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

J. Am. Chem. Soc., Article ASAP

DOI: 10.1021/jacs.5b12004

Publication Date (Web): December 30, 2015

Copyright © 2015 American Chemical Society

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

http://pubs.acs.org/doi/pdf/10.1021/jacs.5b12004

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-Cu_BMb, 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-Cu_BMb 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-Cu_BMb, 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.

 

This work provides a clear example of the importance of H-bond networks involving water in conferring activity to designed metalloenzymes. For enzymes, water is important factor.

I think that  structural features of H-bond networks involving water like this paper will be critical to enhancing the future success of high-activity metalloenzyme design.