An Unusual Ferryl Intermediate and its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO

N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in biosynthetic pathways that provide valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic (nor)loline core. Each reaction requires abstraction of hydrogen (H•) from carbon by an oxoiron(IV) (ferryl) intermediate, but different sites are targeted, and the carbon radicals have different fates. There have been prior indications that the disposition of the substrate and the intermediate in the active site controls the site of H• abstraction and can impact the fate of the radical in related enzymes. These indications led us to determine whether active-site reconfiguration in the second LolO-catalyzed reaction might contribute to the observed change in the outcome. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, the second of two ferryl complexes to accumulate during the oxacyclization step has an isomer shift greater than that of any such complex characterized to date in an Fe/2OG enzyme. Moreover, it abstracts H• from C7 ~20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7–H cleavage. These observations are consistent with coordination of the C2 oxygen to the second ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7–H cleavage and cyclization.