Conformational States of Nitric Oxide Synthase Characterized by Time-Resolved Fluorescence
Conformational States of Nitric Oxide Synthase Characterized by Time-Resolved Fluorescence
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时间分辨荧光表征一氧化氮合酶的构象状态
DOI：
10.1016/j.bpj.2019.11.2534
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发表时间：
2020
影响因子：
3.4
通讯作者：
Smith, Brian C.
中科院分区：
文献类型：
--
作者：
Johnson, Carey K.;Snyder, Alexa A.;Gambill, Alexandria K.;Arnett, David C.;Smith, Brian C.
一氧化氮合酶（NOS）通过一个过程催化一氧化氮的形成，该过程涉及电子顺序地从FAD转移到酶的还原酶结构域中的FMN，然后从FMN转移到同二聚体复合物中伴侣酶的加氧酶结构域中的血红素。通过将钙信号传导蛋白钙调蛋白（CaM）结合到位于还原酶和加氧酶结构域之间的CaM结合结构域来实现有效的NO合酶活性。由于电子转移的速率与距离密切相关，因此电子转移需要电子转移供体和受体非常接近。因此，NOS中的电子转移序列需要酶的多个构象状态，这表明电子转移是构象门控的。我们已经使用荧光标记的钙调素和时间分辨荧光检测存在的多个构象状态的NOS。荧光被FRET猝灭的血红素基团的酶，猝灭的程度取决于构象状态的酶。我们使用的NOS和钙调素的定点突变体分配荧光猝灭状态的构象状态的酶。选择对酶动力学和/或亚结构域相互作用具有已知影响的突变。结果表明，存在多种构象状态，其中钙调素是在接近的加氧酶结构域。我们建议，钙调素对接到加氧酶结构域促进与合作伙伴加氧酶结构域的FMN结构域的相互作用。这种构象将使电子从FMN转移到血红素。
Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide through a process that involves transfer of electrons sequentially from FAD to FMN in the reductase domain of the enzyme and then from FMN to a heme in the oxygenase domain of the partner enzyme in a homodimeric complex. Efficient NO synthase activity is enabled by binding of the calcium signaling protein calmodulin (CaM) to a CaM-binding domain situated between the reductase and oxygenase domains. Because the rate of electron transfer depends sharply on distance, electron transfer requires close proximity of electron-transfer donor and acceptor. The sequence of electron transfers in NOS therefore necessitates multiple conformational states of the enzyme, suggesting that the electron transfers are conformationally gated. We have used fluorescence-labeled CaM and time-resolved fluorescence to detect the presence of multiple conformational states of NOS. Fluorescence is quenched by FRET to the heme groups of the enzyme, and the extent of quenching depends on the conformational state of the enzyme. We used site-directed mutants of both NOS and CaM to assign fluorescence quenching states to conformational states of the enzyme. Mutations were chosen that have known effects on enzyme kinetics and/or subdomain interactions. The results suggest the presence of multiple conformation states in which CaM is in close proximity to the oxygenase domain. We suggest that CaM docking to the oxygenase domain facilitates interaction of the FMN domain with the partner oxygenase domain. Such a conformation would enable electron transfer from FMN to the heme.