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 wh...