# Artificial Hydrogenases Via Rational Metalloprotein Design

> **NIH NIH R15** · UNIVERSITY OF MISSISSIPPI · 2020 · $75,000

## Abstract

PROJECT SUMMARY/ABSTRACT
 Hydrogenases are a class of complex metal containing enzymes that generate energy for certain organisms
by catalyzing reversible interconversion between protons and hydrogen gas. Unraveling the intricate
mechanistic details about the mode of action of this enzyme could lead to significant advances in the alternate
energy research. However, the innate complexity of these enzymes due to the presence of many metallic
cofactors, low yield of purification and oxygen intolerance makes studying these enzymes challenging. Our
long term goals are to design artificial hydrogenases that serve as simpler platforms mimicking these
metalloenzymes.
 Metalloprotein design is an appealing and well-established approach to model complex metalloproteins in
nature using minimal protein scaffolds. Although these designed systems are less complex than native
enzymes, they can serve as structural and functional analogues of the native metalloenzymes allowing rational
engineering of both the primary and secondary shell interactions within the protein scaffold. Employing this
approach, and guided by strong preliminary data, we propose to pursue two Specific Aims describing the
overall design principles of artificial hydrogenases. Furthermore, we will investigate how the metal site and
surrounding protein scaffold work synergistically to influence the physical and catalytic properties of the
designed metalloenzymes. The Specific Aims are: 1) Reengineering a thiolate-rich copper storage protein
(Csp1) into a Ni binding protein (NBP); and 2) Elucidate the role of remote interactions beyond primary
coordination in fine-tuning the thermodynamic, redox, and catalytic properties of NBP. Our data indicate that
the designed NBP is folded and stable in solution, and demonstrates highly cooperative unfolding behavior.
NBP binds 1 equivalent of Ni(II) with micromolar affinity. In addition, Ni(II)-NBP is also active for electrocatalytic
and photocatalytic H+ reduction to H2 gas.
Collectively, results obtained from the proposed research will broadly impact the fields of metalloprotein
design, bioinorganic chemistry, and alternate energy research. Our studies will establish new principles in
rational design of artificial hydrogenases, and provide insight into the likely functional mechanism of these
metalloenzymes. Using this knowledge, it would be possible to prepare new biocatalysts with improved stability
and novel functions.

## Key facts

- **NIH application ID:** 10136364
- **Project number:** 3R15GM131260-01S1
- **Recipient organization:** UNIVERSITY OF MISSISSIPPI
- **Principal Investigator:** Saumen Chakraborty
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $75,000
- **Award type:** 3
- **Project period:** 2018-09-20 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10136364

## Citation

> US National Institutes of Health, RePORTER application 10136364, Artificial Hydrogenases Via Rational Metalloprotein Design (3R15GM131260-01S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10136364. Licensed CC0.

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