Activation of α-Keto Acid-Dependent Dioxygenases: Application of an {FeNO}7/{FeO2}8 Methodology for Characterizing the Initial Steps of O2 Activation

The α-keto acid-dependent dioxygenases are a major subgroup within the O2-activating mononuclear nonheme 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 analogue 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 nonheme ferrous enzymes.