# Probing a novel reaction mechanism of nitrogenase with dynamic active-site rearrangements

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA-IRVINE · 2023 · $310,997

## Abstract

PROJECT SUMMARY
Nitrogenase catalyzes the ambient conversion of N2 to NH3 at its M-cluster site. This reaction
represents a major source of the usable form of nitrogen that supports the existence of human
population. As such, understanding how this enzyme effects ambient conversion of N2 to NH3 is
of significant relevance to human health. Our recent structural observation of asymmetric belt
sulfur displacements with distinct dinitrogen species in the two M-clusters of Mo-nitrogenase
invokes a novel mechanism of N2 reduction that necessitates dynamic structural
rearrangements of the M-clusters during catalysis. Using combined genetic, biochemical,
spectroscopic and structural approaches, we propose to investigate this novel mechanism of N2
reduction by demonstrating the stepwise mechanism of N2 reduction via cluster rotation,
assigning individual catalytic events at the three belt-sulfur sites of the M-cluster, and illustrating
the alternate binding of Fe protein to the two MoFe protein dimers that drives the asynchronous
cluster rotation in these dimers. Through our proposed studies, we hope to illustrate the
dynamic structural rearrangements of the active site of Mo-nitrogenase during N2 reduction and
establish a framework for further mechanistic exploration of this intricate reaction. Investigations
along this line could prove instrumental in (re)calibrating the mechanistic thinking of
nitrogenase, as all mechanistic studies to date have assumed that N2 reduction occurs at a
single site of a uniform M-cluster species that is structurally `static' during catalysis. These
efforts, if successful, will not only lead to a molecular depiction of the intricate catalytic
mechanism of nitrogenase, but also facilitate development of nitrogenase-based applications in
the long run.

## Key facts

- **NIH application ID:** 10557174
- **Project number:** 5R01GM141046-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Yilin Hu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $310,997
- **Award type:** 5
- **Project period:** 2021-04-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10557174

## Citation

> US National Institutes of Health, RePORTER application 10557174, Probing a novel reaction mechanism of nitrogenase with dynamic active-site rearrangements (5R01GM141046-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10557174. Licensed CC0.

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