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 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 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 O(2) reaction coordinate where the strong donation associated with the bound α-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 α-keto acid-dependent dioxygenases and are likely relevant to other subgroups of the O(2) activating nonheme ferrous enzymes.