Price John C, Barr Eric W, Tirupati Bhramara, Bollinger J Martin, Krebs Carsten
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2003 Jun 24;42(24):7497-508. doi: 10.1021/bi030011f.
The Fe(II)- and alpha-ketoglutarate(alphaKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O(2) to a five-coordinate, (His)(2)(Asp)-facially coordinated Fe(II) center to which alphaKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O(2) on the ketone carbonyl of alphaKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O(2)(-)) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O(2)(-) complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O(2) has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature gamma-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related alphaKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O(2)-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O(2)(-) species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.
依赖于Fe(II)和α-酮戊二酸（αKG）的双加氧酶在哺乳动物的胶原蛋白合成和氧感知、微生物的营养获取和抗生素合成以及两者的烷基化DNA修复中发挥作用。这些酶的一种共识机制包括：（i）将O₂添加到一个五配位的、（His）₂（Asp）面配位的Fe(II)中心，αKG也通过其C-1羧酸盐和酮氧与之结合；（ii）结合的O₂的未配位氧攻击αKG的酮羰基，形成双环Fe(IV)-过氧半缩酮配合物；（iii）该配合物脱羧，同时形成氧代铁（Fe(IV)=O₂⁻）中间体；（iv）Fe(IV)=O₂⁻配合物通过底物自由基中间体使底物羟基化，这一机制已被多次提出，但在添加O₂后假定的中间体均未被检测到。在这项工作中，通过快速动力学和光谱方法直接证明了其中一种酶——来自大肠杆菌的牛磺酸/α-酮戊二酸双加氧酶（TauD）反应中的一种氧化态Fe中间体。通过穆斯堡尔光谱和电子顺磁共振光谱对该中间体及其单电子还原形式（通过对捕获的中间体进行低温γ辐解获得）进行表征，确定它是一种高自旋、形式上为Fe(IV)的配合物。然而，其穆斯堡尔同质异能位移明显大于其他已知的Fe(IV)配合物，这表明TauD中间体中的铁配体通过强烈的电子给予赋予高价位点显著的Fe(III)特征。该配合物的性质以及先前关于相关αKG依赖的双加氧酶和其他非血红素Fe(II)依赖的O₂活化酶的结果表明，TauD中间体最有可能是Fe(IV)-过氧半缩酮配合物或牛磺酸羟基化的Fe(IV)=O₂⁻物种。该中间体的检测为比以往报道的该重要酶家族的任何其他成员更详细地剖析TauD反应机制奠定了基础。
