Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane.

@article{Moe2004RemarkableAH,
  title={Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane.},
  author={L. Moe and Zhengbo Hu and Dayi Deng and R. Austin and J. Groves and B. Fox},
  journal={Biochemistry},
  year={2004},
  volume={43 50},
  pages={
          15688-701
        }
}
Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO… Expand
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Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropan, and diethYLcyclopropanes are consistent with a restricted active site for AlkB, and evidence for the presence of a substrate-based radical during the reaction mechanism is clear. Expand
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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It is concluded that a di iron(III,IV)-W* transient, kinetically linked to a preceding diiron(III) intermediate, arises from the one-electron oxidation of W100, and the tryptophan radical is deprotonated. Expand
Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.
TLDR
The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C2 desaturation, and C2 hydroxymethylcyclohexene/radical rearrangement, are parallel and competitive. Expand
Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes.
  • L. Moe, B. Fox
  • Chemistry, Medicine
  • Biochemical and biophysical research communications
  • 2005
TLDR
Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with kcat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluenes, the natural substrate. Expand
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An alkane hydroxylase from the marine organism Alcanivorax borkumensis (AbAlkB) was purified and products formed from a number of differently shaped and sized substrates were characterized to determine the active site constraints of this AlkB. Expand
Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase.
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T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205, which contains a lac promoter instead of a T7 promoter and allows for the recovery of the soluble oxidation in yields that facilitate further biochemical and structural characterizations. Expand
Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein.
Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complexExpand
In-crystal reaction cycle of a toluene-bound diiron hydroxylase
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The structure of a diiron hydroxylase intermediate formed during a reaction with toluene is shown and suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O–O bond by transferring a proton from a metal-bound water. Expand
High-valent iron in chemical and biological oxidations.
  • J. Groves
  • Chemistry, Medicine
  • Journal of inorganic biochemistry
  • 2006
TLDR
Accumulated evidence shows that the ferryl species [Fe(IV)O](2+) can be formed under a variety of conditions including those related to the ferrous ion-hydrogen peroxide system known as Fenton's reagent, indicating that the oxygen rebound pathway is a ubiquitous mechanism for hydrocarbon oxygenation by both heme and non-heme iron enzymes. Expand
Profiling mechanisms of alkane hydroxylase activity in vivo using the diagnostic substrate norcarane.
TLDR
Results from oxidations of this probe with organisms that contain the two most prevalent medium-chain-length alkane-oxidizing metalloenzymes, AlkB and cytochrome P450, indicate that these two classes of enzymes are mechanistically distinguishable and that whole-cell mechanistic assays can identify the active hydroxylase. Expand
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