Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD-Transfer Reaction
Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD-Transfer Reaction
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DOI：
10.1021/bi400763h
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发表时间：
2013-09-03
期刊：
影响因子：
2.9
通讯作者：
Sazinsky, Matthew H.
中科院分区：
文献类型：
--
作者：
Morrison, Eliot;Kantz, Auric;Sazinsky, Matthew H.
来自恶臭假单胞菌S12的双组分黄素蛋白苯乙烯单加氧酶（SMO）催化依赖NADH和FAD的苯乙烯环氧化生成环氧苯乙烷。在本研究中，我们探究了黄素还原以及从还原酶（SMOB）转移到环氧化酶（NSMOA）组分的机制，并根据SMOB的2.2埃晶体结构报告了我们的发现。在与NADH快速混合时，SMOB形成一个NADH→FAD（氧化态）电荷转移中间体，并催化从NADH到FAD的氢负离子转移反应，速率常数为49.1±1.4 s⁻¹，此步骤与NAD⁺的快速解离偶联。电化学和平衡结合研究表明，NSMOA结合FAD（氢醌态）的紧密程度约为SMOB的13倍，这支持了FAD（氢醌态）从还原酶到环氧化酶的定向转移。在与NSMOA结合后，FAD氢醌态迅速与分子氧反应形成一种稳定的C(4a)-氢过氧化物中间体。在FAD转移反应中产生的脱辅基SMOB的半衰期增加了约21倍，这支持了脱辅基SMOB与NSMOA的过氧化物中间体之间的蛋白质 - 蛋白质相互作用。通过监测在有和无吡啶核苷酸存在时细胞色素c的竞争性还原，探究了FAD从SMOB解离和转运到NSMOA的机制。基于这些研究，我们提出一个模型，其中还原态的FAD在无反应的隔离态（S态）和更具反应性的转移态（T态）之间的平衡状态下与SMOB结合。氢负离子转移反应后NAD⁺的解离短暂地使T态占优势，促进FAD（氢醌态）向NSMOA的转移。吡啶核苷酸与SMOB - FAD（氢醌态）的结合使FAD（氢醌态）结合平衡从T态转变为S态。此外，根据吡啶核苷酸门控的黄素转移和电子转移反应，对本工作中报道的SMOB - FAD（氧化态）的2.2埃晶体结构进行了讨论。
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 angstrom 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) similar to 13-times more tightly than SMOB, which supports a vectoral transfer of FAD(hq) from the reductase to the epoxidase. After binding to NSMOA, FADhq 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 similar to 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) (t)o 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 angstrom 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.