EPR and ENDOR of Paramagnetic Biomolecules

超出提供的空间。 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，未配对的电子分布， 蛋白质诱导扰动的证据，以及在形成和相互转化中的动力学变化 顺磁性物种。我们的研究是通过电子核双共振技术进行的 (Endor)和电子顺磁共振(EPR)，包括快速混合流和停流EPR 它们是最近开发出来的。除了EPR-Endor，我们还带来了补充光谱， 根据需要采用动力学和生物化学方法。拟研究的系统包括： 1.连接氧气消耗和电子传输的具有生物能量重要性的金属中心 在末端细胞色素c和喹酚氧化酶中。目的是了解氧的中间体。 血红素-铜中心的化学和对内核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，它们将电子转移耦合到产生 离子梯度。其目的将是探索蛋白质-自由基氢键，并了解 蛋白质扰乱了自由基的自旋密度和电子结构。 3.能从亚硝酸盐或SENSE酶促产生一氧化氮的反硝化蛋白质，以及 结合一氧化氮以保护其免受一氧化氮的毒性。亚硝酸盐还原酶的活性部位铜将是 探讨了利用低温还原技术还原亚硝酸盐的中间体。接下来我们将调查 非结合细胞色素c的不寻常的亚铁血红素位置。 4.选择性攻击DNA的含铁博莱霉素。我们将使用Endor探索 新霉素-靶DNA复合体的相互作用和几何结构，然后，与我们新开发的Flow 和停流EPR装置，我们将探索过氧化氢自由基生成的动力学。 博莱霉素。

EXCEED THE SPACE PROVIDED. 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 ofbioenergetic 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 hinuclear CUA center. 2. Stable radical sites ofbioenergetic 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 Neomycin-target DNA complex, and then, with our newly developed flow and stopped-flow EPR apparatus we will probe the kinetics ofperoxyl radical formation catalyzed by bleomycin.