Fus‐SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency

Abstract The styrene monooxygenase, a two‐component enzymatic system for styrene epoxidation, was characterised through the study of Fus‐SMO – a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH 2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus‐SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well‐distanced arrangement (45–50 Å) facilitated by the flexible linker‘s loopy structure. Pre‐steady‐state kinetics elucidated the FAD ox reduction intricacies (k red =110 s −1 for bound FAD ox ), identifying free FAD ox binding as the rate‐determining step. The aerobic oxidation of FADH 2 (k ox =90 s −1 ) and subsequent decomposition to FAD ox and H 2 O 2 demonstrated StyA′s protective effect on the bound hydroperoxoflavin (k dec =0.2 s −1 ) compared to free cofactor (k dec =1.8 s −1 ). At varied styrene concentrations, k ox for FADH 2 ranged from 80 to 120 s −1 . Studies on NADH consumption vs. styrene epoxidation revealed Fus‐SMO′s ability to achieve quantitative coupling efficiency in solution, surpassing natural two‐component SMOs. The results suggest that Fus‐SMO exhibits enhanced FADH 2 channelling between subunits. This work contributes to comprehending FADH 2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.