Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H-Bond Network in Oxidase Activity of an Engineered Myoglobin

[Image: see text] 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-Cu(B)Mb, a functional model of HCOs engineered in myoglobin (Mb). We find that cryoreduction at 77 K of the O(2)-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-Cu(B)Mb reveals an extensive H-bond network involving H(2)O 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-Cu(B)Mb, an oxygen-activating enzyme that reduces O(2) to H(2)O. These results provide direct evidence of the importance of H-bond networks involving H(2)O 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.