TY - JOUR
T1 - Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase
AU - Song, Woon Ju
AU - McCormick, Michael S.
AU - Behan, Rachel K.
AU - Sazinsky, Matthew H.
AU - Jiang, Wei
AU - Lin, Jeffery
AU - Krebs, Carsten
AU - Lippard, Stephen J.
PY - 2010/10/6
Y1 - 2010/10/6
N2 - 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, T201peroxo, were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O2 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.
AB - 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, T201peroxo, were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O2 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.
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U2 - 10.1021/ja1063795
DO - 10.1021/ja1063795
M3 - Article
C2 - 20839885
AN - SCOPUS:77957297019
SN - 0002-7863
VL - 132
SP - 13582
EP - 13585
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 39
ER -