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

The alpha-keto acid-dependent dioxygenases are a major subgroup within the O-2-activating mononuclear nonheme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the Fe-IV=O states of the reaction have been well studied. The initial O-2-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O-2 analogue to probe the effects of alpha-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 alpha-keto acid bound {FeNO}(7) site that reflect the strong donor interaction of the alpha-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O-2 reaction coordinate where the strong donation associated with the bound alpha-keto acid promotes formation of a new, S = 1 bridged Fe-IV-peroxy species. These studies provide insight into the effects of a strong donor ligand on O-2 binding and activation by Fe-II in the alpha-keto acid-dependent dioxygenases and are likely relevant to other subgroups of the O-2 activating nonheme ferrous enzymes.