Skip to Main Content

Title ImagePublic Abstract


DE-SC0016510: Engineering a Functional Equivalent of Nitrogenase for Mechanistic Investigations of Ammonia Synthesis

Award Status: Active
  • Institution: Regents of the University of California, Irvine, Irvine, CA
  • DUNS: 046705849
  • Most Recent Award Date: 05/12/2023
  • Number of Support Periods: 6
  • PM: Brown, Katherine
  • Current Budget Period: 09/15/2022 - 09/14/2024
  • Current Project Period: 09/15/2020 - 09/14/2024
  • PI: Hu, Yilin
  • Supplement Budget Period: N/A

Public Abstract




Engineering a Functional Equivalent of Nitrogenase for Mechanistic Investigations of Ammonia Synthesis



Yilin Hu, Ph.D. Associate Professor

Department of Molecular Biology & Biochemistry

University of California, Irvine



Markus Ribbe, Ph.D. Chancellor’s Professor

Departments of Molecular Biology & Biochemistry, and Chemistry

University of California, Irvine



Professor David Britt

University of California, Davis, CA (Advanced EPR spectroscopy)

Professors Keith Hodgson and Britt Hedman

Stanford University, Menlo Park, CA (XAS and XES spectroscopy)



Nitrogenase catalyzes the conversion of inert dinitrogen to bioavailable ammonia. Despite major efforts in the past decades, the catalytic mechanism of nitrogenase has not been fully deciphered.  Here, we propose to use NifEN of Azotobacter vinelandii as a mutational platform to construct partially defective or fully functional MoFe protein mimics for mechanistic investigations of ammonia synthesis by nitrogenase. Genetic methods (mutagenesis and homologous recombination) will be used to strategically reconstruct defective or functional mimics of MoFe protein, and biochemical (metal and enzymatic assays) and spectroscopic (EPR and XAS/EXAFS analyses) methods will be employed to monitor and analyze the (re)construction process. Success in generating partially defective variants on a NifEN template will facilitate capture of the reaction intermediates of N2 reduction for mechanistic investigations of nitrogenase; whereas success in generating an active nitrogenase equivalent on a NifEN template will enable identification of all functional determinants for the catalytic activity of nitrogenase and provide a proof-of-concept for minimizing the essential nif gene set for future transgenic expression of nitrogenase via synthetic biology. Together, these efforts not only contribute to a better understanding of the mechanism of ammonia synthesis by nitrogenase, but also have the long-term potential in developing energy-efficient strategies for nitrogenase-based ammonia synthesis and generating modified enzymes with improved efficiency in ammonia or hydrogen production. As such, our proposed research addresses the DOE mission of providing answers to America’s energy and environmental challenges through transformative science and technology solutions.

Scroll to top