Detection of sulfide release from the oxygen-sensing [4Fe-4S] cluster of FNR

The Escherichia coli FNR protein regulates the transcription of > 100 genes in response to environmental O-2, thereby coordinating the response to anoxia. Under O-2-limiting conditions, FNR binds a [4Fe-4S](2+) cluster through four cysteine residues (Cys(20), Cys(23), Cys(29), Cys(122)). The acquisition of the [4Fe-4S](2+) cluster converts FNR into the transcriptionally active dimeric form. Upon exposure to O-2, the cluster converts to a [2Fe-2S](2+) form, generating FNR monomers that no longer bind DNA with high affinity. The mechanism of the cluster conversion reaction and the nature of the released iron and sulfur are of considerable current interest. Here, we report the application of a novel in vitro method, involving 5,5'-dithiobis-(2-nitrobenzoic acid), for determining the oxidation state of the sulfur atoms released during FNR cluster conversion following the addition of O-2. Conversion of [4Fe-4S](2+) to [2Fe-2S](2+) clusters by O-2 for both native and reconstituted FNR results in the release of similar to 2 sulfide ions per [4Fe-4S](2+) cluster. This demonstrates that the reaction between O-2 and the [4Fe-4S](2+) cluster does not require sulfide oxidation and hence must entail iron oxidation.