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

Eiki Yuda,Keiichi Fukuyama,Naoyuki Tanaka,Kei Hirabayashi,Takashi Fujishiro,Nao Yokoyama,Yasuhiro Takahashi,Kei Wada

doi:10.1038/s41598-017-09846-2

Eiki Yuda, Keiichi Fukuyama + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-09846-2

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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.

Highlights

Iron sulfur (Fe-S) proteins contribute to a number of cellular processes such as respiration, photosynthesis, nitrogen fixation and gene regulation[1,2,3,4,5]
Taking advantage of complementation assays using an E. coli mutant strain that can survive without Fe-S clusters[39], we identified eight functional residues in SufB and one residue in SufD (SufDH360) that mapped to two distinct regions in the SufBCD structure
The SufBC254A substitution abolished the activation of SufS cysteine desulfurase in the in vitro assay system composed of SufS, SufE, and the SufBCD complex (Fig. 4a)

Summary

Introduction

Iron sulfur (Fe-S) proteins contribute to a number of cellular processes such as respiration, photosynthesis, nitrogen fixation and gene regulation[1,2,3,4,5]. Mutational studies have underscored the importance of the ATPase activity of SufC for the in vivo biosynthesis of the Fe-S cluster[30, 35], and have further revealed two critical residues, SufBC405 and SufDH360, both of which reside inside the β-helix core domain at the SufB-SufD interface[35, 38]. Six functional residues were found in the N-terminal region of the β-helix core domain of SufB (termed region A) and our biochemical study uncovered that one of them, SufBC254 is essential for the stimulation of SufS-SufE cysteine desulfurase activity and for the accumulation of sulfur moieties in the SufBCD complex, revealing the acceptor site for persulfide from SufE. These findings suggest novel mechanistic implications for the Fe-S cluster assembly by the SufBCD complex

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