Low-Spin Cyanide Complexes of 3-Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues.
Low-Spin Cyanide Complexes of 3-Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues.
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DOI：
10.1021/acs.inorgchem.1c01519
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发表时间：
2021-12-20
影响因子：
4.6
通讯作者：
Pierce, Brad S.
中科院分区：
文献类型：
--
作者：
York, Nicholas J.;Lockart, Molly M.;Pierce, Brad S.
3-巯基丙酸（3 MPA）双加氧酶（MDO）是一种非血红素Fe（II）/O2依赖性的加氧酶，催化巯基底物氧化生成相应的亚磺酸。Fe-位点和一组保守的三个外层残基（Ser-His-Tyr）之间的氢键相互作用在该酶的机制中起着重要的催化作用。统称为SHY基序，这些残基的功能作用仍然知之甚少。在此，用氰化物滴定与3 MPA预络合的无催化活性的Fe（III）-MDO以产生低自旋（S = 1/2）（3 MPA/CN）结合的三元络合物（称为1C）。紫外-可见光谱和电子顺磁共振（EPR）光谱用于监测3 MPA和氰化物的结合。比较从SHY基序变体（H157 N和Y159 F）获得的结果以研究特异性氢键相互作用。对于野生型酶，结合的3 MPA-和氰化物的酶的Fe-网站是选择性的，并在一个均匀的三元复合物的结果。然而，Y159 F变体失去了这种选择性，表明氢键相互作用来自Fe位点处的Tyr 159门配体配位。值得注意的是，低自旋铁位点的g值是Tyr 159 H-键捐赠方向性的诊断。使用计算模型结合CASSCF/NEVPT 2计算的g值来验证野生型和SHY变体之间显示的中心g值（g2）的主要变化可归因于Tyr 159 H键捐赠给Fe结合氰化物的损失。应用于天然共底物，这种H-键捐赠提供了一种稳定Fe结合的分子氧的方法，并可能解释了先前报道的MDO SHY基序变体的催化周转率减弱（约15倍）。
3-Mercaptopropionic acid (3MPA) dioxygenase (MDO) is a non-heme Fe(II)/O2-dependent oxygenase that catalyzes the oxidation of thiol-substrates to yield the corresponding sulfinic acid. Hydrogen-bonding interactions between the Fe-site and a conserved set of three outer-sphere residues (Ser–His–Tyr) play an important catalytic role in the mechanism of this enzyme. Collectively referred to as the SHY-motif, the functional role of these residues remains poorly understood. Here, catalytically inactive Fe(III)-MDO precomplexed with 3MPA was titrated with cyanide to yield a low-spin (S = 1/2) (3MPA/CN)-bound ternary complex (referred to as 1C). UV–visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of 3MPA and cyanide. Comparisons of results obtained from SHY-motif variants (H157N and Y159F) were performed to investigate specific H-bonding interactions. For the wild-type enzyme, the binding of 3MPA- and cyanide to the enzymatic Fe-site is selective and results in a homogeneous ternary complex. However, this selectivity is lost for the Y159F variant, suggesting that H-bonding interactions contributed from Tyr159 gate ligand coordination at the Fe-site. Significantly, the g-values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated g-values were used to verify that a major shift in the central g-value (g2) displayed between wild-type and SHY variants could be attributed to the loss of Tyr159 H-bond donation to the Fe-bound cyanide. Applied to native cosubstrate, this H-bond donation provides a means to stabilize Fe-bound dioxygen and potentially explains the attenuated (~15-fold) rate of catalytic turnover previously reported for MDO SHY-motif variants.
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影响因子：
2.9
作者：
Diebold, Adrienne R.;Neidig, Michael L.;Moran, Graham R.;Straganz, Grit D.;Solomon, Edward I.
通讯作者：
Solomon, Edward I.
DOI：
10.1021/jp404743d
发表时间：
2013-09-12
期刊：
The journal of physical chemistry. B
影响因子：
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通讯作者：
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影响因子：
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作者：
Crowell, Joshua K.;Sardar, Sinjinee;Pierce, Brad S.
通讯作者：
Pierce, Brad S.