Tong W, Burdi D, Riggs-Gelasco P, Chen S, Edmondson D, Huynh B H, Stubbe J, Han S, Arvai A, Tainer J
Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Biochemistry. 1998 Apr 28;37(17):5840-8. doi: 10.1021/bi9728811.
Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of ribonucleotides to deoxyribonucleotides. It is composed of two homodimeric subunits, R1 and R2. R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process. The in vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physical biochemical methods. An intermediate, Fe3+/Fe4+ cluster (intermediate X), has been identified that is thought to be directly involved in the oxidation of Y122 to the tyrosyl radical (*Y122). An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate intermediate X at sufficient levels that it can be studied using a variety of spectroscopic methods. The details of the reconstitution of the apo and diferrous forms of Y122F R2 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic resonance, and Mössbauer spectroscopies. In addition the structure of this mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 A resolution has been determined. These studies suggest that Y122F R2 is an appropriate model for the examination of intermediate X in the assembly process. Studies with two mutants, Y356F and double mutant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR.
来自大肠杆菌的核糖核苷酸还原酶（RNR）催化核糖核苷酸向脱氧核糖核苷酸的转化。它由两个同型二聚体亚基R1和R2组成。R2含有核苷酸还原过程所必需的双铁 - 酪氨酸自由基辅因子。已通过时间分辨物理生化方法研究了从无辅基R2或二价铁形式的R2开始组装该簇的体外机制。已鉴定出一种中间体，即Fe3+/Fe4+簇（中间体X），据认为它直接参与Y122氧化为酪氨酸自由基（*Y122）的过程。一种苯丙氨酸取代了Y122的R2突变体已被用于积累足够水平的中间体X，以便可以使用各种光谱方法对其进行研究。通过停流紫外/可见光谱、快速冷冻淬灭电子顺磁共振和穆斯堡尔光谱研究了Y122F R2的无辅基形式和二价铁形式的重构细节。此外，还确定了该突变体在pH 7.6、无汞条件下结晶时2.46 Å分辨率的结构。这些研究表明，Y122F R2是研究组装过程中中间体X的合适模型。对两个突变体Y356F以及双突变体Y356F和Y122F R2的研究，根据Y356在该RNR的R1和R2亚基之间假定的电子转移反应中的可能作用进行了解释。
