Assembly and Maturation of the Iron-Sulfur Clusters of Nitrogenases

固氮酶系统是一种双组分的酶机械，催化大气中的氮素还原固定为生物可利用的铵。它的亚复合体，ATP水解铁蛋白和催化固氮酶，包含复杂的铁-硫簇，需要专门的、多步骤成熟的机器来组装。虽然人们对固氮酶辅因子成熟的步骤顺序有很大了解，但目前对单个因子的分子细节和机制的了解仍然很少。有了产生模式重氮菌棕色固氮菌的变种和缺失菌株的能力，我们将在本提案中解决这些开放的问题。我们专注于固氮酶辅因子成熟的三个主要步骤，构建碱性[4Fe：4S]片段用于进一步组装的NifSU络合物，将两个这样的簇重新排列为复杂辅因子前体的自由基/SAM酶NifB，以及通过插入顶端异金属、Mo或V完成成熟的EN支架。此外，我们将使用空间精细化的反常色散(扩散)精化来获得对该联盟内调查的几个复杂金属团簇的实验洞察。

The nitrogenase system is a two-component enzyme machinery that catalyzes the reductive fixation of atmospheric dinitrogen as bioavailable ammonium. Its subcomplexes, the ATP-hydrolyzing Fe protein and the catalytic dinitrogenase, contain complex iron-sulfur clusters that require a dedicated, multi-step maturation machinery for their assembly. While the sequence of steps in nitrogenase cofactor maturation is largely understood, current knowledge about the molecular details and mechanisms of the individual factors remains scarce. With the ability to generate variants and deletion strains of the model diazotroph Azotobacter vinelandii, we will address these open question in the present proposal. We focus on the three major steps of nitrogenase cofactor maturation, the NifSU complex that builds a basic [4Fe:4S] fragment for further assembly, the radical/SAM enzyme NifB that rearranges two such clusters into a complex cofactor precursor, and the EN scaffold that completes maturation by inserting an apical heterometal, Mo or V. Affinity-tagged constructs for the three complexes are available of will be generated during the project, to be investigated by X-ray crystallography and spectroscopy, making use of the broad range of methods and tools within PP 1927. In addition, we will use spatially refined anomalous dispersion (SpReAD) refinement to gain experimental insights into several of the complex metal clusters that are investigated within the consortium.