Asido, M. | bioRxiv Science

bioRxiv2025

The impact of carotenoid energy levels on the exciton dynamics and singlet-triplet annihilation in the bacterial Light-Harvesting 2 complex

The light-harvesting 2 (LH2) complex of purple phototrophic bacteria plays a critical role in absorbing solar energy and distributing 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 Rhodobacter sphaeroides, all containing the same BChls but different Cars with increasing conjugation: zeta-carotene (N=7; LH2Zeta), neurosporene (N=9; LH2Neu), spheroidene (N=10; LH2Spher), lycopene (N=11; LH2Lyco), and spirilloxanthin (N=13; LH2Spir). Absorption measurements confirmed that Car excited state energy decreased with increasing conjugation. Similarly, fluorescence spectra showed that the B850 BChl emission peak had an increasing red shift from LH2Zeta[->](LH2Neu/LH2Spher)[->]LH2Lyco[->]LH2Spir. In contrast, time-resolved fluorescence and ultrafast transient absorption (fs-TA) revealed similar excited state lifetimes ([~]1 ns) for all complexes except LH2Spir ([~]0.7 ns). From fs-TA analysis, an additional [~]7 ps non-radiative dissipation step from B850 BChl was observed for LH2Zeta. Further, singlet-singlet and singlet-triplet annihilation studies showed a [~]50% average fluorescence lifetime reduction in LH2Zeta at high laser power and high repetition rate, compared to [~]10-15% reductions in LH2Neu/LH2Spher/LH2Lyco and minimal lifetime change in LH2Spir. In LH2Zeta, 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 s) BChl triplet states in LH2Zeta 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 photo-damage in native membranes.

biophysics↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗