Identification of the S-sulfenylated residues of GSNOR1 in Arabidopsis

The major mechanism by which H2O2 executes its physiological functions is through oxidation of sulfur in the target proteins. Thiol groups of protein cysteine (Cys) could be oxidized by H2O2 to sulfenic, a process also known as S-sulfenylation. Therefore, we reasoned that the activity of GSNOR1 might be regulated by H2O2 through S-sulfenylation. An in vitro S-sulfenylation assay revealed that GSNOR1-His recombinant protein was S-sulfenylated. Because Cys-284 and Cys-285 of GSNOR1 are adjacent and located on the same enzymolysis peptide segment, and also because both of them were identified as S-sulfenylated residues in vivo, to further test whether Cys-284 is S-sulfenylated, Cys-285 was substituted with serine (Ser).