5BWG: Structure of H200C variant of Homoprotocatechuate 2,3-Dioxygenase from B.fuscum at 1.75 Ang resolution

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. Mossbauer 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 = JS1.SR. 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 Mossbauer 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.
PDB ID: 5BWGDownload
MMDB ID: 133705
PDB Deposition Date: 2015/6/8
Updated in MMDB: 2017/12
Experimental Method:
x-ray diffraction
Resolution: 1.75  Å
Source Organism:
Similar Structures:
Biological Unit for 5BWG: tetrameric; determined by author and by software (PISA)
Molecular Components in 5BWG
Label Count Molecule
Proteins (4 molecules)
Homoprotocatechuate 2,3-dioxygenase
Molecule annotation
Chemicals (17 molecules)
* Click molecule labels to explore molecular sequence information.

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