Light-Induced Charge Separation in Photosystem I from Different Biological Species Characterized by Multifrequency Electron Paramagnetic Resonance Spectroscopy

Abstract

Photosystem I (PSI) serves as a model system for studying fundamental processes such as electron transfer (ET) and energy conversion, which are not only central to photosynthesis but also have broader implications for bioenergy production and biomimetic device design. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate key light-induced charge separation steps in PSI isolated from several green algal and cyanobacterial species. Following photoexcitation, rapid sequential ET occurs through either of two quasi-symmetric branches of donor/acceptor cofactors embedded within the protein core, termed the A and B branches. Using high-frequency (130 GHz) time-resolved EPR (TR-EPR) and deuteration techniques to enhance spectral resolution, we observed that at low temperatures prokaryotic PSI exhibits reversible ET in the A branch and irreversible ET in the B branch, while PSI from eukaryotic counterparts displays either reversible ET in both branches or exclusively in the B branch. Furthermore, we observed a notable correlation between low-temperature charge separation to the terminal [4Fe-4S] clusters of PSI, termed F_A and F_B, as reflected in the measured F_A/F_B ratio. These findings enhance our understanding of the mechanistic diversity of PSI's ET across different species and underscore the importance of experimental design in resolving these differences. Though further research is necessary to elucidate the underlying mechanisms and the evolutionary significance of these variations in PSI charge separation, this study sets the stage for future investigations into the complex interplay between protein structure, ET pathways, and the environmental adaptations of photosynthetic organisms.

Keywords: electron paramagnetic resonance spectroscopy; photosynthesis; photosystem I.

Figure 1

(a) Schematic structure and ET pathways in cyanobacterial Photosystem I (PDB ID 1JB0). Following photoexcitation, the excited primary donor, P*₇₀₀, becomes oxidized, transferring one electron to one of two almost identical chains of electron transfer cofactors (chlorophyll A₀ and phylloquinone A₁) and converging at the three [4Fe-4S] clusters, F_X, F_A, and F_B. Photoinduced ET in PSI is bidirectional at ambient temperature, proceeding through both the A and B branches of cofactors as indicated by arrows. (b) Energy diagram and time constants of forward electron transfer reactions in PSI. Information was taken from refs. [12,13].

Figure 2

High-frequency (130 GHz) pulsed EPR spectra of the P₇₀₀⁺ A⁻₁ radical pairs in fully deuterated cyanobacterium, S. leopoliensis. (a) P₇₀₀⁺A_1A⁻ pair in thermal equilibrium (top, purple) and in spin-polarized SCRP state (bottom, red) at 100 K. Green and blue spectra are the simulations for EPR signals of A_1A⁻ and P₇₀₀⁺ in thermal equilibrium, respectively [21]. Positions of the g-tensor main components for A_1A⁻ and P₇₀₀⁺ are shown by arrows. (b) SCRP in A branch, P₇₀₀⁺A_1A⁻, recorded in “native” PSI (red); SCRP in B branch, P₇₀₀⁺A_1B⁻, recorded in PSI sample containing sodium hydrosulfite and pre-reduced by illumination at 205–245 K (blue). Photoaccumulation procedure allows reduction of F_A, F_B, F_X, and A_1A but not A_1B: SCRP in both A and B branches (P₇₀₀⁺A_1A⁻ and P₇₀₀⁺A_1B⁻ with 1:1 ratio) recorded in PSI where the three [4Fe-4S] clusters, F_X, F_A and F_B, were biochemically removed to prevent forward ET from the quinones (green). Arrows indicate absorption (A) and emission (E) contributions to the SCRP spectra. T = 100 K, DAF = 1 μs.

Figure 3

High-frequency D-band (130 GHz) pulsed EPR spectra of the SCRPs recorded in PSI from different biological species. T = 100 K, DAF = 1 μs.

Scheme 1

Schematic presentation of relative redox potential of acceptors A_1A and A_1B with respect to F_x. Note that lower redox midpoint potential means more reducing potential. Dashed arrow indicates recombination reaction to P₇₀₀⁺. (a) Cyclic electron transfer in the A branch. (b) Cyclic electron transfer in the B branch. (c) Cyclic electron transfer in both A and B branches.

Figure 4

Continuous wave (cw) X-band (9.5 GHz) EPR spectra of various PSI samples. Samples were frozen in the dark, cooled down to 10 K in the cavity of the EPR spectrometer, and subsequently illuminated with a white light LED. Microwave power, 3 mW; modulation amplitude, 1.2 mT.