Thiol-disulfide exchange is involved in the catalytic mechanism of peptidemethionine sulfoxide reductase

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) reverses the inac tivation of many proteins due to the oxidation of critical methionine resid ues by reducing methionine sulfoxide. Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equiv alents from either DTT or a thioredoxin-regenerating system. In an effort t o understand these observations. the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzy matic activity of the variants and their free sulfhydryl states by mass spe ctrometry revealed that thiol-disuifide exchange occurs during catalysis. I n particular, the strictly conserved Cys-72 was found to be essential for a ctivity and could form disulfide bonds, only upon incubation with substrate , with either Cys-218 or Cys-227, located at the C terminus. The significan tly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents f rom thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme. Cys-72 acts as a nucleophile and attacks the s ulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the r educed state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.