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.