Lawrence Berkeley National Laboratory

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy was used to obtain (A₁ ^- - A₁) FTIR difference spectra from photosystem I (PSI) samples isolated from eight phylogenetically diverse cyanobacterial strains and one green alga, totaling 13 PSI preparations. These included samples from cells grown under far-red light and PSI in monomeric, dimeric, trimeric, and tetrameric states. Spectral profiles were shown to be independent of oligomeric state. Remarkably, all (A₁ ^- - A₁) FTIR difference spectra exhibited high similarity, underscoring the robustness of the technique and indicating minimal experimental variability. This congruence reveals a highly conserved environment for the phylloquinone cofactor at the A₁ binding site across diverse taxa. Conserved bands associated with the A₀ pigment further suggest structural continuity from A₀ to A₁. To leverage this consistency, we constructed a composite (A₁ ^- - A₁) FTIR difference spectrum by averaging all 13 spectra. This composite spectrum provides enhanced resolution, enabling unambiguous identification of previously unresolved bands. The fact that a highly resolved composite spectrum can be obtained by averaging demonstrates the similarity in the spectra from the different types of samples. Band assignments were refined using prior studies, yielding an improved spectral framework for future investigations of PSI electron transfer cofactors.