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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data.
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Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data.

机译：通过单晶高场EPR和1.4-A X射线数据获得的核糖核苷酸还原酶中酪氨酸自由基辅因子的置换。

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摘要

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122(*)). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-A x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122(*) obtained from high-field EPR with that of the reduced tyrosine Y122-OHreveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.
机译：I类核糖核苷酸还原酶的R2蛋白产生并存储对于核糖核苷酸还原以及DNA合成必不可少的酪氨酰基。蛋白质的X射线结构已经提出了详细的机理建议，但是尚无关于该蛋白质含活性基团状态的结构信息。在这里，我们报告了从大肠杆菌（Y122（*））在R2单晶中生成功能性酪氨酰自由基的方法。我们进一步报告了随后的含自由基晶体的高场EPR实验。收集光谱的完整旋转图案，并确定g张量轴的方向，该方向直接反映了晶格中自由基的方向。与蛋白质的甲基化（氧化）形式的1.42-A X射线结构进行了比较，对EPR数据进行了讨论。从高场EPR获得的自由基Y122（*）与还原的酪氨酸Y122-OH的取向进行比较，可以发现处于活性自由基状态的酪氨酸侧链显着旋转，远离二铁中心。讨论了将R2中的二价铁位点与R1中的底物结合位点进行自由基转移的意义。此外，本研究表明可以通过组合X射线和高场EPR晶体学获得有关活性自由基状态的结构和功能信息。

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