Departments of †Chemistry and ‡Biochemistry and Molecular Biology, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
J Am Chem Soc. 2013 Nov 13;135(45):16758-61. doi: 10.1021/ja407438p. Epub 2013 Oct 31.
A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe2(III/III)/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ~35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe2(II/II) complex with O2 results in accumulation of an Fe2(III/IV) cluster, termed X, which oxidizes the adjacent tyrosine (Y122) to the radical (Y122(•)) as the cluster is converted to the μ-oxo-Fe2(III/III) product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d(Fe-Fe)) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O(2-), HO(-), and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d(Fe-Fe) ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ~2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d(Fe-Fe) = 2.78 Å is fully consistent with computational models containing a (μ-oxo)2-Fe2(III/IV) core. Correction of the d(Fe-Fe) brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y122(•).
一类 Ia 核糖核苷酸还原酶 (RNR) 在其β亚基中使用 μ-氧-Fe2(III/III)/酪氨酸自由基辅因子氧化其α亚基中约 35 Å 远的半胱氨酸残基；所得的半胱氨酸自由基引发底物还原。在大肠杆菌 RNR-β 辅因子的自组装过程中，蛋白质的 Fe2(II/II) 配合物与 O2 的反应导致积累了一个称为 X 的 Fe2(III/IV) 簇，该簇氧化相邻的酪氨酸 (Y122) 为自由基 (Y122(•))，同时簇转化为 μ-氧-Fe2(III/III)产物。作为第一个被鉴定的高核价非血红素铁酶复合物和 Ia 类 RNRs 的关键激活中间体，X 一直是确定其结构的重点。通过扩展 X 射线吸收精细结构 (EXAFS) 光谱的初始特征分析得到了 2.5 Å 的 Fe-Fe 分离 (d(Fe-Fe))，这被解释为表明存在三个单原子桥 (O(2-), HO(-)，和/或 μ-1,1-羧酸盐)。这个短距离与计算和合成模型都不可调和，所有模型的 d(Fe-Fe)≥2.7 Å。为了解决这个难题，我们重新研究了 X 的 EXAFS 特征。假设增加中间产物浓度的样品将产生质量更好的 EXAFS 数据，我们应用了我们最近开发的使用酶亚氯酸盐歧化酶从亚氯酸盐原位生成 O2 的方法，以~2.0 mM 的浓度制备 X，是以前 EXAFS 研究中实现浓度的 2.5 倍以上。测量的 d(Fe-Fe) = 2.78 Å 与含有 (μ-氧)2-Fe2(III/IV) 核的计算模型完全一致。d(Fe-Fe) 的校正使实验数据和计算模型完全一致，并为分析 X 生成 Y122(•)的机制提供了信息。
