An unusual triplet population pathway in the Reaction Centre of the Chlorophyll-d binding Photosystem I of A. marina, as revealed by a combination of TR-EPR and ODMR spectroscopies

Photo-induced Chlorophyll (Chl) triplet states in the isolated Photosystem I (PSI) of Acaryochloris marina, that harbours Chl d as its main pigment, were investigated by Optically Detected Magnetic Resonance (ODMR) and Time-Resolved Electron Paramagnetic Resonance (TR-EPR), and as a function of pre-illumination of the sample under reducing redox poising. Fluorescence Detected Magnetic Resonance (FDMR) allowed resolving four Chl d triplet (³Chl d) populations (T₁-T₄) both in untreated and illuminated samples in the presence of ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The FDMR signals increased following the pre-illumination treatment, particularly for the T₃ and T₄ populations, which are therefore sensitive to the redox state of PSI cofactors. Microwave-induced Triplet minus Singlet (TmS) spectra were detected in the |D|-|E| resonance window of the T₃ and T₄ triplets. These showed a broad singlet bleaching centred at 740 nm and also displayed complex spectral structure with several derivative-like features, indicating that both the T₃ and T₄³Chl d populations are associated with the PSI reaction centre (RC) triplet, P3₇₄₀. Parallel measurements by TR-EPR demonstrated that triplet signals observed under all conditions investigated are dominated by an electron spin polarisation (esp), which is typical of intersystem crossing, differently from what expected for recombination triplet states formed from a radical pair precursor. Moreover, stronger reductant conditions obtained by pre-illumination of the samples in the presence of dithionite and 5-methylphenazinium methyl sulfate (PMS) did not lead to a recombination triplet state esp, but rather to a decrease of the whole signal intensity. The energetics of A. marina PSI and the possible occurrence of distributions of cofactors redox properties are discussed in order to address the unexpected P3₇₄₀ esp.