Ravichandran Kanchana R, Taguchi Alexander T, Wei Yifeng, Tommos Cecilia, Nocera Daniel G, Stubbe JoAnne
Department of Biochemistry and Biophysics, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
J Am Chem Soc. 2016 Oct 19;138(41):13706-13716. doi: 10.1021/jacs.6b08200. Epub 2016 Oct 7.
Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y ⇆ [W] ⇆ Y in β2 to Y ⇆ Y ⇆ C in α2). Equilibration of Y•, Y•, and Y• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y• and Y• has not been detected. Our recent report of Y• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y (FY•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of FY• and Y•. Incubation of FY•-β2, YF-α2 (or YF-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(FY•-Y•) of 20 ± 10 mV at 25 °C. The pH dependence of the FY• ⇆ Y• interconversion (pH 6.8-8.0) reveals that the proton from Y is in rapid exchange with solvent, in contrast to the proton from Y. Insertion of 3,5-difluorotyrosine (FY) at Y and rapid freeze-quench EPR analysis of its reaction with YF-α2, CDP, and ATP at pH 8.2 and 25 °C shows FY• generation by the native Y•. FY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y and C is ∼200 meV uphill.
大肠杆菌I类核糖核苷酸还原酶（RNR）将核糖核苷酸转化为脱氧核苷酸。一个亚基（β2）中的双铁 - 酪氨酰自由基（Y•）通过芳香族氨基酸残基进行远程自由基转移（RT）（β2中Y ⇆ [W] ⇆ Y到α2中Y ⇆ Y ⇆ C），在另一个亚基（α2）中产生一个瞬态硫自由基。最近使用位点特异性掺入的非天然酪氨酸类似物观察到Y•、Y•和Y•的平衡；然而，尚未检测到Y•和Y•之间的平衡。我们最近报道了在Y处含有2,3,5 - 三氟酪氨酸的β2（FY•-β2）与α2、CDP（底物）和ATP（效应物）反应中，以动力学和化学上合适的方式形成Y•，现在这为研究FY•和Y•的平衡提供了机会。在不同温度（2 - 37°C）下孵育FY•-β2、YF-α2（或YF-α2）、CDP和ATP，在25°C时得到ΔE°'(FY•-Y•)为20±10 mV。FY• ⇆ Y•相互转化的pH依赖性（pH 6.8 - 8.0）表明，与来自Y的质子相比，来自Y的质子与溶剂快速交换。在Y处插入3,5 - 二氟酪氨酸（FY）并在pH 8.2和25°C下对其与YF-α2、CDP和ATP的反应进行快速冷冻淬灭EPR分析，显示天然Y•产生FY•。FY-RNRs（n = 2和3）共同为RT途径的热力学态势提供了一个模型，其中Y和C之间的反应向上约200 meV。
