Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: Facile electron transfer between substrates

Substrates homoprotocatechuate (HPCA) and O ₂ bind to the Fe ^II of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O ₂ 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 O ₂. A blue intermediate forms within 20 ms of mixing of O ₂ with H200N-HPCA (H200NInt ₁HPCA). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin Fe ^III (S = ⁵/ ₂) antiferromagnetically coupled to a radical (S _R = 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. H200NInt ₁HPCA decays over the next 2 s, possibly through an Fe ^II intermediate (H200NInt ₂HPCA), to yield the product and the resting Fe ^II enzyme. Reaction of FeHPCD-HPCA with O ₂ results in rapid formation of a colorless Fe ^II intermediate (FeHPCDInt ₁HPCA). 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 FeHPCDInt ₁HPCA suggests it is an intermediate occurring after O ₂ activation and attack. The similar Mössbauer parameters for FeHPCDInt ₁HPCA and H200NInt ₂HPCA suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O ₂ via the iron does occur, leading to aromatic ring cleavage.