Figure 2
Spectral changes associated with oxidation of the photoreduced (a) At64, (b) Xl64, and (c) CraCRY samples. The spectrum was recorded every 10 min after starting the oxidation. Insets for (a) and (c) show the difference spectra in which each spectrum was subtracted by the spectrum at 0 min recorded at wavelengths from 400 to 700 nm. In panel (b), light scattering due to protein aggregation can be seen particularly in the wavelength range of 300–400 nm. This effect may be attributed to Xl64 being dissolved in a buffer optimized for At64 and CraCRY, to maintain consistent buffer conditions across all samples. In our previous experiment using a buffer optimized for Xl64, a similar kinetic behavior to that presented here was observed without the scattering31. This suggests that the fast kinetics of Xl64 does not result from unstable protein folding. An inset for (b) shows the difference spectra in which each spectrum was subtracted by the spectrum of the reduced state, indicating that the considerable amount of FADH− was already reoxidized to FADox immediately after triggering the reaction. The difference spectra, which exhibit a typical change upon reoxidation, also support that the protein quality is sufficient for characterizing the kinetic property of Xl64.
