Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase.
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.
Song
Woon Ju
WJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
McCormick
Michael S
MS
Behan
Rachel K
RK
Sazinsky
Matthew H
MH
Jiang
Wei
W
Lin
Jeffery
J
Krebs
Carsten
C
Lippard
Stephen J
SJ
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*
D005290:Ferric Compounds; D011522:Protons; D013912:Threonine; D010105:Oxygenases; C114360:toluene 2-xylene monooxygenase; D010100:Oxygen