IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA͞SoxS͞Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S] 1؉ cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells. to toxic levels. An important finding in recent years is the discovery of a conserved set of proteins in organisms from bacteria to humans that facilitate assembly of Fe-S clusters into Fe-S proteins (1, 2). In several bacteria, the genes encoding these Fe-S assembly proteins (IscS, IscU, IscA, Hsc66, Hsc20, and ferredoxin) are organized in a cluster, iscSUAhscBAfdx. In this study, we have focused on the regulation of the expression of the genes that encode the Fe-S cluster assembly factors from Escherichia coli.Genetic experiments support the key role of the proteins encoded by iscSUAhscBAfdx in Fe-S cluster assembly, because mutations in the E. coli genes decrease the activity of many Fe-S proteins (3-5). Furthermore, biochemical studies have begun to provide some insight into the process of Fe-S cluster assembly (1, 6-10). IscS is a cysteine desulfurase that procures the sulfur from cysteine for Fe-S cluster assembly (1, 7). IscU can form a complex with IscS and acquire an unstable [2Fe-2S] cluster that has been proposed to be a source of Fe and sulfur for Fe-S protein assembly (6,8,11). IscA can also acquire a Fe-S cluster in vitro (9). Hsc66 and Hsc20, homologs to molecular chaperones, form a complex in vitro with IscU, but the exact physiological function of this interaction has yet to be determined (10).It is also of interest to understand how expression of the Fe-S cluster assembly proteins is controlled. In bacteria, the cellular requirements for Fe-S cluster assembly must vary, because the synthesis of many Fe-S proteins is regulated (12) and environmental conditions arise that lead to the destruction of these metal centers (13). Thus, it would not be surprising to find that expression of the assembly proteins is regulated to accommodate such changes in Fe-S cluster assembly requirements. A clue that the genes encoding Fe-S cluster assembly proteins might be regulated came from the...

SummaryIscR is an iron-sulphur (Fe-S) cluster-containing transcription factor that represses transcription of the operon containing its own gene and the iscSUAhscBA-fdx genes, whose products are involved in Fe-S cluster biogenesis. In this study, global transcriptional profiling of Escherichia coli IscR + and IscR

The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygenstable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S 2؊ /FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. The ability to adapt to changes in oxygen concentrations in the environment is common to many organisms. In the facultative anaerobe, Escherichia coli, the transcription factor FNR ( fumarate nitrate reduction) regulates a network of genes that facilitates adaptation to oxygen deprivation by providing alternative pathways for energy generation (1). Recent data suggest that FNR contains a [4Fe-4S] cluster (2-4) and that this cluster apparently mediates the sensitivity of this transcription factor to oxygen, thus limiting FNR activity to anaerobic conditions. The stoichiometry of iron and labile sulfide relative to the number of cysteine ligands of anaerobically purified FNR is most compatible with the presence of a [4Fe-4S] cluster (3). Iron and sulfide analyses and CD spectra of FNR preparations derived from reconstitution of a cluster into apoprotein also supports this cluster assignment (4). The presence of the Fe-S cluster in the anaerobically purified form of FNR is correlated with an increase in dimerization and specific DNA binding (3), compared with the aerobically purified form that lacks an Fe-S cluster (2, 5, 6). Furthermore, the Fe-S cluster is disrupted by oxygen, and this is correlated with the conversion of FNR into an inactive monomeric protein (3). The loss of this cluster as well as the loss of specific DNA binding upon exposure of FNR to oxygen suggested that the [4Fe-4S] cluster, through its intrinsic instability, serves as an oxygen sensor.This paper reports observations on the path of disassembly of the [4Fe-4S] cluster of FNR in vitro. Little is known about the cluster disassembly mechanism of proteins in the absence of chelators, detergents, chemical oxidants, and other nonphysiological agents. The rapid destruction of the Fe-S cluster of FNR, simply upon exposure of a purified protein solution to air, offers an opportunity to obtain information on the progress and mode of the disassembly of the [4Fe-4S] cluster of this protein. We have used chemical analysis for iron and sulfide as well as electronic, EPR, and Mössbauer spectroscopies to monitor the disassembly process. We report here the unexpected observation that the [4Fe-4S] cluster of FNR is converted in less than 5 min to a [2Fe-2S] cluster in about 60% yield as judged from Mössbauer spectra and this conversion results in a reduction in DNA-binding ability. The [4Fe-4S] 2ϩ cluster can be regenerated from the [2Fe-2S] cluster or its components by reduction with dithionite.