Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.
Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.
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DOI：
10.1021/acs.jpcb.2c04091
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发表时间：
2022-09-15
影响因子：
3.3
通讯作者：
Thielges, Megan C.
中科院分区：
文献类型：
--
作者：
Tumbic, Goran W.;Li, Jinghui;Jiang, Ting;Hossan, Md Yeathad;Feng, Changjian;Thielges, Megan C.
一氧化氮合酶（NOS）是一种同型二聚体的黄素血红蛋白，负责催化L -精氨酸（L - Arg）氧化为瓜氨酸和一氧化氮。反应所需电子通过N末端含血红素的加氧酶结构域和C末端还原酶结构域的含FMN（亚）结构域之间的结构域间电子传递来提供。大量的注意力集中在阐明构象动力学如何调节结构域之间的电子传递。在此，我们研究结构域间FMN -血红素相互作用对诱导型一氧化氮合酶（iNOS）血红素活性位点动力学的影响。采用稳态线性和时间分辨二维红外（2D IR）光谱技术，对人iNOS的全长以及截短或突变构建体的加氧酶结构域内血红素上的CO配体进行探测。虽然CO配体的线性红外光谱在这些构建体之间是相同的，但二维红外光谱揭示了频率动力学的差异。由于同时存在FMN和加氧酶结构域而能够正确形成FMN/加氧酶对接状态的野生型构建体，其动力学比单独的加氧酶结构域更慢。引入预计会扰乱结构域之间静电相互作用的突变（E546N），导致所测得的动力学介于全长构建体和单个加氧酶结构域之间，这与对接/非对接平衡受到扰动是一致的。这些结果表明，FMN结构域与加氧酶结构域的对接不仅使FMN辅因子处于血红素结构域的电子传递距离内，而且还以一种预期会促进高效电子传递的方式调节活性位点内CO配体所感知的动力学。
Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN–heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.
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