Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.
Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.
复制标题
在小鼠核糖核苷酸还原酶蛋白 R2 的 W103Y 变体中，向二铁 (II/II) 簇添加氧是形成酪氨酰自由基的最慢步骤。
DOI：
10.1021/bi7003747
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
BollingerJr,JMartin
中科院分区：
文献类型：
--
作者：
Yun,Danny;Saleh,Lana;Garcia-Serres,Ricardo;Chicalese,BrandonM;An,YoungH;Huynh,BoiHanh;BollingerJr,JMartin
I类核糖核苷酸还原酶蛋白R2中的二铁（II/II）簇激活o2产生该酶必需的酪氨酸自由基。该反应的关键步骤是在生成自由基的双铁（III/IV）中间体时，电子从溶液转移到双铁(II/II)−o2加合物上。R2 fromEscherichia杆菌的反应,这个电子注入是由快速(> 400 s-1at 5°C),瞬态近地表残留的氧化,色氨酸48岁的阳离子自由基和被替代的W48 F, G, Y, L,或问:相比之下,一项研究老鼠的同源蛋白质R2的反应表明电子注入可能是最慢的一步生成的酪氨酰激进,Y177•(施密特,P P Rova, U, Katterle, B。,席兰德,L。和Gräslund， A.（1998）。医学杂志。[j].化学与工程学报，2016,36(2):444 - 444。关键的证据是观察到，在取代W103（电子穿梭W48 inE的同源物）后，Y177•的生成速度减慢了约30倍。coliR2和酪氨酸。在这项工作中，我们对小鼠R2反应应用了止流吸收和冷冻猝灭电子顺磁共振和Mössbauer光谱，以评估通过替代该关键残基将已经缓慢的电子转移步骤减慢30倍的可能性。簇x的积累急剧减少，前体对中间体的积累失败，最重要的是，Y177•的形成速率与o2浓度的一阶依赖性证明，在R2-W103Y反应中，将o2加入到二铁（II/II）簇中，而不是电子注入，是最慢的步骤。这一发现表明，W103Y取代减缓Y177•形成的基础是对蛋白质结构或动力学，其双铁（II/II）簇，或两者的意外二次效应，而不是对电子注入步骤的预期化学效应。
Activation of O2by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)−O2adduct during formation of the radical-generating, diiron(III/IV) intermediateX. In the reaction of R2 fromEscherichia coli, this electron injection is initiated by the rapid (>400 s-1at 5 °C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177• [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998)J. Biol. Chem. 273, 21463−21472]. The crucial evidence was the observation that Y177• production is slowed by ∼30-fold upon substitution of W103, the cognate of the electron-shuttling W48 inE. coliR2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of clusterX, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177• formation on the concentration of O2prove that addition of O2to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177• formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.