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: 07/22/2025
Number of Support Periods: 7
PM: Brown, Katherine

Current Budget Period: 09/15/2025 - 03/14/2027
Current Project Period: 09/15/2025 - 06/15/2028
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., Professor Department of Molecular Biology & Biochemistry University of California, Irvine ABSTRACT Nitrogenase converts the inert dinitrogen to the bioavailable ammonia at ambient conditions. Despite major efforts in the past decades, the catalytic mechanism of nitrogenase has not been fully deciphered. NifEN, a structural and functional homolog of the catalytic NifDK component of nitrogenase, represents a primordial nitrogenase that can be compared with the extant nitrogenase for mechanistic studies of enzymatic N₂ reduction. We hypothesize that nitrogenase preserves the cofactor belt-sulfur mobilization as a key catalytic component but evolves its reaction mechanism from one that involves promiscuous belt-sulfur turnover to one that employs a sequential use of its three belt-sulfur sites for stepwise N₂ reduction via a coordinated rotation of cofactors. Based on this hypothesis, we propose to engineer partially defective or fully functional NifDK mimics (representing the extant nitrogenase) on the template of NifEN for comparative functional and structural analyses with the unmodified NifEN (representing the primordial nitrogenase). Using a combination of genetic, biochemical, spectroscopic, and structural methods, we aim to identify the similarities and distinctions between the reactions catalyzed by the primordial and extant nitrogenases and define the key determinants for the reactivity of the modern-day nitrogenase. These efforts not only contribute to a better mechanistic understanding of the enzymatic ammonia synthesis but also have the long-term potential of developing energy-efficient and environmentally-friendly strategies for nitrogenase-based applications 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., Professor

Department of Molecular Biology & Biochemistry

University of California, Irvine

ABSTRACT

Nitrogenase converts the inert dinitrogen to the bioavailable ammonia at ambient conditions. Despite major efforts in the past decades, the catalytic mechanism of nitrogenase has not been fully deciphered. NifEN, a structural and functional homolog of the catalytic NifDK component of nitrogenase, represents a primordial nitrogenase that can be compared with the extant nitrogenase for mechanistic studies of enzymatic N₂ reduction. We hypothesize that nitrogenase preserves the cofactor belt-sulfur mobilization as a key catalytic component but evolves its reaction mechanism from one that involves promiscuous belt-sulfur turnover to one that employs a sequential use of its three belt-sulfur sites for stepwise N₂ reduction via a coordinated rotation of cofactors. Based on this hypothesis, we propose to engineer partially defective or fully functional NifDK mimics (representing the extant nitrogenase) on the template of NifEN for comparative functional and structural analyses with the unmodified NifEN (representing the primordial nitrogenase). Using a combination of genetic, biochemical, spectroscopic, and structural methods, we aim to identify the similarities and distinctions between the reactions catalyzed by the primordial and extant nitrogenases and define the key determinants for the reactivity of the modern-day nitrogenase. These efforts not only contribute to a better mechanistic understanding of the enzymatic ammonia synthesis but also have the long-term potential of developing energy-efficient and environmentally-friendly strategies for nitrogenase-based applications 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.