Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase
Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase
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DOI：
10.1021/bi036099e
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发表时间：
2004-05-25
期刊：
影响因子：
2.9
通讯作者：
Bollinger, JM
中科院分区：
文献类型：
--
作者：
Saleh, L;Krebs, C;Bollinger, JM
大肠杆菌核糖核苷酸还原酶蛋白R2中催化必需的酪氨酸自由基（Y122(.)）生成的关键步骤是近表面残基色氨酸48 （W48）的电子转移（ET）到羧酸桥接的二铁（II）簇上添加O-2形成的（Fe2O2）（4+）络合物。由于这一步骤是快速的，（Fe2O2）（4+）配合物不会积累，因此没有被表征。ET步骤的产物是“双自由基”中间态，包含表征良好的Fe(IV)Fe（III）簇，X和W48阳离子自由基（W48(+)）。后者可以从溶液中还原，以完成电子向埋藏的二铁位点的两步转移。在本研究中，在野生型蛋白（R2-wt）的反应中，（Fe2O2）（4+）状态可能是X-W48（+）双自由基状态的前体，利用观察发现，在W48被丙氨酸(a)取代的R2变异体中，原本失能的ET步骤可以由吲哚化合物介导。R2-W48A/Y122F的Fe（II）配合物与02的混合导致中间态的积累，在与3-甲基吲哚（3-MI）混合后迅速转化为X。该态包括至少两种，其中每一种都表现出明显的穆斯堡尔四极偶态，具有高自旋Fe（111）离子的参数特征。这些配合物的同分异构体位移和穆斯堡尔谱中磁性超细耦合的缺失表明它们都是反铁磁耦合的双铁（III）团簇。事实上，在前一篇文章中所示的分子（3-MI）处理后，两者都迅速转化为X，以介导W48A R2变体中的ET，这表明它们比X更容易被一个电子氧化，这表明它们具有结合过氧化物等价物。它们没有表现出长波长吸收（在650750 nm）或具有高同分异构体位移（> 0.6 mm/s）的穆斯堡尔双峰，这是在甲烷单加氧酶（P或h -过氧）和R2变异与D84E配体取代的反应中检测到的mu-1,2-过氧桥接双铁（III）中间体所具有的特征，这表明它们具有不同于先前表征的配合物的几何和电子结构。R2-W48A/D84E中积累的过氧化物二铁（III）复合物对3- mi介导的还原反应的反应性远低于R2-W48A/Y122F中的（Fe2O2）（4+）状态。据推测，新的（Fe2O2）（4+）状态要么是氧激活正交途径中的早期加合物，要么更可能是（mu-1,2-过氧）二铁（III）复合物的后继物，该复合物在具有野生型配体集的R2蛋白中非常短暂，但在含有d84e的变体中寿命更长。
A key step in generation of the catalytically essential tyrosyl radical (Y122(.)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe2O2)(4+) complex formed by addition of O-2 to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe2O2)(4+) Complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well -characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+.)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe2O2)(4+) state that is probably the precursor to the X-W48(+.) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with 02 results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mossbauer quadrupole doublet with parameters characteristic of high-spin Fe(111) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mossbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650750 nm) or Mossbauer doublet with high isomer shift (> 0.6 mm/s) that are characteristic of the putatively,mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H-peroxo) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe2O2)(4+) state in R2-W48A/Y122F. It is postulated that the new (Fe2O2)(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.