Rapid-Scan Fourier Transform Infrared Difference Spectroscopy with Two-Dimensional Correlation Analysis to Show the Build-Up of Light-Adapted States in Bacterial Photosynthetic Reaction Centers.

Time-resolved, rapid-scan Fourier transform infrared (FT-IR) difference spectra have been recorded upon illumination on photosynthetic reaction centers (RCs) from 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 PQ to the neutral state PQ, 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 PQ population and its decay to PQ. Comparison between PQ/PQ 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 all evolve synchronously, demonstrating that electron transfer reactions and protein backbone response (at least the one reflected by the 1668 cm 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 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.