Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

摘要

intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe^II enzyme family. Many steps in the O2 activation 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 O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) Fe^III antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm^-1 in ). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by hyperfine interactions (A¹⁷O ≈ 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 at g = 8.05 arising from an S = 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.