Spectral changes during Rif binding and turnover.

(A) Flavin spectral changes as a function of increasing concentration of Rif (0–40 μM). The spectral changes show increases in absorbance at (320, 360, 400, 443, and 525 nm) and the isobestic point at ~ 480 nm. The inset shows spectral differences after subtracting the spectrum of RifMO with 0 μM Rif. (B) Determination of the K_D value of Rif. The change in absorbance of the RifMO-Rif complex at 525 nm was plotted as a function of Rif to determine a K_D value. (C) UV-Vis spectrophotometric monitoring of RifMO product formation representing the decline of the Rif peak at 475 nm (blue), followed by red shifting and an absorbance increase at 493 nm (red).