Characterization of the MethionineS-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst

Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mmmethionine, thed-isomer of methionine sulfoxide was preferentially detected over thel-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mmmethionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. TheVmax/Kmratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine andS-benzyl-l-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinctS-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The findings that the methionineS-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBCS-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionineKmdetermined with the purified methionineS-oxidase was 3.4 mm, whereas the apparent methionineKmdetermined with the purified SBCS-oxidase was 48 mm. The methionine sulfoxided:ldiastereomeric ratio obtained with methionineS-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBCS-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.