Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase
Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase
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中子散射绘制了 NADPH 依赖性同化亚硫酸还原酶的高阶组装图
DOI：
10.1016/j.bpj.2022.04.021
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发表时间：
2022
影响因子：
3.4
通讯作者：
Stroupe, M. Elizabeth
中科院分区：
文献类型：
--
作者：
Murray, Daniel T.;Walia, Nidhi;Weiss, Kevin L.;Stanley, Christopher B.;Randolph, Peter S.;Nagy, Gergely;Stroupe, M. Elizabeth
用于生物质掺入的前体分子必须输入到细胞中，并使其可用于构建细胞的分子机器。含硫大分子需要硫处于S2−氧化态，然后才能被同化为整个生物圈中生物体所必需的氨基酸、辅因子和维生素。在α-变形菌中，NADPH依赖的同化亚硫酸盐还原酶（SiR）进行硫的最后六电子还原。SiR是由八聚体黄素蛋白还原酶（SiRFP）和四种血红素蛋白金属酶氧化酶（SiRHP）组成的十二聚体氧化还原酶。SiR进行电子转移还原反应，通过协调的结构域运动和亚基相互作用从亚硫酸盐产生硫化物，而不释放部分还原的中间体。努力了解负责SiR的效率的电子转移机制被混淆的结构异质性所产生的内在无序的区域在其整个复杂的，包括灵活的接头连接SiRFP的黄素结合域。因此，SiR十二聚体及其亚复合物的高分辨率结构是未知的，在SiR如何进行这种独特的大体积电子转移反应的基本理解中留下了空白。在这里，我们使用氘标记，在体外重建，分析超离心（AUC），小角中子散射（SANS），和中子对比度变化（NCV）观察SiR的高阶组装内的相对亚基位置。AUC和SANS显示SiR是一个灵活的十二聚体，并确认了SiRFP和SiRHP亚基化学计量不匹配。NCV表明复合物是不对称的，SiRHP在复合物的外围，SiRFP和SiRHP组分之间的质量中心相距超过100 μ m。SiRFP在组装成SiR的十二聚体时经历压缩，并且SiRHP在复合物中采用多个位置。SiR的高阶结构的所得地图支持还原酶亚基的结构域之间以及紧密结合或瞬时相互作用的还原酶和氧化酶亚基之间的电子转移的电子转移/transmechanism。
Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2−oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports acis/transmechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.