Abstract 149: S-glutathiolation of Endothelial Nitric Oxide Synthase Modulates its Enzymatic Activity and Protects against Oxidative Damage
Overproduction of superoxide (·O2−) and ·O2−-derived oxidants changes the redox status of the cell. Generation of reactive oxygen species can act as a redox signal trigging cellular events such as apoptosis, proliferation, and senescence. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfides via S-glutathiolation. Protein S-glutathiolation is increased in the post-ischemic heart. This redox based protein modification is thought to play an important role as an adaptive response to oxidative injury in cells, or alternatively in controlling cellular signaling in a manner similar to protein phosphorylation. Human eNOS, which is of critical importance in maintaining cardiovascular function, contains 29 cysteinyl residues. Incubation of heNOS with oxidized glutathione (GSSG) in vitro induced S-glutathiolation of heNOS in a dose dependent manner, confirmed by immunoblotting with an anti-GSH monoclonal antibody. To investigate the molecular function of S-glutathiolation of heNOS, we employed mass spectrometry, DEPMPO EPR spin trapping, and oxyhemoglobin assay for analysis. The LC/MS/MS proteomic analysis revealed that C382 from the oxygenase domain and, C689 and C908 from the reductase domain are sights of S-glutathiolation in human eNOS. All three of these cysteine residues are conserved in all the known mammalian eNOS. C689 is located in FMN-binding domain, whereas C908 is located in FAD-binding domain. The modification of these two conserved residues in the reductase domain could lead to altered electron flow from this domain to the oxygenase domain. Indeed, we observed that S-glutathiolated heNOS had increased ·O2−generation capacity (1.64 ± 0.12 fold) compared to the control heNOS. When the cysteine residues on the surface of heNOS were all alkylated with 1mM N-ethylmaleimide (NEM) there was no change in ·O2− generation. Interestingly, there was little effect of S-glutathiolation on eNOS NO production, with GSSG concentrations up to 5 mM, but with 10 mM levels complete inhibition occurred. Alkylation of the cysteine residues with 1mM NEM also eliminated NO production. Thus, S-glutathiolation provides a unique mechanism for redox regulation of heNOS function.