Illuminating the ultrafast excited state dynamics of protein-bound carotenoids in plants

Abstract

Global energy demands have escalated over the past few decades, creating a necessity for alternative energy sources. Solar technologies inspired by the primary solar energy storing process known on earth, photosynthesis, have subsequently gained popularity. The natural photosynthetic apparatus comprises a network of membrane-bound pigment-protein complexes, with the main plant light-harvesting complex (LHCII) consisting of chlorophyll (Chl) and carotenoid (Car) pigments. Electronic excitation energy transfer (ET) of the harvested energy takes place amongst these pigments on ultrafast timescales. This energy is funnelled towards a photosynthetic reaction centre where charge separation is achieved, creating a Biobattery, which powers the subsequent manufacture of energy-rich chemical compounds for photosynthetic activity. Transient absorption pump-probe spectroscopy has proven to be a useful technique for monitoring the evolution of the excited state dynamics, such as electronic transitions and excitation ET amongst Car and Chl pigments of LHCII trimers isolated from spinach leaves. This method was utilized to probe samples excited under four different conditions: at pump excitation wavelengths (𝜆𝑒𝑥) of 489 nm (preferentially exciting Cars Lutein1 and Neoxanthin) and 506 nm (targeting Cars Lutein2 and Violaxanthin), each with an intensity of either 800 nJ/pulse (relatively high) or 500 nJ/pulse (comparatively low). A global analysis was applied to each dataset using the robust, open-source Glotaran software, from which three kinetic decay lifetimes for the various processes were extracted. General spectral observations encompassed a negative pump ground state bleach (GSB) at each 𝜆𝑒𝑥; negative Chl b and Chl a GSBs, superimposed with negative stimulated emission (SE) signals; and a positive excited state absorption (ESA) band. The first lifetime of a few picoseconds corresponded mainly to Car-S2 depopulation, resulting either from energy relaxation towards Car-S1, or ET to Chls. Small, but distinct Chl b signals of less than 3 mOD were also detected on this timescale. The second lifetime, which is between 10 and 12 ps, was characteristic to the Lutein Car-S1 lifetime, mainly depicting Car-S1 ET to Chl a. The third lifetime, which extended from ~200 ps to the nanosecond timescale, was attributed to Chl a fluorescence. The 𝜆𝑒𝑥 of 489 nm directly excites the Chl Soret region, whilst excitation at 506 nm shows a pump intensity-dependence. Laser pulse photon density values were ~1014 photons·cm-2·pulse-1 for these datasets. Singlet-singlet annihilation calculations performed on the samples excited at 506 nm provided low annihilation probabilities of 9.0% and 11.5% for a low and high pump intensity, respectively, limiting the possibility of sample photobleaching. Optimization and redevelopment of the experimental setup significantly improved both the data quality and various recorded parameters, concluding that pump-probe spectroscopy was successful on the prepared LHCII trimers. Results acquired and calculations performed correlated with literature, where minimal changes were noticed in the timescales and ET pathways. The robustness of plant systems was confirmed through both excitation-wavelength and intensity dependence. This work paves the way for advanced studies on the role Cars play in non-photochemical quenching (NPQ), a self-protection mechanism of plants against over-illumination; and for the tailoring of artificial light-harvesting antennas based on research conducted on their natural counterparts.

Globale energievereistes het oor die afgelope paar dekades toegeneem, wat die ontwikkeling van alternatiewe energiebronne noodsaaklik maak. Sontegnologieë, geïnspireer deur die primêre sonenergiebergingsproses op aarde, fotosintese, het daarom gewild geword. Die natuurlike fotosintetiese apparaat bestaan uit 'n netwerk van membraangebonde pigment-proteïenkomplekse, met die hoof ligversamelingskompleks in plante (LHCII) wat bestaan uit chlorofil- (Chl) en karotenoïed- (Car) pigmente. Die energie wat deur die pigmente geabsorbeer word, word tussen elektroniese opgewekte toestande op verskillende pigmente op ultravinnige tydskale oorgedra. Hierdie energie word na ʼn fotosintetiese reaksiesentrum gekanaliseer, waar 'n ladingskeiding geïnduseer word en 'n Biobattery sodoende geskep word. Die energie wat in dié battery gestoor is, word gebruik om energieryke chemiese verbindings te vervaardig — wat as brandstof vir die plant dien om sy lewensfunksies te verrig. Tydopgeloste-absorpsie-pomp-tasting-spektroskopie is 'n nuttige tegniek om die dinamika tussen opgewekte toestande te volg. ‘n Voorbeeld van sulke dinamika is die elektroniese opwekking en energie-oordrag tussen die Car- en Chl-pigmente van geïsoleerde LHCII-trimere in spinasieblare. Hierdie metode is gebruik om monsters onder vier verskillende toestande te ondersoek by pompgolflengtes (𝜆𝑒𝑥) van 489 nm (waar hoofsaaklik die Cars Luteïne1 en Neoksantine opgewek word) en 506 nm (vir Cars Luteïne2 en Violaksantine), en pompenergieë van ‘n relatief hoë 800 nJ/puls, of 500 nJ/puls vir elke golflengte.