Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.
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 (57)Fe 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 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.
Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Katlyn K
KK
Meier
Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Melanie S
MS
Rogers
Stanford Synchrotron Radiation Lightsource , 2575 Sand Hill Road, Menlo Park, California 94025, United States.
Elena G
EG
Kovaleva
Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, University of Minnesota , Minneapolis, Minnesota 55455, United States.
John D
JD
Lipscomb
Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Emile L
EL
Bominaar
Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Eckard
E
Münck
D049308:Dioxygenases / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D015204:Spectroscopy, Mossbauer / Q000379:methods; D013379:Substrate Specificity
D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase