Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.
Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, New Mexico 87131, United States.
J Phys Chem B. 2022 Sep 15;126(36):6811-6819. doi: 10.1021/acs.jpcb.2c04091. Epub 2022 Sep 3.
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.
一氧化氮合酶（NOS）是一种同二聚体黄素血红素蛋白，负责催化 l-精氨酸（l-Arg）氧化为瓜氨酸和一氧化氮。电子通过 N 端含血红素的加氧酶结构域和 C 端还原酶结构域中含 FMN 的（亚）结构域之间的域间电子转移为反应提供电子。人们广泛关注阐明构象动力学如何调节两个结构域之间的电子转移。在这里，我们研究了结构域间 FMN-血红素相互作用对诱导型 NOS（iNOS）血红素活性位点动力学的影响。稳态线性和时间分辨二维红外（2D IR）光谱被应用于探测氧合酶结构域内血红素中的 CO 配体，用于全长和截短或突变的人 iNOS 构建体。尽管 CO 配体的线性 IR 光谱在构建体之间相同，但 2D IR 光谱显示出频率动力学的变化。由于同时存在 FMN 和加氧酶结构域，能够正确形成 FMN/加氧酶对接状态的野生型构建体表现出比单独的加氧酶结构域更慢的动力学。引入预测会扰乱结构域之间静电相互作用的突变（E546N）导致测量的动力学介于全长和单个加氧酶结构域之间，与对接/非对接平衡的扰动一致。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转移距离内，而且以预期促进有效电子转移的方式调节活性位点内 CO 配体感知的动力学。
