STUDIES OF THE IRON SITE OF RIBONUCLEOTIDE REDUCTASE

核糖核苷酸还原酶（RR）催化一种必需的 脱氧核糖核酸生物合成的一个步骤：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是 对所有细胞的生存至关重要。 非血红素铁依赖性 RR可以从多种来源获得，包括 真核的（例如，人）细胞、原核细胞、哺乳动物 病毒和噬菌体病毒。 从E.杆菌 最好的特点，并作为原型的铁， 依赖性RR。 急诊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位点， 调节细胞的活性和特异性的效应物， 酶，从而保持平衡的池 用于DNA合成的脱氧核糖核苷酸。 也许最 RR的不寻常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，和 这对酶的功能至关重要，因此对DNA 合成和复制。 只有很少的结构信息 直接作用于双核Fe位， 它产生和稳定自由基的物质是未知的。 在这 我们建议扩大我们对当地的调查， B2亚基双核铁位点的结构。杆菌 核糖核苷酸还原酶，相应的M2亚基从 哺乳动物酶和全酶B1 + B2，使用EXAFS 和XANES光谱。 低噪声、高能量分辨率X- 天然蛋白质B2、B1 + B2、M2、氧- 血红蛋白，与亚铁一起孵育的厌氧载脂蛋白B2， 厌氧脱氧Hemerthrin，和合适的模型化合物将是 研究在80 K，120 K，180 K和300 K，并将定量 分析以确定配位数，原子 距离，和EXAFS Debye-Waller因素，第一，第二 和第三配位壳层。 根据这些信息， 桥接角（当存在u-oxo桥时）， 协调的组氨酸残基，以及可能的桥接的数量 将测定羧酸盐。 蛋白质B2的光谱也将 在pH 6.0、7.0、8.0和9.0下在300 K下测量， 全酶（B1 + B2）将在300 K pH下与蛋白质B2进行比较 7.0. 其他相关项目包括剂量和剂量的研究- X-辐射诱导的蛋白B2损伤的速率依赖性，以及 实验XANES和EXAFS光谱与 用理论多次散射波和单次散射波预测 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.