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

Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S⁰‑Based Respiration and a Sulfur-Dependent Antioxidant System in <i>Pyrococcus</i>

FAD and NAD­(P)­H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur-reducing anaerobic hyperthermophiles <i>Pyrococcus horikoshii</i> and <i>Pyrococcus furiosus</i>; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from <i>P. horikoshii</i> to 2.7 Å resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds. These results indicate that the likely <i>in vivo</i> substrates are NAD­(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S₈) <i>in situ</i> is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O₂ and/or H₂O₂. As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S⁰ respiration in <i>Pyrococcus</i> or <i>Thermococcus</i> but appears to participate in oxidative defense in the presence of S⁰. When compared to the homologous Npsr enzyme from <i>Shewanella loihica</i> PV-4 and homologous enzymes known to reduce CoA disulfide, the <i>ph</i>CoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates