# In vitro sulfuration of Rhodobacter capsulatus formate dehydrogenase

## TL;DR

This study shows how to restore the activity of a CO2-reducing enzyme by adding a sulfido ligand in the lab, which could help develop bio-inspired methods for carbon capture.

## Contribution

A new in vitro method for sulfido ligand incorporation in Rhodobacter capsulatus FDH is demonstrated, reactivating the enzyme and confirming the ligand's role.

## Key findings

- Reductive treatment with sulfide or bisulfite under anaerobic conditions reactivates FDH to levels comparable to native enzyme.
- EPR spectroscopy confirmed the insertion of a sulfido ligand into the bis-MGD Mo site, forming a MoV-SH state.
- 33S labeling verified the chemical insertion of the sulfido ligand and its electronic role in the active site.

## Abstract

Metal-dependent formate dehydrogenases (FDHs) are of considerable interest as a bioinspired metalloenzyme target to efficiently reduce the greenhouse gas CO2 into the portable energy carrier formate under physiological conditions. These enzymes were shown to harbor an active site sulfido ligand that is essential for the formate oxidation and CO2 reduction activity and contributes to the oxygen sensitivity of the enzyme, since the ligand is rapidly lost in the presence of O2. Inhibitors like azide or nitrate are routinely employed to protect the active site from oxidative damage. The demonstrated unitary in vitro sulfido ligand incorporation to the active site bis metal-binding pterin guanine dinucleotide (bis-MGD) cofactor in FDH from Rhodobacter capsulatus of this study also completely reactivates the enzyme. Reductive treatment with either sulfide or bisulfite, or with sodium dithionite under weakly acidic conditions in the strict absence of O2 resulted in comparable enzymatic activity to FDH purified after heterologous expression in Escherichia coli. Confirmation of the inserted sulfido ligand was afforded by EPR spectroscopy of a MoV intermediate species associated with MoS6 coordination. Specific insertion of a 33S sulfido ligand to the bis-MGD Mo evidenced the chemical insertion of the sulfido ligand and confirmed its role to serve in defining the electronic character of the sulfurated bis-MGD MoV-SH state. The relevance of these results, in relation to known in vitro sulfuration assays described for other molybdoenzymes, is discussed.