Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system.IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

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