Intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe^IIenzyme family. Many steps in the O₂activation and reaction cycle of Fe^II-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O₂to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S₁ = 5/2) Fe^IIIantiferromagnetically (AF) coupled to anS₂ = 1/2 radical (J ≈ 6 cm^-1in ). It exhibits parallel-mode EPR signals atg = 8.17 from theS = 2 multiplet, andg = 8.8 and 11.6 from theS = 3 multiplet. These signals are broadened significantly by hyperfine interactions (A_¹⁷O ≈ 180 MHz). Thus, Int-1 is an AF-coupled species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O₂. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe^III-radical complex. Int-2 exhibits an EPR signal atg = 8.05 arising from anS = 2 state. The signal is only slightly broadened by (< 3% spin delocalization), suggesting that Int-2 is a peroxo-Fe^III-4NC semiquinone radical species. Our results demonstrate facile electron transfer between Fe^II, O₂, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.

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