Jones, Jason2021-08-162021-08-162021-05https://hdl.handle.net/11299/223166University of Minnesota Ph.D. dissertation. May 2021. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: John Lipscomb. 1 computer file (PDF); xvi, 207 pages.Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that activates molecular oxygen, breaks the 105 kcal/mol C-H bond of CH4, and inserts one atom of O to create methanol at ambient temperature and pressure. This feat of catalytic prowess requires all three protein components of sMMO for efficient multiple turnover catalysis: the hydroxylase (sMMOH), the reductase (MMOR), and the regulatory protein (MMOB). The structural mechanism of how these sMMO components interact to regulate the formation and decay of the chemical intermediates of the reaction cycle is not well understood. Our recent advances in sMMOH expression and purification have allowed us to obtain protein crystals of the sMMOH:MMOB complex. Using X-rays generated by either an X-ray free electron laser at room temperature or a synchrotron at 100 K, we obtained high resolution structures of the Methylosinus trichosporium OB3b sMMOH:MMOB complex for the first time. Analysis of the data shows in great detail how MMOB modulates the structure of sMMOH during the steps leading up to O2 binding. New insight is gained about the path O2 and methane take into the sMMOH active site, and how the selectivity and timing of this entry is controlled by MMOB in the sMMOH:MMOB complex. Additionally, biosynthetic incorporation of 5-fluorotryptophan into MMOB and MMOR, as well as post-translational modification of an MMOB variant with a trifluoroacetone probe, allowed us to use 1D-19F-NMR to investigate the complex series of sMMO protein interactions that regulate the beginning of the sMMO catalytic cycle. A new model emerges describing how sMMO protein component affinities and exchange from protein-protein complexes control the dynamics of reaction cycle intermediates to drive catalysis.enFluorine NMRmethane monooxygenaseX-ray crystallographyThesis or Dissertation