Reaction mechanism and kinetics of the two‐component flavoprotein dimethyl sulfone monooxygenase system: Using hydrogen peroxide for monooxygenation and substrate cleavage

The dimethyl sulfone monooxygenase system is a two‐component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO 2 ) 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 μ m and 4.08 ± 0.8 μ m , respectively. The binding of FMN to DMSMO is required prior to binding DMSO 2 . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO 2 . 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 DMSO 2 did not generate any flavin adducts for monooxygenation. Therefore, H 2 O 2 is likely to be the reactive agent to attack the substrate. The H 2 O 2 assay results demonstrated production of H 2 O 2 from the oxidation of FMNH − :DMSMO, whereas H 2 O 2 was not detected in the presence of DMSO 2 , confirming H 2 O 2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H 2 O 2 rapidly reacts with DMSO 2 , 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.