The α-keto acid dependent dioxygenases are a major subgroup within the O2-activating mononuclear non-heme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the FeIV=O states of the reaction have been well studied. The initial O2-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O2 analog to probe the effects of α-keto acid binding in 4-hydroxyphenylpyruvate dioxygenase (HPPD). A combination of EPR, UV-vis absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies in conjunction with computational models is used to explore the HPPD-NO and HPPD-HPP-NO complexes. New spectroscopic features are present in the α-keto acid bound {FeNO}7 site that reflect the strong donor interaction of the α-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O2 reaction coordinate where the strong donation associated with the bound α-keto acid promotes formation of a new, S=1 bridged FeIV-peroxy species. These studies provide insight into the effects of a strong donor ligand on O2 binding and activation by FeII in the α-keto acid dependent dioxygenases and are likely relevant to other subgroups of the O2 activating non-heme ferrous enzymes.

Isopenicillin N synthase (IPNS) can have both oxidase and oxygenase activity depending on the substrate. For the native substrate, ACV, oxidase activity occurs; however for the substrate analogue ACOV, which lacks an amide nitrogen, IPNS shows oxygenase activity. The potential energy surfaces for the O-O bond elongation and cleavage were calculated for three different reactions: homolytic cleavage via traditional Fenton chemistry, heterolytic cleavage, and nucleophilic attack. These surfaces show that the hydroperoxide-Ferrous intermediate, formed by O 2 activated H atom abstraction from substrate, can undergo different reaction pathways and that interactions with the substrate govern the pathway. The hydroperoxide hydrogen bonds to the amide nitrogen of ACV polarizing the σ* orbital of the peroxide toward the proximal oxygen, facilitating heterolytic cleavage. For the substrate analogue ACOV, this hydrogen bond is no longer present, leading to nucleophilic attack on the substrate intermediate C-S bond. After cleavage of the hydroperoxide, the two reaction pathways proceed with minimal barriers to result in the closure of the β-lactam ring for the oxidase activity (ACV) or formation of the thiocarboxylate for oxygenase activity (ACOV).Isopenicillin N-synthase is a mononuclear non-heme iron enzyme found in fungi and bacteria that catalyzes the formation of isopenicillin N, a bicyclic precursor to the β lactam antibiotics including the penicillins and cephalosporins. (1,2) IPNS binds a tripeptide substrate δ-(L-aaminoadipoyl)-L-cysteinyl-D-valine (ACV) and performs a four electron oxidative double ring closure, fully reducing one equivalent of O 2 to H 2 O and closing the β lactam and thiazolidine rings of isopenicillin N. (3-6) (Scheme 1) This oxidase reactivity is unusual as most non-heme iron enzymes catalyze oxygenation reactions. Previous studies of the IPNS-ACV {FeNO} 7 complex revealed that a major factor contributing to the oxidase reactivity of IPNS is charge donation from the ACV thiolate ligand, which renders the formation of the Fe III -superoxide complex energetically favorable and drives the reaction only at the Fe center. (7) This single center, one electron reaction allows IPNS to avoid the bridged binding of O 2 between the Fe II and the substrate/cofactor required for its two electron reduction; a reaction generally invoked for the non-heme Fe enzymes, that leads to oxygen insertion. The thiolate coordination of the IPNS ACV substrate further activates the reactive Fe III -superoxide complex of the enzyme through a configuration interaction with the bound superoxide π* orbital which creates * To whom correspondence should be addressed: edward.solomon@stanford.edu; phone: 650-723-4694; fax: 650-725-0259.The reorientation of the hydroperoxide was accomplished through contraction of the distance between the ACV amide nitrogen and cysteinyl carbon to a distance of approximately 2.8Å. (15)(16)(17)(18)(19) In one such analogue ACOV, the amide nitrogen of the ACV valine is replaced with an oxyg...