ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase.
ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase.
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DOI：
10.1021/ja3071682
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发表时间：
2012-10-24
影响因子：
15
通讯作者：
Bennati M
中科院分区：
文献类型：
--
作者：
Argirević T;Riplinger C;Stubbe J;Neese F;Bennati M
大肠杆菌 I 类核糖核苷酸还原酶 (RNR) 催化核苷酸转化为脱氧核苷酸，由两个亚基组成：α2 和 β2。 β2 含有稳定的二铁酪氨酰自由基 (Y122•) 辅助因子，需要在 α2 中生成 35 Å 距离的硫基自由基 (C439•)，从而启动还原过程的化学反应。自由基转移过程被认为是通过质子耦合电子转移（PCET）通过特定途径发生的：β2 中的 Y122 ⇆ W48[?] ⇆ Y356，穿过亚基界面到达 α2 中的 Y731⇆ Y730 ⇆ C439。在 α2 内，提出了共线 PCET 模型。为了获得该模型的证据，3-氨基酪氨酸(NH2Y)取代了α2中的Y730，并且将该突变体与β2、CDP和ATP一起孵育以在D2O中生成(NH2Y730•)。获得了该中间体在 94 GHz 的 [2H]-电子-核双共振 (ENDOR) 谱，并与 α2 的 DFT 模型和量子化学计算一起，将突出的 ENDOR 特征分配给可能与 C439 和 Y731 相关的两个氢键。第三个质子被分配给距离残基 730 很近（2.2 Å O-H---O 距离）的水分子。计算还表明，NH2Y730• 测得的异常 g 值与 Cys439 和 Tyr731 氢键的综合效应一致，这两个氢键几乎垂直于 NH2Y730 的环平面。该结果为活性 RNR 复合物 α2 中途径残基之间的氢键网络提供了第一个实验证据，目前尚无可用的结构数据。
E.coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 ⇆ W48[?] ⇆ Y356 in β2, across the subunit interface to Y731⇆ Y730 ⇆ C439 in α2. Within α2 a co-linear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2 and this mutant was incubated with β2, CDP and ATP to generate a (NH2Y730•) in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H---O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen bond network between the pathway residues in α2 of the active RNR complex, for which no structural data is available.