# Chemical Approaches to Studying the Mechanisms and Biophysical Properties of Complex Metallocofactors

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2024 · $306,005

## Abstract

Project Summary/Abstract
Enzymes with complex metallocofactors in their active sites catalyze myriad transformations relevant to human health and
disease. Understanding their reaction mechanisms requires molecular-level characterization of their resting states and
intermediate states, and metal-specific spectroscopic techniques are especially useful in this endeavor. However, the high-
nuclearity of many metallocofactors can limit the usefulness of such techniques; the signals arising from multiple metal
sites can be challenging to resolve, especially in mixtures of reaction intermediates. Moreover, it is often impossible to map
the rich spectroscopic information onto the geometric structure, and this severely limits our understanding of the chemical
bonding—and therefore the reactivity—of complex metallocofactors. We propose to address these challenges by developing
methods for modifying the isotopic and elemental compositions of complex metallocofactors, in particular the nitrogenase
catalytic cofactors. Nitrogenases are responsible for supplying a significant portion of the fixed nitrogen on the planet, and
they therefore play an important role in maintaining a healthy and growing human population. Their catalytic cofactors are
among the most complex in Nature, and as a result their reaction mechanisms have been especially difficult to characterize.
To overcome these challenges and gain new insights into the mechanism of biological nitrogen fixation, we will develop
chemical methods for precisely altering the isotopic and elemental composition of nitrogenase cofactors. Our approach will
be to discover mild protocols for removing specific Fe sites in nitrogenase cofactors and subsequently replacing them with
57Fe. The site-selectivity of the label will allow for the electronic structure (as elucidated spectroscopically) to be connected
to the geometric structure (as defined crystallographically), and will thereby provide unprecedented insights into the
chemical bonding and reactivity of nitrogenase cofactors. Studies of these cofactors in both their resting states and
intermediate states comprise the heart of the proposal. We will also extend the site-selective 57Fe labeling protocol to
incorporating different metals into specific sites of nitrogenase cofactors. This will yield artificial metalloenzymes that will
serve as mechanistic probes with potentially unique properties and/or reactivity. Completion of this project will provide
unprecedented mechanistic insights into biological nitrogen fixation and will articulate concepts and protocols for rendering
complex metallocofactors as mechanistically tractable as mononuclear active sites.

## Key facts

- **NIH application ID:** 10764308
- **Project number:** 5R01GM145787-03
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Daniel Leif Migdow Suess
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $306,005
- **Award type:** 5
- **Project period:** 2022-04-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10764308

## Citation

> US National Institutes of Health, RePORTER application 10764308, Chemical Approaches to Studying the Mechanisms and Biophysical Properties of Complex Metallocofactors (5R01GM145787-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10764308. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*