Reversible S-nitrosylation in an engineered azurin

• Shiliang Tian,
• Jing Liu,
• Ryan E. Cowley,
• Parisa Hosseinzadeh,
• Nicholas M. Marshall,
• Yang Yu,
• Howard Robinson,
• Mark J. Nilges,
• Ninian J. Blackburn,
• Edward I. Solomon
• & Yi Lu
• • http://www.nature.com/nchem/journal/vaop/ncurrent/full/nchem.2489.html

  • Abstract

    • Abstract• 
    • Main• 
    • Results• 
    • Discussion• 
    • Conclusions• 
    • Methods• 
    • Accession codes• 
    • References•
    • Acknowledgements• 
    • Author information• 
    • Supplementary information
    S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper–thiolate bond in an engineered copper centre inPseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)–S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.