ACTIVE SITES OF FREE RADICAL METALLOENZYMES

自由基生物化学是一个重要的新兴的研究领域 有机自由基的参与， 电子，在生物化学过程中。从历史上看， 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， 认识到自由基在酶中的重要作用 氧化还原金属蛋白的生物学功能。早期 自由基酶学领域的研究集中在Fe-和B12-上， 依赖性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重要性 酶的作用机制现在已经很好地确立， 多种功能，如前列腺素和青霉素的生物合成，脂质 过氧化和烷烃氧化。一个主题是从这些 研究的是自由基位点与金属中心的结合， 参与生成、稳定或控制 蛋白质中的自由基两种定义最明确的自由基 金属酶是半乳糖氧化酶和铁依赖性核糖核苷酸 还原酶RRB 2。这些酶中的每一种都稳定蛋白质侧链， 在静息酶中的一种自由基， 催化活性广泛的生化和基础光谱 两种蛋白质的表征现在都完成了，并且两者都是 晶体学定义在近原子分辨率。详细 对这些蛋白质的光谱学研究现在有望扩展到 在结晶学数据中的几何信息， 自由基中心的特殊电子结构特征， 金属的相互作用，导致深入了解的机制， 稳定蛋白质中的自由基并控制其独特的 反应性自由基生物化学的这些关键方面还没有被 以前由蛋白质结构研究定义。这些关键方面 自由基生物化学以前没有用蛋白质来定义 结构研究。我们的研究将集中在所载的信息 在电子顺磁共振（EPR）、电子吸收 圆二色性（CD）和磁性圆二色性（MCD）光谱 半乳糖氧化酶（GO）、核糖核苷酸 还原酶（RRB 2）和异青霉素N合酶（IPNS）。在前者（去， RRB 2）我们将探测自由基位点以及生物金属 复杂.对于IPNS，静息亚铁酶及其底物复合物 将是产生自由基的物种的关键。在所有 无机模型将提供重要的校准。到 探讨自由基金属酶的反应机制， 将探索化学扰动，检查光谱和 电化学后果的协调外源配体在 活性复合物，并研究周转反应的细节，使用 瞬态动力学方法这些基础研究将构成 其他重要和有趣的自由表征的基础 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.