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

Igor D. Petrik, Roman Davydov, Matthew Ross, Xuan Zhao, Brian Hoffman*, Yi Lu

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1134-1137
Number of pages4
JournalJournal of the American Chemical Society
Volume138
Issue number4
DOIs
StatePublished - Feb 3 2016

Funding

We thank Dr. S. Toshkov for assistance with γ-irradiation, Drs. V. Šrajer and R. Henning for assistance with crystal studies at BioCARS, and Drs. P. Hosseinzadeh, S. Tian, and I. Denisov for helpful discussions. Research reported in this publication was supported by the National Institutes of Health (R01GM062211 to Y.L. and R01GM111097 to B.M.H.).

ASJC Scopus subject areas

  • General Chemistry
  • Biochemistry
  • Catalysis
  • Colloid and Surface Chemistry

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