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

• Published in: Journal of the American Chemical Society Vol. 138; no. 4; pp. 1134 - 1137
• Main Authors: Petrik, Igor D, Davydov, Roman, Ross, Matthew, Zhao, Xuan, Hoffman, Brian, Lu, Yi
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.02.2016; American Chemical Society (ACS)
• Subjects: Communication; Crystallography; electron paramagnetic resonance spectroscopy; Electron Spin Resonance Spectroscopy; enzyme activity; enzymes; Hydrogen Bonding; myoglobin; Myoglobin - chemistry; Oxidoreductases - chemistry; oxygen; spectral analysis; Water
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.5b12004

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.