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PDBsum entry 2rdb
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Oxidoreductase
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PDB id
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2rdb
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Contents |
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491 a.a.
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322 a.a.
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83 a.a.
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References listed in PDB file
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Key reference
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Title
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Dioxygen activation at non-Heme diiron centers: oxidation of a proximal residue in the i100w variant of toluene/o-Xylene monooxygenase hydroxylase.
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Authors
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L.J.Murray,
R.García-Serres,
M.S.Mccormick,
R.Davydov,
S.G.Naik,
S.H.Kim,
B.M.Hoffman,
B.H.Huynh,
S.J.Lippard.
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Ref.
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Biochemistry, 2007,
46,
14795-14809.
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PubMed id
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Abstract
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At its carboxylate-bridged diiron active site, the hydroxylase component of
toluene/o-xylene monooxygenase activates dioxygen for subsequent arene
hydroxylation. In an I100W variant of this enzyme, we characterized the
formation and decay of two species formed by addition of dioxygen to the
reduced, diiron(II) state by rapid-freeze quench (RFQ) EPR, Mössbauer, and
ENDOR spectroscopy. The dependence of the formation and decay rates of this
mixed-valent transient on pH and the presence of phenol, propylene, or acetylene
was investigated by double-mixing stopped-flow optical spectroscopy.
Modification of the alpha-subunit of the hydroxylase after reaction of the
reduced protein with dioxygen-saturated buffer was investigated by tryptic
digestion coupled mass spectrometry. From these investigations, we conclude that
(i) a diiron(III,IV)-W* transient, kinetically linked to a preceding diiron(III)
intermediate, arises from the one-electron oxidation of W100, (ii) the
tryptophan radical is deprotonated, (iii) rapid exchange of either a terminal
water or hydroxide ion with water occurs at the ferric ion in the diiron(III,IV)
cluster, and (iv) the diiron(III,IV) core and W* decay to the diiron(III)
product by a common mechanism. No transient radical was observed by stopped-flow
optical spectroscopy for reactions of the reduced hydroxylase variants I100Y,
L208F, and F205W with dioxygen. The absence of such species, and the
deprotonated state of the tryptophanyl radical in the diiron(III,IV)-W*
transient, allow for a conservative estimate of the reduction potential of the
diiron(III) intermediate as lying between 1.1 and 1.3 V. We also describe the
X-ray crystal structure of the I100W variant of ToMOH.
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