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

Research output: Contribution to journalArticlepeer-review


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 in Pseudomonas 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 bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.

Original languageEnglish (US)
Pages (from-to)670-677
Number of pages8
JournalNature Chemistry
Issue number7
StatePublished - Jul 1 2016

ASJC Scopus subject areas

  • General Chemistry
  • General Chemical Engineering


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