The observed rate of conformational change and flavin reduction in CPR are the same.
(A) Example stopped-flow traces of the deconvoluted trace arising from FRET alone for the donor (black) and acceptor (green). Solid lines show the simultaneous fit of both traces to Equation S1. (B) The green line shows the ratio of acceptor to donor emission extracted from the deconvoluted traces (A). The black line shows an example trace for the relative change in CPR absorbance at 600 nm versus a saturating concentration of NADPH (flavin reduction). Solid lines show the fit to Equation S1. The dotted lines show the relative phases of flavin reduction (2e−) and establishment of the internal equilibrium (EQ). Conditions: 50 mM potassium phosphate pH 7, 5 mM NADPH at 15°C. Labelled CPR and unlabelled CPR concentrations were ∼0.5 and 40 µM, respectively.