Rapid-Scan Fourier Transform Infrared Difference Spectroscopy with Two-Dimensional Correlation Analysis to Show the...

Time-resolved, rapid-scan Fourier transform infrared (FT-IR) difference spectra have been recorded upon illumination on photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides under fixed hydration conditions (relative humidity = 76%). Two different illumination schemes were adopted. Whereas the use of a laser flash (duration: 7 ns) made it possible to follow the kinetics of recombination of the light-induced state P⁺Q_A^- to the neutral state PQ_A, the use of a 20.5 s continuous light from a lamp made it possible to follow both the build-up of a steady-state P⁺Q_A^- population and its decay to PQ_A. Comparison between P⁺Q_A^-/PQ_A FT-IR difference spectra obtained under (or 650 ms after) continuous illumination and obtained after one laser flash show small but meaningful differences, reflecting structural changes in the light-adapted state produced by the 20.5 s period of illumination. These differences are strikingly similar to those observed when comparing FT-IR difference spectra reflecting charge separation in photosystem II in light-adapted states and non-light-adapted states (c.f. Sipka et al., "Light-Adapted Charge-Separated State of Photosystem II: Structural and Functional Dynamics of the Closed Reaction Center". Plant Cell. 2021. 33(4): 1286-1302). Two-dimensional correlation spectroscopy analysis revealed that in all the observed series of time-resolved FT-IR difference spectra (under illumination, after illumination, and after a laser flash), marker bands at 1749, 1716, and 1668 cm^-1 all evolve synchronously, demonstrating that electron transfer reactions and protein backbone response (at least the one reflected by the 1668 cm^-1 band) are strongly correlated. Conversely, for spectra under and after continuous illumination, many asynchronicities are observed for (still unassigned) bands throughout the whole 1740-1200 cm^-1 region, reflecting a more complicated molecular scenario in the RC upon build-up of the light-adapted state and during its relaxation to the resting neutral state.