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Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/<i>o</i>-Xylene Monooxygenase Hydroxylase

Cited 28 time in Web of Science Cited 30 time in Scopus
Authors

Song, Woon Ju; McCormick, Michael S.; Behan, Rachel K.; Sazinsky, Matthew H.; Jiang, Wei; Lin, Jeffery; Krebs, Carsten; Lippard, Stephen J.

Issue Date
2010-10
Publisher
AMER CHEMICAL SOC
Citation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol.132 No.39, pp.13582-13585
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, 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 T201 S, T201 C, 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.
ISSN
0002-7863
URI
https://hdl.handle.net/10371/203493
DOI
https://doi.org/10.1021/ja1063795
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  • College of Natural Sciences
  • Department of Chemistry
Research Area Biochemistry, Inorganic, 무기화학, 생화학

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