A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H -: Combined X-ray diffraction and EPR/ENDOR studies
A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H -: Combined X-ray diffraction and EPR/ENDOR studies
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DOI：
10.1074/jbc.m414634200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Lendzian, F
中科院分区：
文献类型：
--
作者：
Kolberg, M;Logan, DT;Lendzian, F
I 类核糖核苷酸还原酶 (RNR) 的 R2 蛋白亚基属于结构相关的氧桥二铁蛋白家族。在野生型大肠杆菌 R2 中，二铁铁中心对分子氧的还原性裂解会在附近的酪氨酸残基 (Tyr122) 上产生自由基，这对于 RNR 的酶活性至关重要，将核糖核苷酸转化为脱氧核糖核苷酸。在这项工作中，我们表征了突变型大肠杆菌蛋白 R2-Y122H，其中自由基位点被组氨酸残基取代。 X射线结构证实了突变。 R2-Y122H 包含一个新的稳定顺磁中心，我们将其命名为 H，我们之前提出将其作为具有强耦合基团 (FeFeR)-Fe-III-R-III 的二铁铁中心。在这里，我们报告了中心 H 的详细表征，使用 H-1/H-2-N-14/N-15- 和 Fe-57-ENDOR 与 R2 的铁重构反应中观察到的 (FeFeIV)-Fe-III 中间体 X 进行比较。苯丙氨酸残基的特异性氘标记表明自由基是由苯丙氨酸产生的。由于 Phe(208) 是铁位点配体球中唯一的苯丙氨酸，并且苯基自由基的产生需要非常高的氧化电位，因此我们提出，在 Y122H 残基中，Phe(208) 被羟基化，正如之前在另一个突变体 (R2-Y122F/E238A) 中观察到的那样，并进一步氧化为与 Fe1 配位的苯氧基自由基。这项工作表明，小的结构变化可以改变二铁位点的反应性，从而导致碳氢化合物的氧化，正如在结构相似的甲烷单加氧酶中观察到的那样，甚至形成稳定的铁配位自由基。
The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, (FeFeR)-Fe-III-R-III . Here we report a detailed characterization of center H, using H-1/H-2-N-14/N-15- and Fe-57-ENDOR in comparison with the (FeFeIV)-Fe-III intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe(208) is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe(208) is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.