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 ) 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 of 17.4 ± 0.9 μM and 4.08 ± 0.8 μM, respectively. The binding of FMN to DMSMO is required prior to binding DMSO . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO . 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 did not generate any flavin adducts for monooxygenation. Therefore, H O is likely to be the reactive agent to attack the substrate. The H O assay results demonstrated production of H O from the oxidation of FMNH :DMSMO, whereas H O was not detected in the presence of DMSO , confirming H O utilization. The rate constant for methanesulfinate formation determined from rapid-quenched flow and the rate constant for flavin oxidation were similar, indicating that H O rapidly reacts with DMSO , 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.