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[Fe.sup.III]-[O.sub.2.sup.*-] intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear [Fe.sup.II] enzyme family. Many steps in the [O.sub.2] activation and reaction cycle of [Fe.sup.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 [O.sub.2] to the H200N-4NC complex is trapped and characterized using EPR and Mossbauer (MB) spectroscopies. Int-1 is a high-spin ([S.sub.1] = 5/2) [Fe.sup.III] antiferromagnetically (AF) coupled to an [S.sub.2] = 1/2 radical (J [approximately equal to] 6 [cm.sup.-1] in H = [JS.sub.1]*[S.sub.2]). 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 [sup.17][O.sub.2] hyperfine interactions ([A17.sub.O] [approximately equal to] 180 MHz). Thus, Int-1 is an AF-coupled [Fe.sup.III]-[O.sub.2.sup.*-] species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound [O.sub.2]. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled [Fe.sup.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 [sup.17][O.sub.2] ( oxygen activation | oxygenase | spectroscopy | superoxide doi/ 10.1073/pnas.1010015107