Deciphering the Path of S-nitrosation of Human Thioredoxin: Evidence of an Internal NO Transfer and Implication for the Cellular Responses to NO

Nitric oxide (NO) is a free radical with a signaling capacity. Its cellular functions are achieved mainly through S-nitrosation where thioredoxin (hTrx) is pivotal in the S-transnitrosation to specific cellular targets. In this study, we use NMR spectroscopy and mass spectrometry to follow the mechanism of S-(trans)nitrosation of hTrx. We describe a site-specific path for S-nitrosation by measuring the reactivity of each of the 5 cysteines of hTrx using cysteine mutants. We showed the interdependence of the three cysteines in the nitrosative site. C73 is the most reactive and is responsible for all S-transnitrosation to other cellular targets. We observed NO internal transfers leading to C62 S-nitrosation, which serves as a storage site for NO. C69-SNO only forms under nitrosative stress, leading to hTrx nuclear translocation.

Automated summary

In this study, the mechanism of S-(trans)nitrosation of thioredoxin (hTrx) by nitric oxide (NO) was investigated using NMR spectroscopy and mass spectrometry. The reactivity of each of the five cysteines of hTrx was measured, revealing that C73 is the most reactive and responsible for S-transnitrosation to other cellular targets. Internal transfers of NO were observed leading to S-nitrosation of C62, which serves as a storage site for NO. Additionally, C69-SNO only forms under nitrosative stress and leads to nuclear translocation of hTrx.