Oxy Intermediates of Homoprotocatechuate 2,3-Dioxygenase: Facile Electron Transfer between Substrates

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Abstract
Substrates homoprotocatechuate (HPCA) and O2 bind to the FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O2 via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD–HPCA and H200N–HPCA, respectively) with O2. A blue intermediate forms within 20 ms of mixing of O2 with H200N–HPCA (H200NInt1HPCA). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin FeIII (S = 5/2) antiferromagnetically coupled to a radical (SR = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200NInt1HPCA decays over the next 2 s, possibly through an FeII intermediate (H200NInt2HPCA), to yield the product and the resting FeII enzyme. Reaction of FeHPCD–HPCA with O2 results in rapid formation of a colorless FeII intermediate (FeHPCDInt1HPCA). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCDInt1HPCA suggests it is an intermediate occurring after O2 activation and attack. The similar Mössbauer parameters for FeHPCDInt1HPCA and H200NInt2HPCA suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O2 via the iron does occur, leading to aromatic ring cleavage.

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