Far‑Red (FR) Light Photoacclimation (FaRLiP) is a physiological program by which some cyanobacteria extend photosynthetic absorption into the far‑red region. FaRLiP produces extensive remodelling of Photosystem I (PSI), including substitution of several core subunits with paralogs that coordinate red‑shifted chlorophyll f (Chl f) pigments. This remodelling alters spectroscopic and functional properties of the reaction center (RC), motivating investigation of how primary photochemistry and electron transfer (ET) operate in FR‑adapted PSI.
The present study directly probes ET in FR‑PSI using spectroscopic measurements sensitive to photogenerated radical pairs. The authors aim to test whether ET remains bidirectional in FR‑PSI or becomes biased, as some structural interpretations of Chl f placement have suggested.
Structural and biochemical work on FaRLiP PSI has left the exact binding positions of Chl f open to discussion. The most recent structural models favor the presence of a single Chl f in the RC, located at the A‑1B site. That placement had been proposed to substantially influence ET directionality, potentially producing an almost monodirectional transfer along the B branch in FR‑PSI. Resolving whether this structural arrangement enforces unidirectional charge separation or whether both branches remain active requires direct functional probes of photochemical ET.
To interrogate primary charge separation and subsequent radical‑pair formation, the investigators used complementary pulse and Time‑Resolved (TR) Electron Paramagnetic Resonance (EPR) spectroscopy at cryogenic temperature. These EPR modalities detect spin‑correlated radical pairs and can resolve distinct electronic states through their relaxation kinetics, dipolar interactions, and spectral signatures. The specific species of interest were radical pairs involving the oxidised primary donor P700 (P700⁺) and reduced A1 acceptors (A1A⁻ or A1B⁻).
Electron spin‑echo decay kinetics measured in FR‑PSI samples showed a distinctly biexponential behaviour. Biexponential decay is interpreted as evidence for formation of two separate charge‑separated states rather than a single homogeneous radical‑pair population. The presence of two kinetic components indicates at least two photochemical pathways or outcomes leading to distinct spin‑correlated radical pairs.
Out‑of‑phase electron spin‑echo envelope modulation (ESEEM) traces were analysed and found to be quantitatively described by two modulation frequencies. These frequencies are consistent with different dipolar interaction strengths arising from spatially distinct spin pairs. The requirement for two modulation components reinforces the interpretation that two distinct radical pairs form after photoexcitation in FR‑PSI.
Time‑Resolved EPR spectra were acquired and compared with spectral simulations. The TR‑EPR data were accurately reproduced by combining the contributions of two radical pairs: [P700⁺A1A⁻] and [P700⁺A1B⁻]. This combined spectral model provides direct spectroscopic evidence that both the A and B branches remain photochemically active in FR‑PSI, rather than activity confined to a single branch.
The coexistence of both [P700⁺A1A⁻] and [P700⁺A1B⁻] radical pairs implies that charge separation events on both branches originate from a common primary electron donor. The data identify P700 as the most likely primary donor in FR‑PSI and argue against a mechanism in which a reaction‑center Chl f acts as the initiating charge separator. Therefore, despite the structural incorporation of red‑shifted Chl f, FR‑PSI preserves bidirectional electron transfer, maintaining functional parity with canonical PSI in this key aspect of primary photochemistry.
These cryogenic EPR measurements provide multiple, converging lines of evidence—biexponential spin‑echo decay, two‑frequency ESEEM modulation, and TR‑EPR spectral simulations—that both A and B ET branches are photochemically competent in FR‑PSI. The findings challenge models that place Chl f as the primary charge separator and instead support retention of P700 as the main donor.
The report is issued as a preprint and has not been certified by peer review; the authors declare no competing interests. The abstract summarises principal observations and interpretations, but detailed experimental parameters, quantitative values, sample preparations, and broader contextual discussion are contained in the full preprint and are not reproduced in the abstract. For complete methodological information and data, readers should consult the full manuscript.