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

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2023 · $59,532

## Abstract

PROJECT SUMMARY/ABSTRACT
 This proposal aims to elucidate how the bacterial metalloenzyme 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
been 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 metal 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 these preliminary findings by a) mapping the ATP-driven conformational
landscape of nitrogenase in unprecedented detail under catalytic turnover conditions 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:** 10795182
- **Project number:** 3R01GM148607-01S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** F. Akif Tezcan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $59,532
- **Award type:** 3
- **Project period:** 2023-01-15 → 2026-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10795182

## Citation

> US National Institutes of Health, RePORTER application 10795182, Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation (3R01GM148607-01S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10795182. Licensed CC0.

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