| Abstract
| - In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlledby 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 theconversion of the cluster to a [2Fe−2S]2+ form, leading to dissociation of the protein into transcriptionallyinactive monomers. The [4Fe−4S]2+ to [2Fe−2S]2+ cluster conversion proceeds in two steps. Step 1 involvesthe one-electron oxidation of the cluster, resulting in the release of Fe2+, generating a [3Fe−4S]1+ clusterintermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to formthe [2Fe−2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, inboth native and reconstituted [4Fe−4S] FNR, the reaction environment and, in particular, the presence ofFe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstratethat while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced byboth Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 isenhanced to the extent that step 1 becomes the rate determining step and the [3Fe−4S]1+ intermediate isno longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presenceof Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reactionin the cell cytoplasm, provides an explanation for apparently contradictory results reported from differentlaboratories.
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