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

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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
Volume55
Issue number12
DOIs
StatePublished - Jun 20 2016

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3,4-dihydroxyphenylacetate 2,3-dioxygenase
Tensors
enzymes
tensors
Electric fields
Substrates
Enzymes
Discrete Fourier transforms
gradients
electric fields
Hamiltonians
rings
symmetry
crystal field theory
integers
Electronic structure
Magnetization
Substitution reactions
Spectroscopy
substitutes

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Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase : Mössbauer and Computational Studies. / Meier, Katlyn K.; Rogers, Melanie S.; Kovaleva, Elena G.; Lipscomb, John D.; Bominaar, Emile L.; Münck, Eckard.

In: Inorganic Chemistry, Vol. 55, No. 12, 20.06.2016, p. 5862-5870.

Research output: Contribution to journalArticle

Meier, Katlyn K. ; Rogers, Melanie S. ; Kovaleva, Elena G. ; Lipscomb, John D. ; Bominaar, Emile L. ; Münck, Eckard. / Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase : Mössbauer and Computational Studies. In: Inorganic Chemistry. 2016 ; Vol. 55, No. 12. pp. 5862-5870.
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title = "Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: M{\"o}ssbauer and Computational Studies",
abstract = "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 M{\"o}ssbauer 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.",
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T2 - Mössbauer and Computational Studies

AU - Meier, Katlyn K.

AU - Rogers, Melanie S.

AU - Kovaleva, Elena G.

AU - Lipscomb, John D.

AU - Bominaar, Emile L.

AU - Münck, Eckard

PY - 2016/6/20

Y1 - 2016/6/20

N2 - 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.

AB - 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.

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