A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mössbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods.
The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.
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.
Michael M
MM
Mbughuni
Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Emile L
EL
Bominaar
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.
Eckard
E
Münck
D049308:Dioxygenases / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D015204:Spectroscopy, Mossbauer
D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase