Annihilation of Excess Excitations along Phycocyanin Rods Precedes Downhill Flow to Allophycocyanin Cores in the Phycobilisome of Synechococcus elongatus PCC 7942

Cyanobacterial phycobilisome complexes absorb visible sunlight and funnel photogenerated excitons to the photosystems where charge separation occurs. In the phycobilisome complex of Synechococcus elongatus PCC 7942, phycocyanin protein rods that absorb bluer wavelengths are assembled on allophycocyanin cores that absorb redder wavelengths. This arrangement creates a natural energy gradient toward the reaction centers of the photosystems. Here, we employ broadband pump–probe spectroscopy to observe the fate of excess excitations in the phycobilisome complex of this organism. We show that excess excitons are quenched through exciton–exciton annihilation along the phycocyanin rods prior to transfer to the allophycocyanin cores. Our observations are especially relevant in comparison to other antenna proteins, where exciton annihilation primarily occurs in the lowest-energy chlorophylls. The observed effect could play a limited photoprotective role in physiological light fluences. The exciton decay dynamics is faster in the intact phycobilisome than in isolated C-phycocyanin trimers studied in earlier work, confirming that this effect is an emergent property of the complex assembly. Using the obtained annihilation data, we calculate exciton hopping times of 2.2–6.4 ps in the phycocyanin rods. This value agrees with earlier FRET calculations of exciton hopping times along phycocyanin hexamers by Sauer and Scheer.

Reproducibility of Pump-probe Spectra S10 Long-time Dynamics in PBS S11 Sucrose gradient unprocessed image S12 Phycobilisome fluorescence spectrum S13 Calculation of the frequency of exciton-exciton annihilation in physiological conditions S14 References S14 S2 PBS integrity confirmation Figure S1. The 1.5 M sucrose gradient fraction with the isolated phycobilisome was tested using SDS-PAGE for integrity. Gel was confirmed with Sato, et al. (1). Table S1: SDS-PAGE lane descriptions: Earlier protocol recommended using a non-reducing Lane Marker buffer as is shown in Lane 3: Figure S2: Circular dichroism spectrum of phycobilisome from cyanobacteria S. elongatus PCC 7942

Number of excitations in pump-probe experiment
To identify the number of excitons generated upon excitation, we perform the calculation adapted from Dostal and co-authors (3). The following equation describes the number of excitations per phycocyanin rod: * * * 1 10 ℎ * 1 Here, is the average energy of the pump pulse collected with Coherent LabMax-TOP powermeter, is the effective area of the pump and probe overlap, is the spectrum of the excitation pulse collected with Ocean Optics USB4000 Spectrometer, is the phycobilisome absorption spectrum shown in Figure 1 of Main Text, C is the molar concentration of the phycocyanin rods in the solution, is the Avogadro's number and d is the thickness of the sample cell used in pump-probe measurements. We use the following values for our calculation: Concentration of our sample is calculated from this ε for an OD of 0.0689 at 568nm in 200μm and comes to 6.86 * 10 20 rods/m 3 .
Our overlap volume is 1.32*10 -11 m 3 giving a concentration of 9.06 * 10 9 rods/excitation volume. Figure S3: Fits to annihilation model at a) 568nm, b) 588nm, c) 596nm and d) 605nm. The quality of the fit decreases with the increased contribution from the allophycocyanin core.

Annihilation model fittings for multiple wavelengths in GSB region
Pump-probe biexponential fitting constants Figure S4: Pump-probe fluence dependent spectral decays plotted at a) 568nm, b) 588nm, c) 596nm and d) 605nm, e) 632nm, f) 642nm, g) 652nm. We have used biexponential fits based on the compartmental model of Berera and co-workers which attributes multiples DADS with photoinduced absorption signals to the overall dynamics of the wild-type and CK mutants. We expect at least biexponential dynamics based on the overlap of photoinduced absorption and the ground state bleach signatures. We only use biexponential dynamics to show that the changing second decay constant suggests that higher-order exciton effects are at play and exponential dynamics cannot describe the underlying photophysics.

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Pump-probe spectra reproducibility Figure S5: Reproducibility of pump-probe spectra at 568nm for a) 1.3 excitations/rod, b) 3.5 excitations/rod, c) 6.1 excitations/rod and d) 14.6 excitations/rod Figure S6: Pump-probe spectra collected up to 1ns delays. a) Excitation spectra for 50ps vs 1ns spectra show the difference in relative excitations at various wavelengths; the overall number of excitations per complex remained the same throughout both measurements. b) 1ns spectra probed at 605nm. c) 1ns spectra probed at 632nm. In b) and c) the relatively low intensity of excitation light at blue wavelengths did not allow for collecting spectra at 568nm.

Calculation of the frequency of exciton-exciton annihilation in physiological conditions
Our experiment uses pulses with energies of 14 nJ/pulse and a beam diameter or290 μm. 14 nJ/pulse corresponds to 1.3 excitations per PC rod per pulse (see section on calculating the Number of excitations in pump-probe experiment).
It is known that excitations move from the PC rods to the APC on the 200ps timescale. However, to be conservative and not overestimate the importance of the observed phenomena, we use an effective lifetime of 100ps in the rods to get the effective power seen by each rod as . 140 .

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The effective fluence of the experiment is then * 2.12 10 .
2.12 10 corresponds to 1.3 excitations per rod per pulse. If we assume a typical physiological fluence of 7 10 and that 15% of solar light is absorbed by the phycocyanin rods based on the solar spectrum at the surface of the earth and the absorption cross section, then it corresponds to 6.4 10 -8 excitations per rod per 100ps.
The Poisson weight for 6.4 10 -8 number of excitations per rod per 100ps corresponds to 2.04 10 -15 occurrences of two excitations per 100ps as would be required for annihilation under ambient solar illumination.
Therefore, the number of occurrences of two excitations in one rod simultaneously in fourteen days at 700 W/m 2 with a 12 hour diurnal photoperiod in each rod: 2.04 10 100 10 100 60 60 ℎ 12 ℎ 14 Therefore, at physiological fluences, this phenomenon can occur in each phycocyanin rod ~10 times over a two-week cell lifetime.