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.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain

“…Regulation of the fumarate and nitrate reduction gene ( fnr ) is involved in the switch between aerobic and anaerobic growth42. Promoters containing FNR binding sites are activated under hypoxia43.…”

Regioselectivity of H Cluster Oxidation

“…48,49,52 A crystal structure of this protein has not yet been solved, and the discussion in the literature is based on a homology model. The distance between Cys 122 and the cubane is proposed to be longer than for three other ligating residues.…”

Origin and phylogenetic relationships of [4Fe–4S]‐containing O2 sensors of bacteria

“…Combination of visible absorbance, EPR spectroscopy and time resolved electrospray ionization mass spectrometry allowed a very detailed analysis of the reactions. The O 2 triggers a reaction in two steps (Crack et al ., ; ; ): …”