5BWH

Structure of H200C variant of Homoprotocatechuate 2,3-Dioxygenase from B.fuscum in complex with HPCA at 1.46 Ang resolution

Summary for 5BWH

• Entry DOI: 10.2210/pdb5bwh/pdb
• Related: 5BWG
• Descriptor: Homoprotocatechuate 2,3-dioxygenase, FE (II) ION, HEXAETHYLENE GLYCOL, ... (7 entities in total)
• Functional Keywords: dioxygenase, 2-his-1-carboxylate facial triad, oxygen activation, acid-base catalysis, oxidoreductase
• Biological source: Brevibacterium fuscum
• Total number of polymer chains: 4
• Total formula weight: 168963.58

Authors

Kovaleva, E.G.,Lipscomb, J.D. (deposition date: 2015-06-08, release date: 2015-10-28, Last modification date: 2023-09-27)

Primary citation

Meier, K.K.,Rogers, M.S.,Kovaleva, E.G.,Mbughuni, M.M.,Bominaar, E.L.,Lipscomb, J.D.,Munck, E.
A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mossbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods.
Inorg.Chem., 54:10269-10280, 2015

PubMed Abstract: 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.

PubMed: 26485328
DOI: 10.1021/acs.inorgchem.5b01576

Experimental method

X-RAY DIFFRACTION (1.46 Å)