Overproduction of superoxide (•O
2
−
) and •O
2
−
-derived oxidants increases cellular oxidative stress. This can lead to cell death, via apoptosis or necrosis. An important response of protein thiols to oxidative stress is reversible formation of protein mixed disulfides via S-glutathiolation. 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 phosphorylation. Protein S-glutathiolation is increased in the post-ischemic heart. Human eNOS, which is of critical importance in maintaining cardiovascular function, contains 29 cysteinyl residues. To investigate the effects of S-glutathiolation on the regulation of eNOS function and its relation to cardiovascular diseases, eNOS functional alterations induced by S-glutathiolation were studied. Additionally, LC/MS/MS was used to determine the precise residues of eNOS involved in this redox-dependent thiol modification. S-glutatiolation significantly reduced NO production from heNOS, with a 63% decrease induced by incubation with 2 mM GSSG in
vitro
. This process was reversible by addition of DTT. Alkylation of the cysteinyl residues with N-ethylmaleimide (NEM) completely inhibited NO production. S-glutathiolation of an uncoupled heNOS increased •O
2
−
generation (> 70%), and this increase was only partially blocked by L-NAME, implicating the reductase site as the source for the increased •O
2
−
generation. When the cysteinyl residues were all alkylated with NEM, the •O
2
−
generation from eNOS was dramatically increased (+2.4-fold), and this increase was not inhibited by L-NAME. We have identified three cysteine residues, C
382
, C
689
and C
908
as sights of S-glutathiolation in heNOS, all three of which are conserved in all known mammalian eNOS enzymes. Therefore, cysteinyl residues are critical for the regulation of eNOS coupling, and S-glutatiolation of specific residues switches eNOS from an NO producing to a •O
2
−
generating enzyme, by inducing electron leakage from the reductase domain. As such, S-glutathiolation provides a novel mechanism for the regulation of heNOS, defining a unique pathway for the redox regulation of cardiovascular function.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
A convenient method to assess chemical modification of protein thiols by electrophilic metals
