# Atomic Models of Nitrogen Fixation by Nitrogenase from CryoEM Structures

> **NIH NIH F32** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2022 · $69,802

## Abstract

PROJECT SUMMARY:
The nitrogenase enzyme complex is the only biological pathway for producing metabolically useful forms of
nitrogen from atmospheric dinitrogen. This two component protein system made up of an obligate reductase,
Fe-protein, and the catalytic protein, MoFe-protein, is only present in a small number of microorganisms, but
produces nearly 50% of all bioavailable nitrogen. Thus, the biological nitrogen fixation mechanism has extremely
important effects on global crop production and human health. Seminal works in the past half century have not
only revealed an intricate kinetic pathway for nitrogenase, but also the most complex metallocluster found in
nature within the MoFe-protein active site. This cluster, known as the FeMo-cofactor, has the composition
[7Fe:9S:1C:1Mo]-R-homocitrate and is only coordinated by two residues within the active site. Electrons are
shuttled through a [4Fe:4S] cluster in the Fe-protein to an [8Fe:7S] center in the MoFe-protein, known as the P-
cluster. These reducing equivalents are then delivered to the active site FeMo-cofactor where substrate reduction
occurs. Four consecutive cycles of electron transfer are required to bind dinitrogen, and four more are required
to fully convert one dinitrogen molecule to two ammonia molecules. It is unknown how the FeMo-cofactor is
primed for substrate binding, how the FeMo-cofactor binds or shuttles electrons to the substrate, or what role
the surrounding active site residues play in substrate reduction. The primary hypothesis of this proposal is
that physical rearrangements of the atoms in the FeMo-cofactor and in the surrounding protein during
substrate reduction are necessary for substrate access and binding to the cofactor, and may further play
a role in the reduction mechanism. The goal of this work is to determine what rearrangements occur in
nitrogenase throughout turnover of substrate by harnessing the power of cryoEM and performing experiments
outlined in two Aims. Aim 1: Structural characterization of resting states of nitrogenase: MoFe protein alone and
the ADP-AlF4- stabilized complex with Fe-protein by single particle cryoEM. Aim 2: Structural characterization of
nitrogenase turnover-related forms by single particle cryoEM. Through the pursual of Aims 1 and 2, I will obtain
training in the field of single particle cryoEM, and each Aim will provide expertise in metalloenzyme chemistry.
The research will be performed at the California Institute of Technology primarily in the well-known CryoEM
Center with ample microscopes and expertise. These Aims will shed much needed light on transient
intermediates within the nitrogenase turnover pathway thereby providing a better foundation for the rational
design of efficient synthetic nitrogen fixation platforms. In addition, the insights and the methodology developed
in this proposal can be applied to other poorly understood metalloenzymes related to human health.

## Key facts

- **NIH application ID:** 10479845
- **Project number:** 5F32GM143836-02
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Rebeccah Warmack
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $69,802
- **Award type:** 5
- **Project period:** 2021-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10479845

## Citation

> US National Institutes of Health, RePORTER application 10479845, Atomic Models of Nitrogen Fixation by Nitrogenase from CryoEM Structures (5F32GM143836-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10479845. Licensed CC0.

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