Sinnecker Sebastian, Svensen Nina, Barr Eric W, Ye Shengfa, Bollinger J Martin, Neese Frank, Krebs Carsten
Max-Planck Institut für Bioanorganische Chemie, D-45470 Mülheim an der Ruhr, Germany.
J Am Chem Soc. 2007 May 16;129(19):6168-79. doi: 10.1021/ja067899q. Epub 2007 Apr 24.
The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases activate O2 for cleavage of unactivated C-H bonds in their substrates. The key intermediate that abstracts hydrogen in the reaction of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, was recently characterized. The intermediate, denoted J, was shown to contain an iron(IV)-oxo unit. Other important structural features of J, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere, are not yet understood. To probe these important structural features, a series of models for J with the Fe(IV) ion coordinated by the expected two imidazole (from His99 and His255), two carboxylate (succinate and Asp101), and oxo ligands have been generated by density functional theory (DFT) calculations, and spectroscopic parameters (Mössbauer isomer shift, quadrupole splitting, and asymmetry parameter, 57Fe hyperfine coupling tensor, and zero field splitting parameters, D and E/D) have been calculated for each model. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data. Similar analysis of the Fe(IV) complex generated in the variant protein with His99, the residue that contributes the imidazole ligand cis to the oxo group, replaced by alanine suggests that the deleted imidazole is replaced by a water ligand. This work lends credence to the idea that the combination of Mössbauer spectroscopy and DFT calculations can provide detailed structural information for reactive intermediates in the catalytic cycles of iron enzymes.
依赖于亚铁（Fe(II)）和α-酮戊二酸（αKG）的双加氧酶激活氧气，用于切割其底物中未活化的碳氢键。该酶家族成员之一的牛磺酸：αKG双加氧酶（TauD）反应中提取氢的关键中间体最近得到了表征。该中间体，记为J，显示含有一个铁（IV）-氧单元。J的其他重要结构特征，如铁（IV）配位球中配体的数量、身份和排列，尚不清楚。为了探究这些重要的结构特征，通过密度泛函理论（DFT）计算生成了一系列J的模型，其中铁（IV）离子由预期的两个咪唑（来自His99和His255）、两个羧酸盐（琥珀酸盐和Asp101）和氧配体配位，并为每个模型计算了光谱参数（穆斯堡尔同质异能位移、四极分裂和不对称参数、57Fe超精细耦合张量以及零场分裂参数D和E/D）。对于J的扭曲八面体模型（其中一个羧酸盐作为单齿配体，另一个作为双齿配体）和三角双锥模型（其中两个羧酸盐都作为单齿配体）的计算参数与实验参数吻合良好，而氧配体位于赤道平面的四方锥模型的计算参数与数据不一致。对变体蛋白中生成的铁（IV）配合物进行类似分析，其中将贡献咪唑配体与氧基团顺式的His99残基替换为丙氨酸，结果表明缺失的咪唑被水配体取代。这项工作支持了这样一种观点，即穆斯堡尔光谱和DFT计算相结合可以为铁酶催化循环中的反应中间体提供详细的结构信息。
