Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl <i>a</i> and β-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the CC stretch band of the 2A_g^– (S₁) state of β-carotene upon Chl <i>a</i> excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct β-carotene conformers by the CC stretch frequency of the 2A_g^– (S₁) state, but only the β-carotene whose 2A_g^– energy level is significantly lowered and has a lower CC stretch frequency is involved in quenching. It implies that the low carotenoid S₁ energy that results from specific pigment–protein or pigment–pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state’s properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state’s properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach