Jasniewski, Andrew2019-09-172019-09-172017-05https://hdl.handle.net/11299/206656University of Minnesota Ph.D. dissertation. May 2017. Major: Chemistry. Advisor: Lawrence Que. 1 computer file (PDF); xviii, 337 pages.The general mechanism of O2 activation by nonheme diiron enzymes begins when the diferrous iron cluster binds dioxygen. The diiron cluster is oxidized to a peroxo-diferric intermediate that in some cases reacts directly with substrates, and in others becomes further activated via the cleavage of the O–O bond, leading to the generation of a potent high-valent oxidant that is the active oxidant for the cycle. Peroxo-diferric intermediates are of high interest because they are crossroads between the use of peroxo-diferric or high-valent oxo intermediate as the active oxidant in diiron-cluster-mediated oxidase and oxygenase chemistry. Understanding this O2 activation process requires structural characterization of enzymatic peroxo-diferric species. Spectroscopic methods, like electronic absorbance, X-ray absorption (XAS), and resonance Raman (rR) spectroscopies are used to probe a rich landscape of oxygen-activated intermediates and obtain detailed structures of these species. Through systematic study, insight can be gained into the mechanisms of these biological systems and ultimately this insight can be used to understand how Nature has chosen to use peroxo-diferric intermediates for a variety of different functions. In Chapter 2, X-ray diffraction and XAS were used to characterize various form of the enzyme CmlA to understand how O2 is regulated in the presence and in the absence of its non-ribosomal peptide synthetase (NRPS) bound substrate. In Chapter 3, the intermediate species on the O2 activation pathway of the human enzyme deoxyhypusine hydroxylase (hDOHH), including the µ-1,2-peroxo species, were studied using XAS. The structural analysis of the active sites of the various hDOHH species provided insight into the reaction mechanism for the system. In Chapter 4, XAS and rR studies on the unusual peroxo-diferric species of the N-oxygenase CmlI were carried out. The spectroscopic analysis of the peroxo intermediate describes a new peroxo binding geometry for diiron enzymes, a µ-1,1-peroxo species. In Chapter 5, detailed XAS characterization of various synthetic peroxo-diferric and oxoiron(IV) model complexes is described. Overall, this thesis demonstrates the power of structural characterization by complementary spectroscopic methods to support and generate enzymatic mechanistic hypotheses.enbioinorganicnonhemespectroscopyThesis or Dissertation