title: Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase.
abstract: Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201(peroxo), were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O(2) activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation, and possible structures of oxygenated intermediates are discussed.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Woon Ju
authors.initials: WJ
authors.lastname: Song
authors.forename: Michael S
authors.initials: MS
authors.lastname: McCormick
authors.forename: Rachel K
authors.initials: RK
authors.lastname: Behan
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Sazinsky
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Jeffery
authors.initials: J
authors.lastname: Lin
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005290:Ferric Compounds; D011522:Protons; D013912:Threonine; D010105:Oxygenases; C114360:toluene 2-xylene monooxygenase; D010100:Oxygen