Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/o-Xylene Monooxygenase Hydroxylase
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Song et al JACS.pdf
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Author(s)
Song, Woon Ju
McCormick, Michael S.
Behan, Rachel K.
Sazinsky, Matthew H.
Jiang, Wei
Lin, Jeffery
Krebs, Carsten
Lippard, Stephen J.
Date Issued
October 2010
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society
Citation
Song, Woon Ju et al. “Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/o-xylene Monooxygenase Hydroxylase.” Journal of the American Chemical Society 132.39 (2010): 13582-13585.
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Author's final manuscript
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 Mossbauer 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.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/ja1063795
