Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) clusterto oxygen

To understand the role of the [4Fe-4S](2+) cluster in controlling the activ ity of the Escherichia coli transcription factor FNR (fumarate nitrate redu ction) during changes in O-2 availability, we have characterized a mutant F NR protein containing a substitution of Leu-28 with His (FNR-L28H) which, u nlike its wild type (WT) counterpart, is functional under aerobic growth co nditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) clu ster of FNR-L28H in the presence of O-2 because air-exposed FNR-L28H did no t undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or conc omitant loss in site-specific DNA binding and dimerization, which are chara cteristic of WT-FNR under these conditions. This increased cluster stabilit y was not a result of His-28 replacing the WT-FNR cluster ligands because s ubstitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L2 8H. The Mossbauer spectra of FNR-L28H indicated that the coordination envir onment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whol e cell Mossbauer spectroscopy showed that aerobically grown cells overexpre ssing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cl uster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [ 4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under ae robic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.