Superoxide destroys the [2Fe-2S]2+ cluster of FNR from Escherichia coli

The oxygen sensing ability of the transcription factor FNR depends on the presence of a [4Fe-4S](2+) cluster. In the presence Of O-2, conversion of the [4Fe-4S](2+) cluster to a [2Fe-2S](2+) cluster inactivates FNR, but the fate of the [2Fe-2S](2+) cluster in cells grown under aerobic conditions is unknown. The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the [2Fe-2S](2+) cluster, like the [4Fe-4S](2+) cluster, is not stable under these conditions. By quantifying the amount of [2Fe-2S](2+) cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O-2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the [2Fe-2S](2+) cluster. Mossbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the [2Fe-2S](2+) cluster. We conclude that the [2Fe-2S](2+) cluster of FNR is labile to superoxide both in vitro and in vivo. This lability may explain the absence of the [2Fe-2S](2+) cluster form of FNR under aerobic growth conditions.