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

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.