Abstract
S-nitrosothiols are known as reagents for NO storage and transportation, and as regulators in many physiological processes. While the S-nitrosylation catalyzed by heme 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 center in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential via 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 within 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.
Accepted Version
Published Version
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