Mononuclear non-heme iron enzymes catalyze a wide variety of medically, pharmaceutically and biologically relevant reactions through the activation of dioxygen at a single iron center for reaction with an organic substrate. One class of these enzymes utilizes α-ketoglutarate (α-KG) as a redox-active cosubstrate, which supplies two electrons for the activation of dioxygen at an Fe^II center. A combination of spectroscopic techniques including EPR, absorbance, circular dichroism (CD), magnetic CD, and variable-temperature, variable-field MCD (VTVH-MCD), in conjunction with Density Function Theory (DFT) calculations correlated to the spectroscopic data, are used to explore the reaction mechanisms of a subclass of the α-KG-dependent enzymes that bind a fused α-KG cofactor and substrate or a redox-active substrate. Nitric oxide is used as a dioxygen analogue to bind to Fe ^II and create {FeNO}⁷ complexes that are chromophoric and allow for the application of these spectroscopic techniques. Specifically focusing on the segment of the reaction mechanism between the formation of the Fe^II-enzyme substrate complex and the formation of experimentally observed oxygen intermediates, these studies explore how modification of the 2-His-1-carboxylate facial triad iron-binding motif and the binding of these redox-active substrates affect Fe^II reactivity with dioxygen, the mechanism of decarboxylation of the α-KG-cofactor, and how these modifications tune the reactivity of the enzyme.

Halogenating enzymes. Recently, a new sub-class of α-KG-dependent oxygenases has been identified which exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl ^- ligand is coordinated in place of the carboxylate. Studies of the chlorinating enzymes CytC3 combined with parallel studies of Taurine Dioxygenase (TauD) and past studies of Clavaminate Synthase (CS2), define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the non-coordinating oxygen of the carboxylate and the coordinated water. These studies also provide initial insight into the active site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

4-Hydroxyphenylpyruvate dioxygenase. 4-hydroxyphenylpyruvate dioxygenase (HPPD) uses the substrate 4-hydroxyphenylpyruvate (HPP), which contains an α-keto acid that is covalently attached to the substrate, to catalyze an electrophilic attack that results in aromatic hydroxylation. 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. The general view of the O₂ reaction of the α-keto acid dependent enzymes required the presence of a five-coordinate Fe^II site and bidentate coordination of α-keto acid. However, studies of HPPD complexed with an inhibitor reveal additional substrate interactions at the ferrous site that contribute to the decarboxylation of the α-keto acid. Studies of the Fe-HPPD-HPP-NO complex probe the interaction of HPP with the {FeNO}⁷ moiety and reveal strong σ donation from HPP, which leads to a new bridged binding mode for the Fe-HPPD-HPP-O ₂ complex and a new pathway for the formation of the Fe^IV-oxo intermediate.

Isopenicillin N-synthase. Isopenicillin N synthase (IPNS) is a unique mononuclear non-heme Fe enzyme that catalyzes the four electron oxidative double ring closure of its tripeptide substrate ACV. Studies of the Fe-IPNS-ACV-NO complex show an interaction between the thiolate of the ACV and the {FeNO}⁷ unit that creates a Frontier Molecular Orbital for the initial step of the reaction. Evaluation of the energetics of NO/O₂ binding to Fe-IPNS-ACV demonstrates that charge donation from the ACV thiolate ligand renders the formation of the Fe ^III-superoxide complex energetically favorable, driving the reaction at the Fe center. This single center reaction allows IPNS to avoid the O ₂ bridged binding generally invoked in other non-heme Fe enzymes that leads to oxygen insertion (i.e. oxygenase function) and determines the oxidase activity of IPNS. In order to explore formation of the Fe^IV-oxo complex of IPNS and the initial ring closure of ACV, studies of the Fe-HPPD-HPP-hydroperoxide complex were made that reveal three pathways for the reaction of an Fe ^II-hydroperoxide species. Evaluation of these pathways shows that interactions between the Fe^II-hydroperoxide unit and the ACV substrate govern the choice of reaction pathways and control the reactivity of the enzyme.

Taken together, these studies probe oxygen activation and the formation of oxygen intermediates for α-KG-dependent enzymes on the molecular level and give new insights into the reaction mechanisms of this important class of enzymes.