Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD-Transfer Reaction

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FAD_ox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s^–1, in a step that is coupled to the rapid dissociation of NAD⁺. Electrochemical and equilibrium-binding studies indicate that NSMOA binds FAD_hq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FAD_hq from the reductase to the epoxidase. After binding to NSMOA, FAD_hq rapidly reacts with molecular oxygen to form a stable C­(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein–protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD⁺ after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FAD_hq to NSMOA. The binding of pyridine nucleotides to SMOB–FAD_hq shifts the FAD_hq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB–FAD_ox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.