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 O-2, FNR binds a [4Fe-4S](2+) cluster, generating a transcriptionally active dimeric form. Exposure to O-2 results in the conversion of the cluster to a [2Fe-2 S](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.