Nitrogenase - From mechanistic investigation to heterologous expression

Nitrogenase is a complex metalloenzyme central to the global nitrogen cycle, catalyzing the reduction of atmospheric nitrogen to bioavailable ammonia. Termed biological nitrogen fixation, this fundamental process provides ~200 million tons of reduced nitrogen annually, sustaining Earth's ecosystems. Despite decades of research, the (1) enzymatic mechanism and (2) heterologous expression of nitrogenase remain elusive, presenting opportunities for innovation. With respect to 1, the traditional mechanistic thinking of nitrogenase has been driven by the presumption of a structurally ‘static’ cofactor during catalysis. Our recent structural observation of asymmetric belt-sulfur displacements with distinct dinitrogen species in the two cofactors of Mo-nitrogenase invokes a novel mechanism of N2 reduction that necessitates dynamic structural rearrangements of the cofactors during catalysis. In the next 5 years, we will use genetic, biochemical, spectroscopic and structural approaches to probe this novel reaction mechanism, demonstrating the stepwise N2 reduction at the three belt-sulfur sites of each cofactor and illustrating the alternate interaction between the two components of Mo-nitrogenase that drives the coordinated reaction sequences via asynchronous rotation of the two cofactors. These studies will establish a much-needed framework for further exploration of the intricate reaction mechanism of nitrogenase and, in the long run, facilitate the development of nitrogenase-based applications. With respect to 2, efforts to heterologous express nitrogenase have been focused on introducing a complete gene set into a foreign host like E. coli via a ‘top-down’ approach. However, this approach faces challenges in ensuring proper assembly of the key intermediates that eventually give rise to a functional nitrogenase. Our recently developed ‘bottom-up’ strategy allowed a systematic reconstruction of the nitrogenase assembly pathway in E. coli and the ultimate expression of an active prototype nitrogenase, albeit with reduced activity under anaerobic conditions. In the next 5 years, we will use protein-engineering and synthetic-biology methods to enhance the functionality of our prototype nitrogen-fixing system through optimization of the nitrogenase assembly pathway, engineering of the electron and energy supplies in the E. coli host, and implementation of the mechanism of oxygen tolerance in this expression system. These efforts will provide strategies that can also be applied to the heterologous expression of other complex metalloenzymes, thereby advancing both fundamental science and general biotechnological innovation. Project Number: 1R35GM161208-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Yilin Hu | Institution: UNIVERSITY OF CALIFORNIA-IRVINE, IRVINE, CA | Award Amount: $503,280 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11258227