Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, USA.
College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA.
J Biol Inorg Chem. 2024 Sep;29(6):611-623. doi: 10.1007/s00775-024-02068-8. Epub 2024 Aug 13.
Nitric oxide synthases (NOSs), a family of flavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNH) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimental analysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN-heme domain docking energies for nNOS and iNOS were similar (-3.8 kcal/mol), in agreement with the high degree of conservation of the FMN-heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN-heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN-heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN-heme IET rates between the two NOS isoforms.
一氧化氮合酶（NOSs）是一类具有相对刚性结构域的黄素-血红素蛋白，通过柔性区域连接，需要 FMN 结构域与血红素结构域最佳对接，以实现有效的结构域间电子转移（IET）。为了探测 FMN-血红素结构域间的对接，使用弛豫诱导偶极调制增强（RIDME）技术测量了神经元型和诱导型 NOS（nNOS 和 iNOS）的氧合酶/FMN（oxyFMN）构建体中 FMN 半醌自由基（FMNH）与低自旋高铁血红素中心之间的磁偶极相互作用。使用 NOS 构象分布的介观蒙特卡罗计算分析了 FMNH RIDME 数据，该计算方法得到了改进，可以考虑构成柔性结构域连接的氨基酸残基的天然自由度。这种组合计算和实验分析允许估计钙调蛋白（CaM）和 FMN 结构域与血红素结构域的对接复合物的稳定能和种群。此外，将五脉冲和缩放四脉冲 RIDME 数据结合到单个迹线中，显著降低了估计对接概率的不确定性。nNOS 和 iNOS 的 FMN-血红素结构域对接能相似（-3.8 kcal/mol），与 NOS 同工型之间 FMN-血红素结构域对接界面的高度保守性一致。尽管能量相似，但 nNOS 和 iNOS oxyFMN 中的 FMN-血红素结构域对接概率明显不同（~0.19 和 0.23），可能是由于 FMN-血红素结构域间连接的长度和对接界面形貌的差异所致。基于 IET 理论和 RIDME 实验的分析表明，构象动力学的变化可能占两个 NOS 同工型之间 FMN-血红素 IET 速率差异的一半。
