ACTIVE SITES OF FREE RADICAL METALLOENZYMES

摘要自由基生物化学是一个重要的新兴领域。 有机自由基的参与，分子中没有配对 电子，在生化过程中。从历史上看，对免费的研究 生物学中的自由基强调了自由基的有害作用。 在毒性机制和辐射损伤方面，但有越来越多的 认识自由基在酶中的重要作用 氧化还原金属蛋白的催化作用和生物学功能。早些时候 自由基酶学领域的研究主要集中在Fe-和B12- 某些厌氧菌产生的依赖核糖核苷酸还原酶和变位酶 细菌。然而，自由基在生物化学中的意义 机制现在在酶中得到了很好的确立，具有同样重要的和 前列腺素和青霉素生物合成、脂质等多种功能 过氧化和烷烃氧化。一个从这些主题中涌现出来的主题 研究是自由基位点与金属中心的关联 参与反应的产生、稳定或控制 蛋白质中的自由基。两个定义最明确的自由基 金属酶是半乳糖氧化酶和铁依赖的核糖核苷酸 还原酶RRB2。这些酶中的每一种都将蛋白质侧链稳定为 休眠酶中的一种自由基，它是 催化活性。广泛的生化和基础光谱学 这两种蛋白质的特性现在都完成了，两种蛋白质也都是 以接近原子分辨率定义的结晶学。详细 这些蛋白质的光谱研究现在有望扩展到 可利用的几何信息在结晶学数据中开发 自由基位的特殊电子结构特征和 金属相互作用，导致对 蛋白质中自由基的稳定化及其特有的调控 反应性。自由基生物化学的这些关键方面还没有 以前由蛋白质结构研究定义的。这些关键方面 自由基生物化学以前并不是由蛋白质定义的 结构研究。我们的研究将集中在所包含的信息上 在电子顺磁共振(EPR)、电子吸收、 化合物的圆二色谱(CD)和磁性圆二色谱(MCD) 半乳糖氧化酶(GO)、核糖核苷酸中的活性中心复合体 还原酶(RRB2)和异青霉素N合成酶(IPNS)。在前者(Go， RRB2)我们将探索自由基位点以及生物金属 很复杂。对IPNS而言，静息亚铁酶及其底物复合体 将是产生自由基的物种的关键利益所在。总而言之， 三项研究中，无机模型将提供重要的校准。至 自由基金属酶反应机理的探讨 将探索化学微扰，检查光谱和 配位外源配体在细胞内的电化学效应 活性络合物，并研究了转化反应的细节 瞬变动力学方法。这些基础性研究将形成 描述其他重要和有趣的自由的基础 自由基金属酶。

Free radical biochemistry is an important and emerging new area probing the involvement of organic free radicals, molecules having unpaired electrons, in biochemical processes. Historically, studies on free radicals in biology have emphasized the deleterious effects of radicals in toxicity mechanisms and radiation damage, but there is a growing recognition of the essential role that free radicals play in enzyme catalysis and the biological function of redox metalloproteins. Early studies in the area of free radical enzymology focussed on Fe- and B12- dependent ribonucleotide reductases and mutases from certain anaerobic bacteria. However, the significance of radicals in biochemical mechanisms is now well established in enzymes with as important and diverse functions as prostaglandin and penicillin biosynthesis, lipid peroxidation and alkane oxidation. A motif which is emerging from these studies is the association of the radical site with a metal center that is involved in generation, stabilization or control of reactivity of radicals in proteins. Two of the most well-defined free radical metalloenzymes are galactose oxidase and the Fe-dependent ribonucleotide reductase RRB2. Each of these enzymes stabilize a protein side chain as a free radical in the resting enzyme, a radical which is required for catalytic activity. Extensive biochemical and basic spectroscopic characterization is now complete for both proteins, and both are also crystallographically defined at near atomic resolution. Detailed spectroscopic studies of these proteins can now be expected to extend to geometric information available in the crystalligraphic data to develop the special electronic structural features of the radical sites and metal interactions, leading to insights into the mechanism of stabilization of radicals in proteins and the control of their unique reactivity. these key aspects of free radical biochemistry have not been previously defined by protein structural studies. These key aspects of free radical biochemistry have not been previously defined by protein structural studies. Our studies will focus on the information contained in electron paramagnetic resonance (EPR), electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the active site complexes in galactose oxidase (GO), ribonucleotide reductase (RRB2) and isopenicillin N synthase (IPNS). In the former (GO, RRB2) we will probe the radical sites as well as the biological metal complex. For IPNS the resting ferrous enzyme and its substrate complex will be of key interest in terms of radical-generating species. In all three studies, inorganic models will provide important calibration. To probe the mechanisms of reactivity for free radical metalloenzymes we will explore chemical perturbations, examining the spectroscopic and electrochemical consequences of coordinating exogenous ligands in the active complexes, and investigate the details of turnover reaction using transient kinetics methods. These fundamental studies will form the basis for characterization of other important and interesting free radical metalloenzymes.