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1 H-HYSCORE Reveals Structural Details at the Fe(II) Active Site of Taurine:2-Oxoglutarate Dioxygenase
Applied Magnetic Resonance ( IF 1 ) Pub Date : 2020-10-28 , DOI: 10.1007/s00723-020-01288-w
John McCracken 1 , Thomas M Casey 2 , Robert P Hausinger 3
Affiliation  

Proton Hyperfine Sublevel Correlation (1H-HYSCORE) experiments have been used to probe the ligation structure of the Fe(II) active site of taurine:2-oxoglutarate dioxygenase (TauD), a non-heme Fe(II) hydroxylase. To facilitate Electron Paramagnetic Resonance (EPR) experiments, Fe(II) derivatives of the enzyme were studied using nitric oxide as a substitute for molecular oxygen. The addition of NO to the enzyme yields an S = 3/2 {FeNO}7 paramagnetic center characterized by nearly axial EPR spectra with g = 4 and g||= 2. Using results from (i) an X-ray crystallographic study of TauD crystallized under anaerobic conditions in the presence of both cosubstrate 2-oxoglutarate and substrate taurine, (ii) a published theoretical description of the {FeNO}7 derivative of this form of the enzyme, and (iii) previous 2H-Electron Spin Echo Envelope Modulation (ESEEM) studies, we were able to assign the proton cross peaks detected in orientation-selected 1H-HYSCORE spectra. Discrete contributions from the protons of two coordinated histidine ligands were resolved. If substrate taurine is absent from the complex, orientation-selective HYSCORE spectra show cross peaks that are less resolved and when combined with information obtained from continuous wave EPR, support an alternate binding scheme for 2-oxoglutarate. HYSCORE studies of TauD in the absence of 2-oxoglutarate show additional 1H cross peaks that can be assigned to two distinct bound water molecules. In addition, 1H and 14N cross peaks that arise from the coordinated histidine side chains show a change in NO coordination for this species. For all of the TauD species, 1H hyperfine couplings and their orientations are sensitive to the detailed electronic structure of the {FeNO}7 center.



中文翻译:

1 H-HYSCORE 揭示了牛磺酸 Fe(II) 活性位点的结构细节:2-氧戊二酸双加氧酶

质子超精细亚水平相关 ( 1 H-HYSCORE) 实验已用于探测牛磺酸的 Fe(II) 活性位点的连接结构:2-氧戊二酸双加氧酶 (TauD),一种非血红素 Fe(II) 羟化酶。为了促进电子顺磁共振 (EPR) 实验,使用一氧化氮作为分子氧的替代品研究了酶的 Fe(II) 衍生物。向酶中添加 NO 会产生一个S  = 3/2 {FeNO} 7顺磁中心,其特征在于具有g  = 4 和g ||的近轴向 EPR 光谱。= 2. 使用 (i) 在共底物 2-氧代戊二酸和底物牛磺酸存在下在厌氧条件下结晶的 TauD 的 X 射线晶体学研究结果,(ii) 已发表的关于 {FeNO} 7衍生物的理论描述酶的形式,和 (iii) 之前的2 个H-电子自旋回波包络调制 (ESEEM) 研究,我们能够分配在方向选择1中检测到的质子交叉峰H-HYSCORE 光谱。解决了来自两个配位组氨酸配体的质子的离散贡献。如果复合物中不存在底物牛磺酸,则定向选择性 HYSCORE 光谱显示出分辨率较低的交叉峰,并且当与从连续波 EPR 获得的信息相结合时,支持 2-酮戊二酸的替代结合方案。在没有 2-酮戊二酸的情况下对 TauD 的 HYSCORE 研究显示额外的1 H 交叉峰可以分配给两个不同的结合水分子。此外,由配位组氨酸侧链产生的1 H 和14 N 交叉峰表明该物种的 NO 配位发生了变化。对于所有 TauD 物种,1H 超精细耦合及其方向对{FeNO} 7中心的详细电子结构敏感。

更新日期:2020-10-30
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