title: Dioxygen activation at non-heme diiron centers: characterization of intermediates in a mutant form of toluene/o-xylene monooxygenase hydroxylase.
abstract: We report the generation and characterization of an intermediate in a mutant form of the toluene/o-xylene monooxygenase hydroxylase component from Pseudomonas stutzeri OX1. The reaction of chemically reduced I100W variant in the presence of the coupling protein, ToMOD, with dioxygen was monitored by stopped-flow UV/visible spectroscopy. Rapid-freeze quench (RFQ) samples were also generated for EPR and Mössbauer spectroscopy. A transient species is observed in the UV/visible spectrum with an absorption maximum at 500 nm. EPR and Mössbauer spectra of RFQ samples identified this species as a diiron(III,IV) cluster spin-coupled to a neutral W radical. A diamagnetic precursor to the mixed-valent diiron(III,IV) was also observed at an earlier time point, with Mössbauer parameters typical of high-spin FeIII. We have tentatively assigned this antiferromagnetically coupled diiron(III) intermediate as a peroxo-bridged cluster, and this complex has also been observed in preliminary studies of the wild-type hydroxylase.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Murray
authors.forename: Ricardo
authors.initials: R
authors.lastname: García-Serres
authors.forename: Sunil
authors.initials: S
authors.lastname: Naik
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D009154:Mutation; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D014050:Toluene / Q000737:chemistry*; D014992:Xylenes / Q000737:chemistry*
chemical_list: D019159:Nonheme Iron Proteins; D014992:Xylenes; D014050:Toluene; D010105:Oxygenases; C067624:xylene monooxygenase; D010100:Oxygen; C026114:2-xylene