Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, United States.
J Am Chem Soc. 2011 Nov 16;133(45):18420-32. doi: 10.1021/ja207455k. Epub 2011 Oct 26.
Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) within β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(•) proposed to be located on Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(•) delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y(•) radicals in the PCET pathway and their conformations.
大肠杆菌核糖核苷酸还原酶是一种 α2β2 复合物，它利用 β2 中的二价铁酪氨酸自由基 (Y(122)(•)) 辅因子将核苷酸转化为脱氧核苷酸，以启动 α2 中的催化作用。每个周转率都需要通过特定途径在两个亚基之间进行可逆的长程质子耦合电子转移 (PCET)，距离为 35 Å（β 中的 Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) ⇆ α 中的 Y(731) ⇆ Y(730) ⇆ C(439)）。之前，我们报道了一种 β2 突变体，其中 3-硝基酪氨酸自由基 (NO(2)Y(•)；每个 β2 中有 1.2 个自由基) 取代 Y(122)(•)，在存在 α2、CDP 和 ATP 的情况下，催化形成 0.6 当量的 dCDP，并积累 0.6 当量的新 Y(•)，该 Y(•)被提议位于 β2 中的 Y(356)上。我们现在报告了三种独立的方法，这些方法确定 Y(356)是自由基的主要位置（85-90%），其余 10-15% 离域到 α2 中的 Y(731)和 Y(730)上。通过快速冷冻淬火 (RFQ) 方法制备样品的脉冲电子-电子双共振光谱鉴定了三个距离：30 ± 0.4 Å（88% ± 3%）和 33 ± 0.4 和 38 ± 0.5 Å（12% ± 3%），分别表示 NO(2)Y(122)(•)-Y(356)(•)、NO(2)Y(122)(•)-NO(2)Y(122)(•) 和 NO(2)Y(122)(•)-Y(731(730))(•)。RFQ 电子顺磁共振 (EPR) 研究支持 α2 中的自由基分布，该研究使用 Y(731)(3,5-F(2)Y) 或 Y(730)(3,5-F(2)Y)-α2，其揭示了 F(2)Y(•)，使用全局掺入的 [β-(2)H(2)]Y-α2 的研究，以及使用 140 GHz EPR 光谱获得的参数进行的分析。这两项研究中 α2 中离域的 Y(•)量从 6%到 15%不等。这些研究共同首次深入了解 PCET 途径中三个瞬态 Y(•)自由基的相对氧化还原电位及其构象。
