Cysteine-125 is the catalytic residue of Saccharomyces cerevisiae free methionine-R-sulfoxide reductase

Free methionine-R-sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine-R-sulfoxide to methionine. This enzyme cannot reduce oxidized methionine residues in proteins. While three Cys residues, Cys-91, Cys-101 and Cys-125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys-125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1-2% and 9-10%, respectively, of the activity of the wild-type fRMsr. These data show that Cys-125 is the catalytic residue in fRMsr.