Department of Chemistry and Biochemistry, San Francisco State University , San Francisco, California, United States.
Biochemistry. 2013 Sep 3;52(35):6063-75. doi: 10.1021/bi400763h. Epub 2013 Aug 20.
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 → FADox 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 FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq 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 ∼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 FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.
两成分黄素蛋白苯乙烯单加氧酶（SMO）来自恶臭假单胞菌 S12 催化 NADH 和 FAD 依赖的苯乙烯环氧化为苯乙烯氧化物。在这项研究中，我们研究了黄素还原和从还原酶（SMOB）到环氧化酶（NSMOA）组分转移的机制，并根据 SMOB 的 2.2 Å 晶体结构报告了我们的发现。在与 NADH 快速混合后，SMOB 形成 NADH→FADox 电荷转移中间体，并催化 NADH 向 FAD 的氢转移反应，速率常数为 49.1±1.4 s(-1)，这一步与 NAD(+)的快速解离偶联。电化学和平衡结合研究表明，NSMOA 与 SMOB 相比，结合 FADhq 约强 13 倍，这支持了 FADhq 从还原酶到环氧化酶的向量转移。与 NSMOA 结合后，FADhq 与分子氧快速反应形成稳定的 C(4a)-过氧化物中间物。在 FAD 转移反应中生成的 apoSMOB 的半衰期增加了约 21 倍，支持 apoSMOB 与 NSMOA 的过氧化物中间物之间的蛋白质-蛋白质相互作用。通过监测在有或没有吡啶核苷酸存在的情况下细胞色素 c 的竞争还原，探测了 FAD 从 SMOB 向 NSMOA 解离和转运的机制。基于这些研究，我们提出了一个模型，其中还原的 FAD 在反应平衡中与 SMOB 结合，在不反应的隔离状态（S 状态）和更反应的转移状态（T 状态）之间。氢转移反应后 NAD(+)的解离瞬时使 T 态增加，促进 FADhq 向 NSMOA 的转移。吡啶核苷酸与 SMOB-FADhq 的结合将 FADhq 结合平衡从 T 态转移到 S 态。此外，本文报道的 SMOB-FADox 的 2.2 Å 晶体结构根据吡啶核苷酸门控黄素转移和电子转移反应进行了讨论。
