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

Abstract

The α-ketoglutate (α-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe II, 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 Fe IV O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O 2 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 Fe II and the presence of a 5C Fe II site for the O 2 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 Fe II 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 ∼10 5-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n → π ∗ transition of the HPPD/Fe II/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 CO 2 is disfavored.