title: Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.
abstract: 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-d(4), 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.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States.
authors.forename: Harriet L R
authors.initials: HL
authors.lastname: Cooper
authors.forename: Girish
authors.initials: G
authors.lastname: Mishra
authors.forename: Xiongyi
authors.initials: X
authors.lastname: Huang
authors.forename: Marilla
authors.initials: M
authors.lastname: Pender-Cudlip
authors.forename: Rachel N
authors.initials: RN
authors.lastname: Austin
authors.forename: John
authors.initials: J
authors.lastname: Shanklin
authors.forename: John T
authors.initials: JT
authors.lastname: Groves
mesh_terms: D006900:Hydroxylation; D007501:Iron / Q000378:metabolism*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D007501:Iron; D006899:Mixed Function Oxygenases