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. 2014 Jan 8;289(7):4307–4315. doi: 10.1074/jbc.M113.542050

FIGURE 3.

FIGURE 3.

Reduction of mitoNEET [2Fe-2S] clusters by the thioredoxin reductase system in vitro. Purified mitoNEET (mito) [2Fe-2S] clusters (10 μm) (spectrum 1) were incubated with NADPH (500 μm) (spectrum 2); E. coli thioredoxin (TrxA, 10 μm) and thioredoxin reductase (TrxB, 10 μm) (spectrum 3); or E. coli thioredoxin (10 μm), thioredoxin reductase (10 μm), and NADPH (500 μm) (spectrum 4) at 37 °C for 20 min under anaerobic conditions. Spectrum 5 shows purified mitoNEET [2Fe-2S] clusters reduced with dithiothreitol (2 mm). Spectrum 6 shows the mitoNEET mutant H87C [2Fe-2S] clusters (10 μm) incubated with E. coli thioredoxin-1 (10 μm), thioredoxin reductase (10 μm), and NADPH (500 μm) at 37 °C for 20 min under anaerobic conditions. After incubation, the samples were transferred to EPR tubes for EPR measurements. The results are representative of three independent experiments. mT, millitesla.