Tuning the F695 fluorescent state in photosystem II using site-directed mutagenesis in Synechocystis sp. PCC 6803.

In photosynthetic proteins, pigments at a higher energy level funnel excitation energy to pigments at a lower energy level. Specifically, in Photosystem II (PSII), energy is transferred downhill to the reaction center (RC), where water splitting occurs. However, the lowest-energy state in PSII is not the RC, but the F695 state, which can be observed using low-temperature spectroscopy. This lowest-energy state is typically assigned to a monomeric pigment, Chl B16 (ligated by His114), but this assignment has been called into question based on theoretical fits to low-temperature spectra. In this study, we set out to test concretely whether the F695 state is localized on Chl B16 using site-directed mutagenesis and 77 K fluorescence spectroscopy. To reduce spectral congestion for whole-cell PSII studies, we developed a background strain (PSI-kd/ΔPBS) that combines a Photosystem I (PSI) knockdown with a Phycobilisome (PBS) knockout. In this background strain, we made site-directed mutations at site Thr5 in the PsbH subunit, which forms a hydrogen bond with the [Formula: see text]-keto group of Chl B16. All mutants were capable of heterotrophic growth (without noticeable differences from wild-type), indicating the PSII function remains intact. As expected for Chl B16-localized fluorescence, the Thr5→Arg mutation red-shifted the F695 state due to the strengthening of the hydrogen bond, while the Thr5→Ala mutation exhibits a blue shift as the hydrogen bond is eliminated. Taken together, these findings provide strong confirmation that Chl B16 is responsible for the lowest-energy state.