Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: Using...

The dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO₂ ) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH^- with a K_d of 17.4 ± 0.9 μm and 4.08 ± 0.8 μm, respectively. The binding of FMN to DMSMO is required prior to binding DMSO₂ . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO₂ . Substituting reduced DMSR with FMNH^- demonstrated the same oxidation kinetics, indicating that FMNH^- from DMSR was transferred to DMSMO. The oxidation of FMNH^- :DMSMO, with and without DMSO₂ did not generate any flavin adducts for monooxygenation. Therefore, H₂ O₂ is likely to be the reactive agent to attack the substrate. The H₂ O₂ assay results demonstrated production of H₂ O₂ from the oxidation of FMNH^- :DMSMO, whereas H₂ O₂ was not detected in the presence of DMSO₂ , confirming H₂ O₂ utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H₂ O₂ rapidly reacts with DMSO₂ , with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC-MS.