Approaches to determination of the mechanism of the Rieske monooxygenase salicylate 5-hydroxylase

Rieske oxygenases catalyze an exceptionally broad range of discrete types of reactions despite the utilization of a highly conserved quaternary structure and metal cofactor complement. Oxygen activation within this family occurs at a mononuclear Fe^II site, which is located approximately 12 Å from a one-electron reduced Rieske-type iron-sulfur cluster. Electron transfer from the Rieske cluster to the mononuclear iron site occurs during O₂ activation and product formation. A key question is whether all Rieske oxygenase reactions involve the same type of activated oxygen species. This question has been explored using the Rieske oxygenase salicylate 5-hydroxylase, which catalyzes both aromatic hydroxylation of salicylate and aromatic methyl hydroxylation when a methyl substituent is placed in the normal position of aromatic ring hydroxylation. We show here that the combined application of kinetic, biophysical, computational, and isotope effect methods reveals a uniform mechanism for initial O₂ activation and substrate attack for both types of reactivity. However, the mechanism diverges during the later phases of the reactions in response to the electronic nature and geometry of the substrates as well as the lifetime of intermediates. Similar factors may be encountered broadly in the Rieske oxygenase family.