The plant nonheme 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, are 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. Against 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 analyzed 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 by-products. Our computational modelling studies shows 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.