Structure and Mechanism of Styrene Monooxygenase Reductase:
New Insight into the FAD-Transfer Reaction
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Abstract
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<sub>ox</sub> charge-transfer
intermediate and catalyzes a hydride-transfer reaction from NADH to
FAD, with a rate constant of 49.1 ± 1.4 s<sup>–1</sup>, in a step that is coupled to the rapid dissociation of NAD<sup>+</sup>. Electrochemical and equilibrium-binding studies indicate
that NSMOA binds FAD<sub>hq</sub> ∼13-times more tightly than
SMOB, which supports a vectoral transfer of FAD<sub>hq</sub> from
the reductase to the epoxidase. After binding to NSMOA, FAD<sub>hq</sub> 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<sup>+</sup> after the hydride-transfer reaction
transiently populates the T state, promoting the transfer of FAD<sub>hq</sub> to NSMOA. The binding of pyridine nucleotides to SMOB–FAD<sub>hq</sub> shifts the FAD<sub>hq</sub>-binding equilibrium from the
T state to the S state. Additionally, the 2.2 Å crystal structure
of SMOB–FAD<sub>ox</sub> reported in this work is discussed
in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer
reactions