Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1). Single turnover studies of the reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance characteristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find that the observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer and passive diffusion of reduced FAD from SMOB to SMOA.