Public 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
UEI: MJC5FCYQTPE6
DUNS: 046705849

Most Recent Award Date: 08/08/2024
Number of Support Periods: 6
PM: Brown, Katherine

Current Budget Period: 09/15/2022 - 09/14/2025
Current Project Period: 09/15/2020 - 09/14/2025
PI: Hu, Yilin

Supplement Budget Period: N/A

Public Abstract

PROJECT SUMMARY/ABSTRACT PROJECT TITLE Engineering a Functional Equivalent of Nitrogenase for Mechanistic Investigations of Ammonia Synthesis PRINCIPAL INVESTIGATOR (PI) Yilin Hu, Ph.D. Associate Professor Department of Molecular Biology & Biochemistry University of California, Irvine CO-PRINCIPAL INVESTIGATOR (co-PI) Markus Ribbe, Ph.D. Chancellor’s Professor Departments of Molecular Biology & Biochemistry, and Chemistry University of California, Irvine COLLABORATOR 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) ABSTRACT 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.

PROJECT SUMMARY/ABSTRACT

PROJECT TITLE

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

PRINCIPAL INVESTIGATOR (PI)

Yilin Hu, Ph.D. Associate Professor

Department of Molecular Biology & Biochemistry

University of California, Irvine

CO-PRINCIPAL INVESTIGATOR (co-PI)

Markus Ribbe, Ph.D. Chancellor’s Professor

Departments of Molecular Biology & Biochemistry, and Chemistry

University of California, Irvine

COLLABORATOR

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)

ABSTRACT

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.