Crystal structures of Fe²⁺ dioxygenase superoxo, alkylperoxo, and bound product intermediates

We report the structures of three intermediates in the O₂ activation and insertion reactions of an extradiol ring-cleaving dioxygenase. A crystal of Fe²⁺-containing homoprotocatechuate 2,3-dioxygenase was soaked in the slow substrate 4-nitrocatechol in a low O₂ atmosphere. The x-ray crystal structure shows that three different intermediates reside in different subunits of a single homotetrameric enzyme molecule. One of these is the key substrate-alkylperoxo-Fe²⁺ intermediate, which has been predicted, but not structurally characterized, in an oxygenase. The intermediates define the major chemical steps of the dioxygenase mechanism and point to a general mechanistic strategy for the diverse 2-His-1-carboxylate enzyme family.