Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

Abstract

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.

Identification of IscU residues critical for de novo iron–sulfur cluster assembly

“…The results suggest that D39 is also essential for IscU, which is consistent with the role proposed for this residue in controlling the stability/lability of the Fe–S cluster required for its delivery to apo‐proteins (Raulfs et al , ). This contrasts with B. subtilis SufU in which the corresponding aspartate residue can be functionally substituted with glutamate or cysteine (Yokoyama et al , ).…”

“…Our attempts to isolate a revertant from cells carrying mutations at C37, C63, C106, H105 or D39 were unsuccessful, indicating stringent constraints on these residues. This contrasts with SufU in which D43 (corresponding to IscU D39) has been functionally substituted with cysteine or glutamate and C128 (corresponding to IscU C106) with aspartate, underscoring that the roles of IscU and SufU are distinct (Yokoyama et al , ).…”

“…SufU, a paralogous protein of IscU, is distributed in Gram‐positive bacteria, and serves as a Zn‐dependent sulfur carrier protein from SufS cysteine desulfurase to the SufBCD scaffold complex in the SUF‐like Fe–S cluster biosynthesis machinery (Selbach et al , ; Yokoyama et al , ). The three cysteines of IscU responsible for Fe–S cluster binding are conserved in SufU (C41, C66 and C128 in Bacillus subtilis SufU), but, characteristically, these residues of SufU together with the nearby aspartate residue (D43) form a binding site for a Zn 2+ ion.…”

“…Upon complexation with SufS, ligand exchange from SufU C41 to SufS H342 occurs, which facilitates the association of SufS and SufU with concomitant release of C41 to receive persulfide from SufS C361 (Fujishiro et al , ). Intriguingly, two residues in SufU can be functionally replaced with other amino acids: D43 with glutamate or cysteine and C128 with aspartate (Yokoyama et al , ). These observations prompted us to reinvestigate the effect of amino acid substitutions in IscU, with a focus on the Fe–S binding site and nearby residues.…”

Snapshots of PLP‐substrate and PLP‐product external aldimines as intermediates in two types of cysteine desulfurase enzymes

“…S1). The SUF‐like machinery is characterized to exclusively utilize SufU , a zinc‐containing sulfur‐transferring protein, with SufS, whereas the SUF machinery utilizes SufE with SufS .…”