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. 2015 Dec 7;291(6):2712–2730. doi: 10.1074/jbc.M115.676619

FIGURE 4.

FIGURE 4.

Literature-derived equilibrium pig heart E3 FAD spectral titrations. A, pig heart E3 FAD absorption spectra were obtained from Fig. 3 in Ref. 46. In this experiment, pig heart E3 was anaerobically reduced with dithionite (initial red spectrum) and then titrated with NAD+ (0, 0.151, 0.303, 0.602, and 1.757 mm; red to black lines) at a pH of 5.8. B, semi-log plot of the singular values from a singular value decomposition of the spectra in A. C, fraction of NAD+ bound to the 2e− reduced E3 state was calculated assuming the spectra in A are composed of only bound and unbound species and solving for this fraction using linear least squares. The resulting fractions, or α values, are shown as a function of total NAD+. D, pig heart E3 FAD absorption spectra were obtained from Fig. 4 in Ref. 47. In this experiment, oxidized pig heart E3 was anaerobically titrated with dihydrolipoamide (0, 10.6, 21.2, 31.8, 44.3, 65.3, and 84.67 μm; black to green). E, pig heart E3 FAD absorption spectra were from oxidized (light green), 2e− reduced (dark green), and 4e− reduced (black) obtained from Fig. 1 in Ref. 47. F, pig heart E3 absorption spectra of the different redox states in E were used to solve (see “Experimental Procedures”) the fraction of oxidized, 2e−, and 4e− reduced enzyme states as a function of the dihydrolipoamide titration in D. Extracted fractional enzyme states (oxidized, 2e− reduced, and 4e− reduced) are shown as a function of dihydrolipoamide.