Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of α-keto acid-dependent dioxygenases

The α-ketoglutate (α-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require FeII, α-KG and dioxygen for catalysis, with the α-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the α-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an FeIVO intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O2 to generate this species is the decarboxylation of the α-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the α-keto acid to FeII and the presence of a 5C FeII site for the O2 reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate α-KG coordination and a 5C FeII site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an ∼105-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n → π∗ transition of the HPPD/FeII/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the α-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO2 is disfavored.