# Biosynthesis and Reactivity of the Active Site of the [FeFe]-Hydrogenases

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2020 · $308,676

## Abstract

Hydrogenase enzymes are pervasive, being found in bacteria, archaea, and some higher organisms.
Hydrogenases are found in many pathogens, including some that inhabit the human gut. The [FeFe] and
[NiFe]-hydrogenases mediate the most fundamental chemical reaction: the interconversion of H2 with
protons and reducing equivalents. Since H2 is an unusual substrate, the enzymes are also structurally
exceptional with an array of distinctive cofactors, especially the site of H2 binding and release. Similarly,
the biosynthesis of these active sites involves elaborate and novel biochemistry. In addition to
biophysical approaches, elucidation of these mechanisms relies on organometallic chemistry, especially
since the substrates (H2, H+, H-) are often invisible to conventional biophysical methods.
This project aims to elucidate the biosynthesis and mechanism of action of the [FeFe] enzymes, the faster
of the two main hydrogenases and the one most amenable to development for other applications. The
work involves synthesis of proposed intermediates, spectroscopic and electrochemical characterization,
in vitro assays, and isotopic labeling. It also relies on extensive collaborations with groups that offer
specialized skills in theory, synchrotron-based spectroscopy, spin resonance, and in vitro testing.
This project specifically focuses on the recently confirmed azadithiolate (adt) cofactor, which is the
enzyme's most remarkable component from a mechanistic perspective. Although the adt cofactor is
unstable in the free state, methods are being developed to stabilize it in protected form. It will be
isotopically labeled and incorporated into an apo enzyme. These experiments will allow us to identify the
precursor to the adt (main hypothesis: radical SAM induced reactions of cysteine-Fe conjugates). Prior to
construction of the Fe2(adt) ensemble, two Fe-cysteine-CO centers are supposed to be preorganized in a
scaffold protein HydF. This work will produce the first Fe-cysteine-CO complexes for testing in vitro and
by chemical methods. Finally, the work will evaluate the role of adt on the catalytic mechanism. Of
specific interest is the influence of the protonation state of the amine cofactor on the behavior of the [2Fe]
subunit. The latter studies will be conducted on model complexes containing Fe2 with authentic and
modified amine cofactors. Of specific interest is the influence of N-protonation on the binding of H2 and
the inhibitors CO and formaldehyde.
Overall, the work focuses on understanding the biosynthesis and mechanism of action of the fastest
catalysts known for producing H2 from water.

## Key facts

- **NIH application ID:** 9912778
- **Project number:** 5R01GM061153-19
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Alison R Fout
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $308,676
- **Award type:** 5
- **Project period:** 2000-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9912778

## Citation

> US National Institutes of Health, RePORTER application 9912778, Biosynthesis and Reactivity of the Active Site of the [FeFe]-Hydrogenases (5R01GM061153-19). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9912778. Licensed CC0.

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