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

Katlyn K. Meier, Melanie S. Rogers, Elena G. Kovaleva, John D. Lipscomb, Emile L. Bominaar, Eckard Münck

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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 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.

Original languageEnglish (US)
Pages (from-to)5862-5870
Number of pages9
JournalInorganic chemistry
Issue number12
StatePublished - Jun 20 2016

Bibliographical note

Funding Information:
National Institutes of Health Grant GM024689

Publisher Copyright:
© 2016 American Chemical Society.


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