Mapping the key residues of SufB and SufD essential for biosynthesis of iron-sulfur clusters

Abstract

Biogenesis of iron-sulfur (Fe-S) clusters is an indispensable process in living cells. In Escherichia coli, the SUF biosynthetic system consists of six proteins among which SufB, SufC and SufD form the SufBCD complex, which serves as a scaffold for the assembly of nascent Fe-S cluster. Despite recent progress in biochemical and structural studies, little is known about the specific regions providing the scaffold. Here we present a systematic mutational analysis of SufB and SufD and map their critical residues in two distinct regions. One region is located on the N-terminal side of the β-helix core domain of SufB, where biochemical studies revealed that Cys254 of SufB (SufBC254) is essential for sulfur-transfer from SufE. Another functional region resides at an interface between SufB and SufD, where three residues (SufBC405, SufBE434, and SufDH360) appear to comprise the site for de novo cluster formation. Furthermore, we demonstrate a plausible tunnel in the β-helix core domain of SufB through which the sulfur species may be transferred from SufBC254 to SufBC405. In contrast, a canonical Fe-S cluster binding motif (CxxCxxxC) of SufB is dispensable. These findings provide new insights into the mechanism of Fe-S cluster assembly by the SufBCD complex.

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

“…These findings strongly suggest that the three B. subtilis components form an SufB 1 C 2 D 1 complex as observed in E. coli , and work in concert to assemble Fe–S clusters. According to recent studies on the E. coli complex, the mechanism seems to involve several steps: (i) persulfide sulfur is transferred from SufE to SufB Cys254, (ii) the sulfur is released and migrates through the tunnel in the β‐helix core domain of SufB, (iii) conformational change of SufBD is induced by ATP‐driven dimerization of two subunits of SufC and (iv) the Fe–S cluster is assembled at the interface of SufB and SufD using three essential residues (Cys405 and Glu434 of SufB and His360 of SufD) (Hirabayashi et al ., ; Yuda et al ., ). Importantly, amino acid residues critical for these functions (Lys40, Glu171 and His203 of SufC, Arg226, Asn228, Cys254, Gln285, Trp287, Lys303, Cys405 and Glu434 of SufB, and His360 of SufD; E. coli numbering) are all conserved in the B. subtilis components.…”

“…S10). We have recently demonstrated that, in the E. coli SUF machinery, the persulfide sulfur is transferred from SufE Cys51 to SufB Cys254, a residue located at the N‐terminal side of the β‐helix core domain (Yuda et al ., ). This cysteine is conserved in B. subtilis SufB (Cys231) together with the nearby residues (Supporting Information Fig.…”

“…SufB forms a stable complex with SufC and SufD with a 1:2:1 stoichiometry, and the complex acts as a scaffold for the de novo Fe–S cluster assembly (Chahal et al ., ; Gupta et al ., ; Saini et al ., ; Wollers et al ., ). Our recent structural, biochemical and mutational studies have provided insights into the unique mechanism of Fe–S cluster assembly by the SufBCD complex including conformational change of SufBD triggered by dimerization of two subunits of SufC on ATP binding (Hirabayashi et al ., ; Yuda et al ., ). However, the intermediate states, in particular the Fe–S cluster‐containing form, have yet to be clarified.…”

The SUF system: an ABC ATPase-dependent protein complex with a role in Fe–S cluster biogenesis

“…Moreover both in vivo and structural studies pointed to the invariant Cys405 residue and Glu434, His433 and/or Glu432 residues as proposed FeeS ligands ( Fig. 3A, B) [40,44]. It is worth noting that the only available structure of SufB (PDB: 5AWF) corresponds to the form complexed with SufC and SufD (see below).…”

“…Importantly, in vitro, both the [2Fee2S] and [4Fee4S] holoforms of SufB are able to transfer their cluster to [2Fee2S] and [4Fee4S] client proteins [41e43]. The N-terminal region of SufB contains a putative FeeS cluster motif (CxxCxxxC) that was proposed to provide the ligands of the FeeS cluster [38], but such a prediction was ruled out by in vivo mutagenesis analysis [44]. Moreover both in vivo and structural studies pointed to the invariant Cys405 residue and Glu434, His433 and/or Glu432 residues as proposed FeeS ligands ( Fig.…”