Measuring the orientation of taurine in the active site of the non-heme Fe(II)/α-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy.
Measuring the orientation of taurine in the active site of the non-heme Fe(II)/α-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy.
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DOI：
10.1021/jp404743d
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发表时间：
2013-09-12
期刊：
影响因子：
--
通讯作者：
McCracken J
中科院分区：
文献类型：
--
作者：
Casey TM;Grzyska PK;Hausinger RP;McCracken J
用电子自旋回波包络调制（ESEEM）技术测定了牛磺酸在α-酮戊二酸（α-KG）依赖的牛磺酸羟化酶（TauD）的非血红素Fe（II）中心附近的位置和取向。在厌氧条件下制备含有Fe（II）、α-KG和天然丰度牛磺酸或特别是氘代牛磺酸的TauD溶液，并用一氧化氮（NO）处理，以制备S=3/2 {FeNO}7络合物，该络合物适用于EPR光谱的稳健分析。使用具有天然丰度的牛磺酸或氘代牛磺酸的TauD样品收集的ESEEM光谱的比率，从光谱中过滤出1H和14 N调制，并且可以分别研究与牛磺酸上的特定氘的相互作用。采用全局优化算法，得到了计算含氘原子ESEEM频谱振幅和线型的哈密顿参数。进行了额外的统计分析，以验证优化参数的解释。最强的2 H超精细偶联是与牛磺酸C1位上的氘的偶联，其特征在于与{FeNO}7顺磁中心的有效偶极距为3.90 ± 0.25 μ m。C1- 2 H超精细耦合张量和核四极相互作用张量的主轴分别与{FeNO}7零场分裂张量的主轴成26 ± 5°和52 ± 17°角。这些结果进行了讨论的上下文中的基板牛磺酸的取向开始前的氢提取。
The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG) dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S=3/2 {FeNO}7 complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of ESEEM spectra collected for TauD samples having natural abundance taurine or deuterated taurine, 1H and 14N modulations were filtered out of the spectra and interactions with specific deuterons on taurine could be studied separately. The Hamiltonian parameters used to calculate the amplitudes and line shapes of frequency spectra containing isolated deuterium ESEEM were obtained with global optimization algorithms. Additional statistical analysis was performed to validate the interpretation of the optimized parameters. The strongest 2H hyperfine coupling was to a deuteron on the C1 position of taurine and was characterized by an effective dipolar distance of 3.90 ± 0.25 Å from the {FeNO}7 paramagnetic center. The principal axes of this C1-2H hyperfine coupling and nuclear quadrupole interaction tensors were found to make angles of 26 ± 5° and 52 ± 17°, respectively, with the principal axis of the {FeNO}7 zero-field splitting tensor. These results are discussed within the context of the orientation of substrate taurine prior to the initiation of hydrogen abstraction.
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影响因子：
4.8
作者：
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通讯作者：
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作者：
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