DNA-BINDING AND DIMERIZATION OF THE FE-S-CONTAINING FNR PROTEIN FROM ESCHERICHIA-COLI ARE REGULATED BY OXYGEN

The transcription factor FNR from Escherichia coli regulates transcrip tion 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 isolat ed that had properties similar to those observed in vivo, This was acc omplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen, Iron and sulfide analyses of thi s FNR fraction indicated the presence of an Fe-S cluster, To determine the type of Fe-S cluster present, an oxygen-stable mutant protein LH2 8-DA154 was also analyzed since FNR LH28-DA154 purified anoxically con tained almost 3-fold more iron and sulfide than the wild-type protein, Based on the sulfide analysis, the stoichiometry (3.3 mol of S2-/FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clust ers per mutant FNR monomer, However, since FNR has only four Cys resid ues 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 pa rtial occupancy, Consistent with this, the Fe-S cluster in the wild-ty pe protein was found to be extremely oxygen-labile. In addition, molec ular-sieve chromatographic analysis showed that the majority of the an oxically purified protein was a dimer as compared to aerobically purif ied FNR which is a monomer, The loss of the Fe-S cluster by exposure t o oxygen was associated with a conversion to the monomeric form and de creased DNA binding, Taken together, these observations suggest that o xygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.