IRON SITE OF RIBONUCLEOTIDE REDUCTASE

核糖核苷酸还原酶(RR)催化一种必需的 DNA生物合成的一步：核糖核苷酸的还原 到脱氧核糖核苷酸。核糖核苷酸还原酶活性为 对所有细胞的生存至关重要。非血红素铁依赖 RRS可从多种来源获得，包括 真核(例如，人类)细胞、原核细胞、哺乳动物 病毒和噬菌体病毒。来自E.Coli的酶是 最具特色的，并作为熨斗的原型 依赖RRS。大肠杆菌酶由两个亚基组成，B1 和B2。B1亚基含有变构的结合部位 调节细胞活性和特异性的效应器 酶，从而维持一个平衡的池 用于DNA合成的脱氧核糖核苷酸。也许是最多的 RR的不同寻常的特征是稳定的酪氨酸自由基和 包含在B2亚基中的双核铁络合物，以及 它们对酶的功能是必不可少的，因此对DNA也是如此 合成和复制。几乎没有结构性信息 直接在双核Fe位上可用，并且以 它产生并稳定了自由基，目前还不清楚。在这 我们建议将调查范围扩大到当地 大肠杆菌B2亚基双核铁中心的结构 核苷酸还原酶，相应的M2亚基来自 哺乳动物酶和全酶B1+B2的EXAFS分析 和XANES光谱分析。低噪音、高能量分辨率X- 天然蛋白B2，B1+B2，M2，oxy-1的射线吸收光谱 高氯氰菊酯，厌氧载脂蛋白B2与亚铁孵育， 厌氧脱氧菊酯，合适的模型化合物将是 在80K、120K、180K和300K下研究，将定量 分析确定了配位数、原子 距离和第一、第二个的EXAFS德拜-沃勒系数 和第三个配位壳。根据这一信息，Fe-O-Fe 桥接角度(当存在u-oxo桥时)， 协调的组氨酸残基，以及可能的桥联数量 将测定羧酸盐。蛋白质B2的光谱也将 在300K、pH 6.0、7.0、8.0和9.0下测量，并且 将全酶(B1+B2)与蛋白质B2在300K pH下进行比较 7.0版本。其他相关项目包括研究剂量和剂量- X射线对蛋白质B2损伤的速率依赖性，以及 XANES和EXAFS实验光谱与实验结果的比较 用理论多次散射波和单散射波预测 计算。

The enzyme ribonucleotide reductase (RR) catalyzes an essential step in the biosynthesis of DNA: the reduction of ribonucleotides to deoxyribonucleotides. Ribonucleotide reductase activity is essential for the survival of all cells. Non-heme iron dependent RRs can be obtained from a wide variety of sources including eucaryotic (e.g., human) cells, procaryotic cells, mammalian viruses, and bacteriophage viruses. The enzyme from E. Coli is the best characterized, and serves as a prototype for the iron dependent RRs. The E. Coli enzyme consists of two subunits, B1 and B2. The B1 subunit contains binding sites for allosteric effectors which regulate the activity and specificity of the enzyme and thereby maintain a balanced pool of deoxyribonucleotides for DNA synthesis. Perhaps the most unusual features of RR are a stable tyrosine free radical and a binuclear iron complex that are contained in the B2 subunit, and which are essential for enzyme function, and hence for DNA synthesis and replication. Very little structural information has been directly available on the binuclear Fe site, and the manner in which it generates and stabilizes the radical is not known. In this application we propose to extend our investigations of the local structures of the binuclear iron sites of the B2 subunit of E. Coli ribonucleotide reductase, the corresponding M2 subunit from the mammalian enzyme, and the holoenzyme B1 + B2 using EXAFS and XANES spectroscopy. Low noise, high energy resolution X- ray absorption spectra of native protein B2, B1 + B2, M2, oxy- hemerythrin, anaerobic apoprotein B2 incubated with ferrous iron, anaerobic deoxyhemerthrin, and suitable model compounds will be studied at 80 K, 120 K, 180 K and 300 K and will be quantitatively analyzed to determined the coordination numbers, atomic distances, and EXAFS Debye-Waller factors for the first, second and third coordination shells. From this information the Fe-O-Fe bridging angle (when the u-oxo bridge is present), the number of coordinated histidine residues, and possibly the number of bridging carboxylates will be determined. Spectra of protein B2 will also be measured at 300 K at pH 6.0, 7.0, 8.0 and 9.0, and the holoenzyme (B1 + B2) will be compared to protein B2 at 300 K pH 7.0. Other related projects include studies of the dose and dose- rate dependence of X-radiation induced damage to protein B2, and comparison of experimental XANES and EXAFS spectra to those predicted by theoretical multiple- and single-scattered wave calculations.