EPR and ENDOR of Paramagnetic Biomolecules

描述(由申请人提供)：我们的总体目标是了解包含顺磁中心的生物系统是如何工作的。我们探测这些顺磁中心以确定局部配位结构、未配对电子分布、蛋白质诱导扰动的证据以及顺磁物种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)技术进行的，包括最近发展起来的快速混合流和停流EPR。除了EPR-Endor，我们还根据需要提供补充的光谱、动力学和生化方法。要研究的体系是：1.在末端细胞色素c和喹酚氧化酶中连接氧消耗和电子传递的具有生物能量重要性的金属中心。目的是了解血红素-铜中心的氧化学中间体以及对双核CUA中心的扰动影响。2.具有生物能量重要性的稳定的自由基位置，它将电子转移耦合到离子梯度的产生。其目的将是探索蛋白质-自由基氢键，并了解蛋白质如何扰乱自由基自旋密度和电子结构。3.反硝化蛋白质，它能从亚硝酸盐或正义的亚硝酸盐中酶促产生一氧化氮，并与一氧化氮结合，以保护一氧化氮的毒性。利用低温还原技术，探索亚硝酸还原酶的活性中心铜作为亚硝酸根还原的中间体。接下来，我们将研究非结合细胞色素c.4.选择性攻击DNA的含铁博莱霉素的不寻常的血红素位置。我们将使用Endor来探索博莱霉素与靶DNA复合物的相互作用和几何结构，然后利用我们新开发的流动和停流EPR装置来探索博莱霉素催化的过氧化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.