title: Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H-Bond Network in Oxidase Activity of an Engineered Myoglobin.
abstract: Heme-copper oxidases (HCOs) catalyze efficient reduction of oxygen to water in biological respiration. Despite progress in studying native enzymes and their models, the roles of non-covalent interactions in promoting this activity are still not well understood. Here we report EPR spectroscopic studies of cryoreduced oxy-F33Y-CuBMb, a functional model of HCOs engineered in myoglobin (Mb). We find that cryoreduction at 77 K of the O2-bound form, trapped in the conformation of the parent oxyferrous form, displays a ferric-hydroperoxo EPR signal, in contrast to the cryoreduced oxy-wild-type (WT) Mb, which is unable to deliver a proton and shows a signal from the peroxo-ferric state. Crystallography of oxy-F33Y-CuBMb reveals an extensive H-bond network involving H2O molecules, which is absent from oxy-WTMb. This H-bonding proton-delivery network is the key structural feature that transforms the reversible oxygen-binding protein, WTMb, into F33Y-CuBMb, an oxygen-activating enzyme that reduces O2 to H2O. These results provide direct evidence of the importance of H-bond networks involving H2O in conferring enzymatic activity to a designed protein. Incorporating such extended H-bond networks in designing other metalloenzymes may allow us to confer and fine-tune their enzymatic activities.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Igor D
authors.initials: ID
authors.lastname: Petrik
authors.affiliation: The Department of Chemistry, Northwestern University , Evanston, Illinois 60201, United States.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|The Department of Chemistry, Northwestern University , Evanston, Illinois 60201, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Ross
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhao
authors.affiliation: The Department of Chemistry, Northwestern University , Evanston, Illinois 60201, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Hoffman
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D003461:Crystallography; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D009211:Myoglobin / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D014867:Water
chemical_list: D009211:Myoglobin; D014867:Water; D010088:Oxidoreductases