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. 2013 Dec 30;289(8):5145–5157. doi: 10.1074/jbc.M113.521013

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FIGURE 2. — Bifurcation assays. See “Experimental Procedures” for details. A, stoichiometry between NADH and crotonyl-CoA of 2.1:1. The assays were performed with limiting amounts of crotonyl-CoA. B, dependence of the specific activity of NADH oxidation (μmol of NADH/mg of Etf_Af) on the concentration of the Bcd_Af monomer. Each assay contained 0.5 μm Etf_Af, 1 μm ferredoxin and hydrogenase (30 μg/ml). C, dependence of the specific activity of NADH oxidation (μmol of NADH/mg of Etf_Af) on the concentration of ferredoxin used in the assay. Each assay contained 0.5 μm Etf_Af, 1 μm Bcd_Af monomer and hydrogenase; K_m (ferredoxin) = 0.2 μm. D, dependence of the specific activity of NADH oxidation (μmol of NADH/mg of Etf_Af) on the concentration of NADH used in the assay. Each bifurcation assay contained 0.5 μm Etf_Af, 1 μm Bcd_Af monomer, 1 μm ferredoxin and hydrogenase. E, complete reduction of 15 μm ferredoxin in the bifurcation assay without hydrogenase. (a) Spectrum of oxidized ferredoxin before starting the bifurcation; (b) excess dithionite was added; (c) after bifurcation has terminated due to limiting amounts of ferredoxin.