title: Influence of the environment on the [4Fe-4S]2+ to [2Fe-2S]2+ cluster switch in the transcriptional regulator FNR.
abstract: In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+, generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories.
authors.affiliation: Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.forename: Alisa A
authors.initials: AA
authors.lastname: Gaskell
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Nick E
authors.initials: NE
authors.lastname: Le Brun
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
mesh_terms: D002614:Chelating Agents / Q000737:chemistry; D004583:Electrons; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000737:chemistry* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D002614:Chelating Agents; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D007501:Iron