Rivard, Brent2017-07-182017-07-182016-03https://hdl.handle.net/11299/188898University of Minnesota Ph.D. dissertation. March 2016. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: John Lipscomb. 1 computer file (PDF); iv, 125 pages.Rieske dearomatizing dioxygenases are multicomponent enzymes that catalyze a biochemically unique regio and stereospecific cis-dihydroxylation of aromatic compounds. The active site of the terminal oxygenase component contains a nonheme mononuclear iron and a [2Fe-2S] Rieske cluster. The isolated oxygenase component (hereafter RDD) can rapidly form product in a single turnover (STO) reaction after stoichiometric reduction of the metal centers and exposure to substrate and O2. After product formation, both metal centers are oxidized, indicating that two non-substrate-derived electrons are required for the reaction. The normal O2-driven STO reaction is complete in ≪1 second and no reaction cycle intermediates have been detected. Past studies have also shown that the fully oxidized RDDs can form product by utilizing H2O2 as the source of both oxygen and electrons. In the specific case of the RDD benzoate 1,2-dioxygenase, product formation during H2O2-driven reactions is much slower (completion requires ≥ 60 min), and a kinetically competent Fe3+-hydroperoxo species has been detected. These results, combined with several other logical and experimentally supported arguments, engendered the hypothesis that an Fe3+-hydroperoxo or an electronically equivalent Fe5+-oxo/hydroxo was the initial substrate oxidant of the RDD reaction. This thesis presents the most complete presteady-state kinetic analysis of O2-driven RDD cis-dihydroxylation to date. In contrast to the previous mechanistic hypotheses, the results support a model in which an Fe3+-superoxo-like species is the initial substrate oxidant. The use of this oxidant significantly changes the predicted reaction coordinate utilized by RDD for cis-dihydroxylation under O2-driven conditions. Additionally, the structure of the Fe3+-hydroperoxo species formed during H2O2-driven turnover and the conditions that allow its formation are further defined. In total, the new insights gained from the studies herein provide the first evidence that O2- and H2O2-driven turnover reactions utilize different reaction coordinates, but nevertheless lead to formation of the same unique cis-diol product.endioxygenaseironoxygen activationoxygenaseRieksesuperoxoThesis or Dissertation