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Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation, and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB

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    Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation, and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB
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    § Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
    † Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, United States
    ‡ Department of Chemistry, Bates College, Lewiston, Maine 04240, United States
    [email protected]
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2012, 134, 50, 20365–20375
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    https://doi.org/10.1021/ja3059149
    Published November 16, 2012
    Copyright © 2012 American Chemical Society
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    Abstract

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    A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C–H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C–H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. 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, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C–H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

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    Mass spectra of starting materials; total ion current plots and mass spectra of products; plot of NADPH consumption; potential energy plots for 2- and 3-norcaranyl radicals; comparisons of experimental and simulated product ratios. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2012, 134, 50, 20365–20375
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ja3059149
    Published November 16, 2012
    Copyright © 2012 American Chemical Society
    Request reuse permissions

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