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

Luke A. Moe, Zhengbo Hu, Dayi Deng, Rachel N. Austin, John Taylor Groves, Brian G. Fox

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

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 (T4moH) with different regiospecificities for toluene hydroxylation (kcat ≈ 1.9-2.3 s-1 and coupling efficiency ≈ 81-96%) revealed similar catalytic parameters for norcarane oxidation (kcat ≈ 0.3-0.5 s-1 and coupling efficiency ≈ 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with ∼50-fold slower kcat values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O2 in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H2O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.

Original languageEnglish (US)
Pages (from-to)15688-15701
Number of pages14
JournalBiochemistry
Volume43
Issue number50
DOIs
StatePublished - Dec 21 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry

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