# Hydrogen sulfide acts as a sulfur source for iron sulfur cluster biosynthesis in cysteine desulfurase-deficient Escherichia coli under anaerobic conditions

## TL;DR

Hydrogen sulfide helps bacteria lacking a key sulfur-releasing enzyme by providing sulfur for iron-sulfur cluster formation, restoring growth under low oxygen conditions.

## Contribution

The study reveals that H₂S acts as a sulfur source for Fe-S cluster biosynthesis in IscS-deficient E. coli, compensating for the loss of the cysteine desulfurase.

## Key findings

- H₂S exposure rescues growth defects in ΔiscS E. coli by restoring Fe-S cluster-dependent enzyme activity.
- H₂S promotes Fe-S cluster biosynthesis on IscU, not directly on apoproteins, as shown by ΔiscU mutant insensitivity to H₂S.
- Sulfur supplementation during recombinant expression increases Fe-S cluster abundance and activity in ΔiscS mutants.

## Abstract

The cysteine desulfurase (IscS) is a core component of the ISC iron–sulfur (Fe-S) cluster assembly system in Escherichia coli. Deficiency of IscS leads to serious growth defects in E. coli, along with reduced activity of Fe-S cluster-dependent enzymes. We previously demonstrated that the growth defect of IscS-deficient E. coli (ΔiscS) is completely restored by H₂S exposure, but the underlying molecular mechanism was not fully understood. Here, based on proteomic analysis, we identified 19 up-regulated Fe-S proteins in the ΔiscS mutant upon H₂S exposure, 13 of which are involved with energy metabolism. Correspondingly, H₂S exposure also enhanced the activity of Fe-S enzymes in the mutant. Metabolomic analysis revealed a remarkable increase in the levels of the energy metabolites NAD+, succinate, and leucine. These results implied that H2S could restore cell proliferation and Fe-S cluster biosynthesis by compensating for the functional loss of IscS. We also constructed a series of mutants, each lacking a single component of the ISC assembly system. A key observation was that the ΔiscU mutant, deficient in the Fe-S cluster scaffold protein IscU, failed to have its growth defect rescued by H₂S exposure. These findings indicated that H2S promotes Fe-S cluster biosynthesis on IscU, ruling out direct assembly on apoproteins. Moreover, Na₂S supplementation during recombinant expression of aconitase B in the ΔiscS mutant significantly increased its Fe-S cluster abundance and enzymatic activity. We also demonstrated that, unlike the ΔiscS mutant, deletion of sufS, which encodes the cysteine desulfurase of the SUF Fe-S cluster biogenesis system, did not significantly impair bacterial growth, and the resulting mutant’s proliferation was not affected by H₂S exposure. Our study elucidates the mechanism by which H₂S exposure rescues the proliferation impairment of the ΔiscS mutant. Specifically, we demonstrate that H₂S functions as a sulfur donor for Fe-S cluster assembly, thereby compensating for the biosynthetic deficit.