NSF/BIO-DFG: Biological Fe-S intermediates in the synthesis of nitrogenase metalloclusters

固氮酶催化氮气生物转化为NH4+，这是生物地球化学氮循环中的关键步骤。尽管固氮对地球上的生命很重要，但固氮是一种仅限于一小部分微生物的特征，固定氮气的能力在机制、遗传学和调节方面是复杂的。固氮酶的活性取决于参与其相关金属辅因子合成的因素，以及支持这种高度赋能反应的代谢条件。因此，定义这些因素所执行的反应具有内在的智力价值，并有助于理解它们的演变。这对于制定可持续的、节能的战略来生产含氮粮食作物补充剂具有重要意义。这一建议旨在阐明初始反应步骤涉及合成由NifU和Nifs促进的用于固氮酶的Fe-S簇合物。在其他好氧固氮微生物中，NifU和Nifs的存在占主导地位，这表明当时需要专用的Fe-S簇生物合成系统来维持固氮。在模式细菌棕色固氮菌中，NifUS是三种固氮酶异构体(NIF、ANF和VNF)中的任何一种固氮所必需的。我们的初步数据现在显示，当在其本土生物A.vinelandii中表达时，NifU上有一个以前未描述的集群物种。我们还表明，Fpr2是一种合适的生理还原剂，首次能够检测到与NifU相关的新的EPR活性物种。我们推测，这种短暂的簇状物种代表了固氮酶金属簇合成过程中的一个功能中间体。该研究计划旨在通过建立1)与NiFU相关的新簇合物的位置和光谱特征，2)参与这一过程的附加因素，以及3)新簇合物作为合成固氮酶金属簇合物的基石的反应性，来研究Fe-S簇合物合成的机理和化学步骤。确定使固氮成为可能的分子和进化细节有可能产生重大的农学、经济和环境影响。这些原理还可以指导在更复杂的体系中对铁-S簇合物的基础研究，而不仅仅限于固氮。这个跨学科、协作的国际项目将通过研究经验和职业发展机会吸引本科生和研究生。德国和美国的PI伙伴关系将支持两项协同研究和专业发展努力：(A)研究生在国外的合作研究经验；(B)为当地本科生提供的指导性研究奖学金。

Nitrogenase catalyzes the biological conversion of N2 gas into NH4+, a critical step in the biogeochemical nitrogen cycle. Despite its importance for life on Earth, nitrogen fixation is a trait limited to a subset of microbes, and the capacity to fix N2 is complex in mechanism, genetics, and regulation. The activity of nitrogenase depends on factors involved in the synthesis of its associated metallocofactors and on metabolic conditions that support this highly endergonic reaction. Therefore, defining the reactions performed by these factors has intrinsic intellectual merit and helps to understand their evolution. It is significant for developing sustainable, energy-saving strategies to produce nitrogen-based food crop supplements. This proposal aims to elucidate initial reaction steps involve the synthesis of Fe-S clusters destined for nitrogenase that are promoted by NifU and NifS. The presence of NifU and NifS is dominant in other aerobic nitrogen-fixing microbes, suggesting the requirement of a dedicated Fe-S cluster biosynthesis system for sustaining nitrogen fixation exactly then. In the model bacterium Azotobacter vinelandii, NifUS is required for nitrogen fixation by any of the three nitrogenase isoforms (Nif, Anf, and Vnf). Our preliminary data now show a previously uncharacterized cluster species on NifU when expressed in its native organism, A. vinelandii. We also show that Fpr2 is a suitable physiological reductant enabling for the first time the detection of novel, EPR-active species associated with NifU. We hypothesize that this transient cluster species represents a functional intermediate in the synthesis of nitrogenase metalloclusters. The research plan is aimed at investigating the mechanistic and chemical steps of Fe-S cluster synthesis by establishing 1) the location and spectroscopic features of novel cluster species associated with NifU, 2) the involvement additional factors in this process, and 3) the reactivity of the new cluster species as building blocks for the synthesis of nitrogenase metalloclusters. Defining the molecular and evolutionary details that enable nitrogen fixation has a prospect for a major agronomic, economic, and environmental impact. These principles can also guide foundational studies on Fe-S clusters on more complex systems not limited to nitrogen fixation. The interdisciplinary, collaborative international project will engage undergraduate and graduate students through research experiences and professional development opportunities. A German and an American PI partnership will support two synergistic research and professional development efforts: (a) collaborative research experiences abroad for graduate students; and (b) mentored research fellowships for local undergraduate students.