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-bound 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 [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 the environmental temperatures. Iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and self-assembly 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 systems. 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. A 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 may act as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

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Aims Irisin is a newly discovered actin that has been shown to be effective against inflammation-related symptoms. The aim of this study was to investigate the influence of Irisin on LPS-induced inflammation in vitro and to investigate the role of the mitogen-activated protein kinase (MAPK) pathway in the protecting effect of Irisin in LPS-stimulated RAW 264.7 macrophages.Methods Cell viability was assessed using MTT assay and flow cytometry, cell morphology was measured by optical microscope, IL-12 and IL-23 were detected by Elisa, and the levels of related proteins were analyzed by western blot. Phagocytosis of zymosan and clearance of apoptotic cells were measured.Results MTT assay and flow cytometry results showed that LPS-induced cytotoxicity was reversed by Irisin(p<0.001), after coincubation with Irisin, the level of IL-12 and IL-23 decreased in LPS-stimulated RAW264.7 macrophages(p>0.05). Irisin pretreatment significantly inhibited the phosphorylation of ERK and AKT and increased the expression of PPAR α and PPAR γ(p<0.05). LPS-induced enhancement of phagocytosis(p<0.05) and cell clearance was reversed by Irisin pretreatment(p<0.001).Conclusions Irisin ameliorated LPS-induced inflammation by inhibiting cytotoxicity and apoptosis, and this protective effect may be mediated through the MAPK pathway.

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