DNA Binding and Dimerization of the Fe−S-containing FNR Protein from Escherichia coli Are Regulated by Oxygen

Abstract

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.

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“…This sequence matches the known consensus binding site for the oxygenresponsive transcriptional regulator FNR (16,41). FNR directly senses a range of low-oxygen conditions and binds to DNA to either repress genes required for aerobic growth or activate those needed for growth under low-oxygen conditions (28,35).…”

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“…They may mediate electron transfer as in the ferredoxins; they may be structural elements, as in E. coli endonuclease III (24);, or they may be at the active site of enzymes, as in aconitase. More recently, some [4Fe-4S] centers have been found to be important for the control of gene expression (25)(26)(27). The discovery of the ferredoxin-like [2Fe-2S] clusters in the SoxR protein, which controls an oxidative stress regulon, immediately suggested that these groups are the redox sensor for the regulon and that the ability of SoxR to serve as a 2 J. Wu and B. Weiss, unpublished result.…”

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