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 O2, thereby coordinating the response to anoxia. Under O2-limiting conditions, FNR binds a [4Fe-4S]2+ cluster through four cysteine residues (Cys20, Cys23, Cys29, Cys122). The acquisition of the [4Fe-4S]2+ cluster converts FNR into the transcriptionally active dimeric form. Upon exposure to O2, 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 O2. Conversion of [4Fe-4S]2+ to [2Fe-2S]2+ clusters by O2 for both native and reconstituted FNR results in the release of approximately 2 sulfide ions per [4Fe-4S]2+ cluster. This demonstrates that the reaction between O2 and the [4Fe-4S]2+ cluster does not require sulfide oxidation and hence must entail iron oxidation.
Crack
Jason C
JC
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom. J.Crack@uea.ac.uk
Green
Jeffrey
J
Le Brun
Nick E
NE
Thomson
Andrew J
AJ
D001426:Bacterial Proteins / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D000860:Hypoxia; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D013440:Sulfides; D004247:DNA; D010100:Oxygen