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

Abstract

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 Kd 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, H2O2 is likely to be the reactive agent to attack the substrate. The H2O2 assay results demonstrated production of H2O2 from the oxidation of FMNH−:DMSMO, whereas H2O2 was not detected in the presence of DMSO2, confirming H2O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2O2 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.