原酶的活性中心铜进行了研究。 接下来我们将研究NO结合细胞色素c的不寻常的血红素位点。 4. 含铁博莱霉素，选择性攻击DNA。 我们将使用ENDOR探索博来霉素-靶DNA复合物的相互作用和几何形状，然后，用我们新开发的流动和停流EPR装置，我们将探测博来霉素催化的过氧自由基形成的动力学。

DESCRIPTION (provided by applicant): Our overall goal is to understand how biological systems that contain paramagnetic centers work. We probe these paramagnetic centers to determine local liganding structure, unpaired electron distribution, evidence for protein-induced perturbation, and kinetic change in the formation and interconversion of paramagnetic species. Our investigation is through the techniques of electron nuclear double resonance (ENDOR) and electron paramagnetic resonance (EPR), including rapid-mix flow and stopped-flow EPR which have been recently developed. Besides EPR-ENDOR, we bring complementary spectroscopic, kinetic, and biochemical methods as needed. The systems to be studied are: 1. Metal centers of bioenergetic importance that couple oxygen consumption and electron transport in terminal cytochrome c and quinol oxidases. The purpose will be to understand intermediates of oxygen chemistry at the heme-copper center and perturbing influences on the binuclear CUA center. 2. Stable radical sites of bioenergetic importance that couple electron transfer to the production of ion gradients. The purpose will be to probe protein-radical hydrogen bonding and to understand how the protein perturbs radical spin density and electronic structure. 3. Proteins of denitrification which enzymatically produce nitric oxide from nitrite or sense and bind nitric oxide to protect against nitric oxide toxicity. The active site copper of nitrite reductase will be probed for intermediates of nitrite reduction using cryoreduction techniques. We will next investigate the unusual heme locale of NO-binding cytochrome c. 4. Iron-containing Bleomycin that selectively attacks DNA. We will use ENDOR to explore the interaction and geometry of the bleomycin-target DNA complex, and then, with our newly developed flow and stopped-flow EPR apparatus we will probe the kinetics of peroxyl radical formation catalyzed by bleomycin.