The Impact of Carotenoid Energy Levels on the Exciton Dynamics and Singlet–Triplet Annihilation in Isolated Bacterial Light-Harvesting 2 Complexes

The light-harvesting 2 (LH2) complex of purple phototrophic bacteria plays a critical role in absorbing solar energy and distributing the excitation energy. Exciton dynamics within LH2 complexes are controlled by the structural arrangement and energy levels of the bacteriochlorophyll (BChl) and carotenoid (Car) pigments. However, there is still debate over the competing light-harvesting versus energy-dissipation pathways. In this work, we compared five variants of the LH2 complex from genetically modified strains of <i>Rhodobacter sphaeroides</i>, all containing the same BChls but different Cars with increasing conjugation: zeta-carotene (<i>N</i> = 7; LH2_Zeta), neurosporene (<i>N</i> = 9; LH2_Neu), spheroidene (<i>N</i> = 10; LH2_Spher), lycopene (<i>N</i> = 11; LH2_Lyco), and spirilloxanthin (<i>N</i> = 13; LH2_Spir). Absorption measurements confirmed that the Car excited-state energy decreased with increasing conjugation. Similarly, fluorescence spectra showed that the B850 BChl emission peak had an increasing red shift from LH2_Zeta→(LH2_Neu/LH2_Spher)→LH2_Lyco→LH2_Spir. In contrast, time-resolved fluorescence and ultrafast transient absorption (fs-TA) revealed similar excited-state lifetimes (∼1 ns) for all complexes except LH2_Spir (∼0.7 ns). From fs-TA analysis, an additional ∼7 ps nonradiative dissipation step from B850 BChl was observed for LH2_Zeta. Further, singlet-singlet and singlet-triplet annihilation studies showed a ∼50% average fluorescence lifetime reduction in LH2_Zeta at high laser power and high repetition rate, compared to ∼10-15% reductions in LH2_Neu/LH2_Spher/LH2_Lyco and minimal lifetime change in LH2_Spir. In LH2_Zeta, the fastest decay component (<50 ps) became prominent at high repetition rates, consistent with strong singlet-triplet annihilation. Nanosecond TA measurements revealed long-lived (>40 μs) BChl triplet states in LH2_Zeta and signs of damage caused by singlet oxygen, whereas other LH2s showed faster triplet quenching (∼18 ns) by Cars. These findings highlight a key design principle of LH2 complexes: the Car triplet energy must be significantly lower than the BChl triplet energy to efficiently quench BChl triplets that otherwise act as potent "trap states," causing exciton annihilation in laser-based experiments or photodamage in native membranes.