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

• Applicant: Professor Dr. Oliver Einsle
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 536145634

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.

• DFG Programme: Research Grants
• International Connection: USA
• Cooperation Partner: Professorin Patricia dos Santos, Ph.D.