Flavin Coenzyme Exchange and Inter-Subunit Dynamics in Styrene Monooxygenase from Pseudomonas putida
Tymesha Bovell
Department of Chemistry & Biochemistry
Faculty Supervisor: George Gassner
Styrene Monooxygenase (SMO) is a two-component flavoprotein system from Pseudomonas putida that catalyzes the epoxidation of styrene and has applications in biocatalysis and environmental remediation. SMO consists of a reductase (SMOB) that reduces FAD using NADH and an epoxidase (SMOA) that uses reduced FAD to generate enantiopure styrene oxide via a C4a-hydroxyflavin intermediate. Efficient transfer of FAD between these subunits is essential for catalysis, yet the mechanism of flavin exchange remains unresolved.
This work investigates how protein–protein interactions mediate flavin transfer. Fluorescent fusion constructs of SMOA and SMOB were engineered to enable Förster resonance energy transfer (FRET) measurements through spectral overlap with flavin intermediates. Using stopped-flow spectroscopy, we monitor single- and multiple-turnover kinetics of FAD reduction, exchange, and epoxidation in real time. These studies provide mechanistic insight into coenzyme channeling and support engineering of SMO for biocatalytic applications.