Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.
Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.
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DOI：
10.1021/ja207455k
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发表时间：
2011-11-16
影响因子：
15
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Yokoyama, Kenichi;Smith, Albert A.;Corzilius, Bjoern;Griffin, Robert G.;Stubbe, JoAnne
大肠杆菌核糖核苷酸还原酶是一种α2β2复合物，利用β2中的二铁酪氨酰自由基（Y122·）辅因子启动α2中的催化作用，催化核苷酸转化为脱氧核苷酸。每一次转换都需要在两个亚基之间通过特定的途径进行超过35 μ m的可逆长程质子耦合电子转移（PCET）（Y122·E [W 48？]β内的Y356至Y 731、Y 730和α内的C439）。以前，我们报道了在α2、CDP和ATP存在下，用3-硝基酪氨酰基自由基（NO2 Y·，1.2自由基/β2）代替Y122·的β2突变体催化形成0.6当量的dCDP，并积累0.6当量的新Y·，推测其位于β2中的Y356上。我们现在报告了三种独立的方法，确定Y356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。对通过快速冷冻淬火（Rapid Freequench，简称骤冷）方法制备的样品进行脉冲电子-电子双共振光谱分析，确定了三个距离：30 ± 0.4 μ m（88 ± 3%）、33 ± 0.4 μ m和38 ± 0.5 μ m（12 ± 3%），分别表示NO2 Y122·-Y356·、NO2 Y122·-NO2 Y122·和NO2 Y122·-Y 731（730）·。使用Y 731（3，5-F2 Y）或Y 730（3，5-F2 Y）-α2的EPR研究显示了F2 Y·，使用全局结合的[β-2H2]Y-α2的研究和使用140 GHz EPR光谱获得的参数进行的分析支持了α2中的自由基分布。这两项研究中α2中离域的Y·量从6%到15%不等。这些研究首次揭示了PCET途径中三种瞬态Y·s的相对氧化还原电位及其构象。
Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric-tyrosyl radical (Y122•) 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 (Y122• ⇆ [W48?] ⇆ Y356 within β to Y731 ⇆ Y730 ⇆ C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y•, 1.2 radicals/β2) in place of Y122• in the presence of α2, CDP and ATP catalyzes formation of 0.6 equiv dCDP and accumulates 0.6 equiv of a new Y• proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85 – 90%) of the radical with the remaining 10 – 15% delocalized onto Y731 and Y730 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%), 33 ± 0.4 Å and 38 ± 0.5 Å (12 ± 3%) indicative of NO2Y122•-Y356•, NO2Y122•- NO2Y122•, and NO2Y122•-Y731(730)•, respectively. Radical distribution in α2 was supported by RFQ EPR studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2 which revealed F2Y•, and by studies using globally incorporated [β-2H2]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•s in the PCET pathway and their conformations.
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影响因子：
15
作者：
Minnihan, Ellen C.;Seyedsayamdost, Mohammad R.;Uhlin, Ulla;Stubbe, JoAnne
通讯作者：
Stubbe, JoAnne
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