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. 2023 Apr 24;299(6):104748. doi: 10.1016/j.jbc.2023.104748

Figure 3.

Figure 3

Red-Fur loses its Fur-box binding activity upon removal of the [2Fe-2S] cluster.A, UV-Vis absorption spectra of Red-Fur after being treated with EDTA (0.1 mM) or sodium dithionite (4 mM), followed by the dialysis against buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0). Spectrum 1, purified Red-Fur (50 μM); spectrum 2, Red-Fur (50 μM) after being treated with EDTA and dialysis; spectrum 3, Red-Fur (50 μM) after being treated with sodium dithionite and dialysis. B, the restriction site protection assays of the EDTA-treated Red-Fur. Lane 1, pUC18-iuc (3.2 nM) only. Lane 2, no Fur protein was added before the HinfI digestion. Lanes 3 to 6, pUC18-iuc (3.2 nM) was preincubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM Red-Fur, respectively, followed by the HinfI digestion. Lanes 7 to 10, pUC18-iuc (3.2 nM) was preincubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM the EDTA-treated Red-Fur, respectively, followed by the HinfI digestion. C, the Fur-box binding activity of Red-Fur after removal of the [2Fe-2S] cluster. Biotin-labeled Fur-box DNA (0.7 nM) was incubated with increasing concentrations of Red-Fur before and after removal of the [2Fe-2S] cluster. Lane 1, no Fur protein was added. Lanes 2 to 5, the biotin-labeled Fur-box DNA (0.7 nM) was incubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM Red-Fur, respectively. Lanes 6 to 9, the biotin-labeled Fur-box DNA (0.7 nM) was incubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM Fur without the [2Fe-2S] cluster, respectively. D, the restriction site protection assays of Red-Fur after removal of the [2Fe-2S] cluster. pUC18-iuc (3.2 nM) was preincubated with increasing concentrations of Red-Fur with or without the [2Fe-2S] cluster, followed by digestion with HinfI at 37 °C for 10 min. The digested DNA products were separated on 1.5% agarose gel electrophoresis. Lane 1, pUC18-iuc (3.2 nM) only. Lane 2, no Fur protein was added. Lanes 3 to 6, pUC18-iuc (3.2 nM) was preincubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM Red-Fur, respectively. Lanes 7 to 10, pUC18-iuc (3.2 nM) was preincubated with 0.25 μM, 0.5 μM, 1.0 μM, and 2.0 μM Fur without the [2Fe-2S] cluster, respectively. The data are representative of three independent experiments. Fur, Ferric uptake regulator.