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

Abstract

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 C 2 -H bond rather than the weaker C 3 -H bond of the substrate first. We analyzed the origin of this unexpected selective hydrogen atom abstraction pathway and find that the alternative C 3 -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 C 2 -H group leading to desaturation products efficiently.

Deciphering the origin of million-fold reactivity observed for the open core diiron [HO–FeIII–O–FeIVO]2+species towards C–H bond activation: role of spin-states, spin-coupling, and spin-cooperation

“…S3 and Table S2 in the ESI †). 128 Among all the states computed, the 2 1 hs-hs doublet state is found to be the ground state and this is followed by 10 1 hs-hs , 8 1 hs-is , 4 1 hs-is , 6 1 hs-ls , 8 1 ishs , 2 1 is-hs , 6 1 is-is , 2 1 is-is , 4 1 is-ls , 6 1 ls-hs , 4 1 ls-hs , 4 1 ls-is , 2 Table 1). Our energy calculations reveal that the Fe III (S a ) and Fe IV (S b ) centres possess a high spin state as their ground state, and these states are antiferromagnetically coupled leading to the S T ¼ 1/2 ground state, and this is also consistent with the experiments.…”

“…In the rst step, a weak reactant complex formation is assumed with the substrate, and the O/H interaction between O(1) and a hydrogen atom of the substrate. We have computed nine possible spin states with 2 RC hs-hs as the ground state, with the 10 RC hs-hs , 8 RC hs-is , 4 RC hs-is , 6 RC is-is , 2 RC is-is , 4 , respectively. Formation of species RC from the reactant is found to be slightly exothermic by À18.1 kJ mol À1 justifying the need to invoke such complex formation.…”

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…Thus, in plants, flavonols are synthesized from dihydroflavonol through a desaturation reaction by a nonheme iron dioxygenase using dioxygen and α‐ketoglutarate . A recent computational study showed that, against chemical intuition, the enzyme abstracts the strongest of the two C−H bonds of the substrate first . It was shown that substrate positioning in the active site guided the reaction to the thermodynamically less favorable pathway, which is important as it drives the reaction to selective desaturation, whereas an initial hydrogen atom abstraction from the other position would lead to dominant substrate hydroxylation instead.…”

“…Consequently, substrate and oxidant positioning in flavonol synthase leads to dominant desaturated product rather than the thermodynamically more stable hydroxylated product. As such, nature has a range of nonheme iron enzymes that perform negative catalysis, whereby the thermodynamically favored pathway is blocked through stereochemical interactions and substrate and oxidant positioning in the active site …”

“…Usually, OH rebound has a small and sometimes negligible barrier and efficiently leads to alcohol products. However, when the rebound barrier is high, the iron(III)‐hydroxo radical intermediate has a finite lifetime and can undergo rearrangement leading to byproducts and isomerizations . In nonheme iron halogenases, therefore, the protein will need to perturb the OH rebound barriers and destabilize them so that they are well above the barrier for Cl transfer in energy.…”