Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies

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• 作者：Katlyn K. Meier ; Melanie S. Rogers ; Elena G. Kovaleva ; John D. Lipscomb ; Emile L. Bominaar ; Eckard Münck
• 刊名：Inorganic Chemistry
• 出版年：2016
• 出版时间：June 20, 2016
• 年：2016
• 卷：55
• 期：12
• 页码：5862-5870
• 全文大小：437K
• 年卷期：0
• ISSN：1520-510X

文摘

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 second-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 Fe^II 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 Fe^II 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 Fe^II system is nearly parallel to the symmetry axis, z, of the doubly occupied d_xy 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.