Selective Hydrogen Atom Abstraction from Dihydroflavonol by a Nonheme Iron Center Is the Key Step in the Enzymatic Flavonol Synthesis and Avoids Byproducts.

The plant non-heme iron dioxygenase flavonol synthase performs a regioselective desaturation reaction as part of the biosynthesis of the signaling molecule flavonol that triggers the growing of leaves and flowers. These compounds also have health benefits for humans. Desaturation of aliphatic compounds generally proceeds through two consecutive hydrogen atom abstraction steps from two adjacent carbon atoms and in nature often is performed by a high-valent iron(IV)-oxo species. We show that the order of the hydrogen atom abstraction steps, however, is opposite of those expected from the C-H bond strengths in the substrate and determines the product distributions. As such, flavonol synthase follows a negative catalysis mechanism. Using density functional theory methods on large active-site model complexes, we investigated pathways for desaturation and hydroxylation by an iron(IV)-oxo active-site model. Contrary to thermochemical predictions, we find that the oxidant abstracts the hydrogen atom from the strong C2-H bond rather than the weaker C3-H bond of the substrate first. We analyze the origin of this unexpected selective hydrogen atom abstraction pathway and find that the alternative C3-H hydrogen atom abstraction would be followed by a low-energy and competitive substrate hydroxylation mechanism hence, should give considerable amount of byproducts. Our computational modeling studies show that substrate positioning in flavonol synthase is essential, as it guides the reactivity to a chemo- and regioselective substrate desaturation from the C2-H group, leading to desaturation products efficiently.

Flavonol biosynthesis by nonheme iron dioxygenases: A computational study into the structure and mechanism.

Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo- and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3ß-hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with α-ketoglutarate co-substrate gives succinate, an iron(IV)-oxo species and CO2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic CH bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol-1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary CH bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger CH bond. As such, the flavanone-3ß-hydroxylase enzyme reacts regioselectively with one specific CH bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.