In vivo evidence for the iron-binding activity of an iron–sulfur cluster assembly protein IscA in Escherichia coli

Abstract

SYNOPSIS IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. Here we report the in vivo evidence demonstrating the iron binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1μM) in the M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralog SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions but not under anaerobic growth conditions. The results provide the in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.

Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins

“…We provide in vivo evidence that the yeast Isa1 and Isa2 bind iron, in analogy to some of their bacterial orthologues (3,5,6,32). Iron binding to the Isa proteins appears to be intimately linked to their physiological function, because mutation of the conserved C-terminal cysteine pair resulted in both loss of function of Isa1 and loss of iron binding (25).…”

“…In contrast, the suggested role of the A-type ISC assembly proteins as alternative Fe/S scaffold proteins that work in parallel to the Isu proteins seems unlikely in yeast. Rather, the Isa proteins and Iba57 form a specialized ISC subcomplex that operates downstream of the central components of the mitochondrial ISC assembly system and is specifically required for maturation of mitochondrial (27,32).…”

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

“…These results led us to propose that IscA and its homologs may act as iron chaperones for iron-sulfur cluster biogenesis. Interestingly, although IscA and its homologs are essential for iron-sulfur cluster biogenesis in bacteria and eukaryotic cells under aerobic conditions (42, 49 -56), deletion of IscA and its homolog SufA has very little or no effect on iron-sulfur proteins in E. coli cells under anaerobic growth conditions (31). One of the simplest explanation is that, under anaerobic conditions, the intracellular iron concentration may be sufficient to facilitate iron-sulfur cluster assembly in proteins without IscA and its homologs (31).…”

“…To test this idea, wild-type E. coli cells expressing recombinant IscS were grown in LB medium supplemented with or without 2,2Ј-dipyridyl, a membrane-permeable iron chelator (31), under aerobic growth conditions. Fig.…”

The Role of Mitochondria in Cellular Iron-Sulfur Protein Biogenesis: Mechanisms, Connected Processes, and Diseases

“…However, by which mechanism the iron-binding Isa proteins may assist in the conversion of the Isu1-generated [2Fe-2S] cluster into a [4Fe-4S] moiety remains unclear. In bacteria, the related A-type ISC proteins IscA, ErpA, and SufA were shown to bind either iron or a [2Fe-2S] cluster (see, e.g., Gupta et al 2009;Py and Barras 2010;Wang et al 2010;Mapolelo et al 2013). However, the physiological relevance of the two different bound iron cofactors remains to be resolved.…”