FREE RADICALS IN BIOLOGICAL REDOX REACTIONS

自由基在许多生物氧化还原和代谢中起着关键作用 反应。在这里提出的研究中，我们将集中在两类 这些顺磁性中间体：(A)发生在 线粒体和光合作用电子传递的几个步骤 和(B)酪氨酸自由基，作为蛋白质氧化普遍存在 产物，也是催化核酸形成所必需的 核糖核苷酸还原酶(RDPR)。在它们的蛋白质结合部位， 这些自由基被固定，EPR线形被严重扭曲 未平均的各向异性相互作用。我们的初步数据 表明这些粉末样品的Endor谱可用于 同时提取各向同性和各向异性相互作用参数 以取代基的形式存在于醌环上的各种质子。 此外，Endor还能够探索蛋白质结合部位 短距离(小于6埃)并识别结合部位/自由基 互动。我们计划通过研究一系列 苯二酚和酪氨酸模型化合物，包含各种 生物上相关的环取代基。光谱技术 补充取向选择和重离子的赋值 我们现在使用的交易所将被开发。这些包括Q波段EPR、Endor 三重共振、二维样品取向和计算 接近了。待研究的原位体系包括Z+/D+自由基 参与水的氧化，RDPR中的酪氨酸自由基和多种 呼吸和光合作用系统中的苯醌自由基中间体。 为模型化合物研究开发的技术将用于 这项工作；我们还将用特定的同位素标记的氨基 酸转化为一些正在研究的蛋白质，特别是RDPR和水 拆分组装，以便于频谱分配。这个 这项工作的基本假设是自由基的控制 这些系统中的功能是通过蛋白质/自由基相互作用实现的； 这项工作的长期目标是描述这些联系的特点。

Free radicals play key roles in a number of biological redox and metabolic reactions. In the research proposed here we will focus on two classes of these paramagnetic intermediates: (a) quinone free radicals which occur in several of the steps in mitochondrial and photosynthetic electron transfer and (b) tyrosine free radicals, which are prevalent as protein oxidation products, are also essential to catalysis in nucleic acid formation by the enzyme, ribonucleotide reductase (RDPR). In their protein binding sites, these radicals are immobilized and EPR lineshapes are badly distorted by anisotropic interactions which are not averaged. Our preliminary data indicate that ENDOR spectroscopy of these powder samples can be used to extract both isotropic and anisotropic interaction parameters for several kinds of protons which are present as substituents on the quinone ring. Moreover, ENDOR has the capability to explore the protein binding site at short range (less than 6 Angstrom) and to identify binding site/radical interactions. We plan to extend this approach by studying a series of quinone and tyrosine model compounds which contain a variety of biologically relevant ring substituents. Techniques for spectral assignment which complement the orientation selection and deuterium exchange we now use will be developed. These include Q-band EPR, ENDOR triple resonance, two-dimensional sample orientation and computational approaches. The in situ systems to be studied include the Z+/D+ radical involved in water oxidation, the tyrosine radical in RDPR and a variety of quinone radical intermediates in respiratory and photosynthetic systems. The techniques developed for the model compound studies will be used in this work; we will also substitute specific, isotopically labeled amino acids into some of the proteins under study, notably RDPR and the water splitting assembly, in order to facilitate spectral assignment. The underlying assumption in this work is that the control of free radical function in these systems is achieved through protein/radical interaction; the long term goal of this work is to characterize these contacts.