# Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $55,999

## Abstract

PROJECT SUMMARY/ABSTRACT
 This proposal aims to elucidate how the bacterial enzyme nitrogenase catalyzes the
chemically difficult transformation of atmospheric dinitrogen into a bioavailable form, ammonia,
and why/how it utilizes ATP hydrolysis to drive this reaction. Being the only enzyme responsible
for reductive nitrogen fixation, nitrogenase sustains the agricultural/nutritional needs of ~40% of
the human population. Aside from its global importance, nitrogenase is a unique model system
with broad relevance to biological redox catalysis as well as ATP/GTP-dependent energy
transduction processes, which are both central to proper cellular functioning and thus directly
relevant to human health.
 Despite nearly five decades of extensive biochemical, biophysical and structural
characterization, the two most important questions about nitrogenase mechanism have not
answered in detail: a) Why and how ATP hydrolysis is ultimately utilized for the reduction of N2
or alternative substrates? b) What is the intimate mechanism of dinitrogen reduction on the
nitrogenase active site cluster, FeMoco? The major experimental challenge in the investigations
of nitrogenase arises from the fact that the catalytic activity of nitrogenase depends on
continuous ATP turnover, which leads to a heterogeneous mixture of redox and nucleotide-
bound states of nitrogenase that are difficult to distinguish from one another. To circumvent this
challenge, we have initiated a research program in cryogenic electron microscopy (cryoEM) to
structurally characterize dynamic states of nitrogenase at atomic resolution under enzymatic
turnover conditions. Preliminary experiments have not only established the feasibility of this
approach but also revealed unexpected structural features of nitrogenase which have fueled
new mechanistic hypotheses. In the proposed project, we aim to build upon on our preliminary
findings by a) mapping the ATP-driven conformational landscape of nitrogenase in
unprecedented detail and b) elucidating FeMoco structural dynamics and FeMoco-small
molecule interactions in atomic resolution, while also c) contributing to the development of
cutting-edge cryoEM methodologies for the structural interrogation of highly complex/dynamic
protein assemblies and metallocofactors.

## Key facts

- **NIH application ID:** 11097607
- **Project number:** 3R01GM148607-02S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** F. Akif Tezcan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $55,999
- **Award type:** 3
- **Project period:** 2023-01-15 → 2026-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11097607

## Citation

> US National Institutes of Health, RePORTER application 11097607, Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation (3R01GM148607-02S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/11097607. Licensed CC0.

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