Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.
Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.
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DOI：
10.1021/acs.inorgchem.6b00148
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发表时间：
2016-06-20
影响因子：
4.6
通讯作者：
Münck E
中科院分区：
文献类型：
--
作者：
Meier KK;Rogers MS;Kovaleva EG;Lipscomb JD;Bominaar EL;Münck E
额外二醇芳香环裂解酶同原儿茶酸 2,3-双加氧酶 (HPCD) 催化涉及活性位点第二球残基的复杂反应链。第二球残基 His200 的重要性在 HPCD 变体（例如 His200Cys (H200C)）的研究中得到了证明，它揭示了由于取代而催化过程中某些步骤的显着延迟，从而允许捕获新的反应循环中间体以进行光谱表征。由于 H200C 变体在很大程度上保留了野生型活性位点结构并产生正确的环裂解产物，因此该变体为机械 HPCD 研究提供了一个有价值的目标。这里，利用穆斯堡尔光谱法对静止 H200C 和 H200C-同原儿茶酸酶-底物 (ES) 复合物的高自旋 FeII 态进行了表征，以评估这些状态下活性位点的电子结构。分析揭示了低对称环境中的高自旋 FeII 中心，这反映在零场分裂 (ZFS)（D ≈ -8 cm−1，ES 中 E/D ≈ 1/3）的值以及 57Fe 磁超精细 (A) 和电场梯度 (EFG) 张量主轴相对于 ZFS 张量轴的相对方向上。 ES 复合体光谱的自旋哈密顿分析表明，整数自旋 S = 2 FeII 系统的磁化轴几乎平行于从 EFG 和 A 值推导出的双占据 dxy 地面轨道的对称轴 z，这一观察结果无法通过 DFT 辅助晶体场理论合理化。相比之下，ZFS 张量的 ORCA/CASSCF 计算与 EFG 张量和 A 张量的 DFT 计算相结合，可以很好地描述实验数据。高原儿茶酸-2,3-双加氧酶的 H200C 变体可产生天然底物的正确环裂解产物。穆斯堡尔 (Mössbauer) 对 (S = 2) FeII 酶-底物复合物的研究表明存在零场分裂（ZFS，D = −8 cm−1，E/D ≈ 1/3），且易磁化轴沿 dxy 地面轨道的 z 轴。晶体场理论和 DFT 都没有对此观察结果提供解释。然而，CASSCF 计算给出的 ZFS 张量与我们的实验数据非常吻合。
The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the 2nd-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here the high-spin FeII states of resting H200C and the H200C–homoprotocatechuate enzyme–substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin FeII center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ −8 cm−1, E/D ≈ 1/3 in ES) as well as the relative orientations of the principal axes of the 57Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 FeII system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well. The H200C variant of homoprotocatechuate-2,3-dioxygenase yields the correct ring-cleaved product of the native substrate. Mössbauer studies of the (S = 2) FeII enzyme–substrate complex indicate a zero-field splitting (ZFS, D = −8 cm−1, E/D ≈ 1/3) with an easy axis of magnetization along the z axis of dxy ground orbital. Neither crystal-field theory nor DFT provide an explanation for this observation. However, CASSCF calculations give a ZFS tensor in excellent agreement with our experimental data.