Photogeneration and quenching of singlet molecular oxygen by bacterial C40 carotenoids with long chain of conjugated double bonds.

Measurement of photosensitized luminescence of singlet oxygen has been applied to studies of singlet oxygen generation and quenching by C40 carotenoids (neurosporene, lycopene, rhodopin, and spirilloxanthin) with long chain of conjugated double bonds (CDB) using hexafluorobenzene as a solvent. It has been found that neurosporene, lycopene, and rhodopin are capable of the low efficient singlet oxygen generation in aerated solutions upon photoexcitation in the spectral region of their main absorption maxima. The quantum yield of this process was estimated to be (1.5-3.0) × 10-2. This value is near the singlet oxygen yields in solutions of ζ-carotene (7 CDB) and phytoene (3 CDB) and many-fold smaller than in solutions of phytofluene (5 CDB) (Ashikhmin et al. Biochemistry (Mosc) 85:773-780, https://doi.org/10.1134/S0006297920070056 , 2020, Biochemistry (Mosc) 87:1169-1178, 2022, https://doi.org/10.1134/S00062979221001082022 ). Photogeneration of singlet oxygen was not observed in spirilloxanthin solutions. A correlation was found between the singlet oxygen yields and the quantum yields and lifetimes of the fluorescence of the carotenoid molecules. All carotenoids were shown to be strong physical quenchers of singlet oxygen. The rate constants of 1O2 quenching by the carotenoids with long chain of CDB (9-13) were close to the rate constant of the diffusion-limited reactions ≈1010 M-1 s-1, being many-fold greater than the rate constants of 1O2 quenching by the carotenoids with the short chain of CDB (3-7) phytoene, phytofluene, and ζ-carotene studied in prior papers of our group (Ashikhmin et al. 2020, 2022). To our knowledge, the quenching rate constants of rhodopin and spirilloxanthin have been obtained in this paper for the first time. The mechanisms of 1O2 photogeneration by carotenoids in solution and in the LH2 complexes of photosynthetic cells, as well as the efficiencies of their protective action are discussed.

Phytofluene as a Highly Efficient UVA Photosensitizer of Singlet Oxygen Generation.

Phytoene and phytofluene - uncolored C40 carotenoids with short chain of conjugated double bonds (3 and 5, respectively) - are known to be universal precursors in biosynthesis of colored carotenoids in photosynthesizing organisms. It is commonly recognized that C40 carotenoids are photoprotectors of cells and tissues. We have shown that phytofluene is an exception to this rule. By measuring photosensitized phosphorescence of singlet oxygen (1O2) we found out that phytofluene was very effective photosensitizer of 1O2 formation in aerated solutions under UVA irradiation (quantum yield of 85 ± 5%), whereas phytoene was almost inactive in this process. It was demonstrated that both carotenoids quench singlet oxygen in the dark. The obtained quenching rate constants [(4 ± 1) × 106 M-1·s-1 for phytoene and (2 ± 0.5) × 107 M-1·s-1 for phytofluene] were smaller than the rate constant of the diffusion-controlled reactions by 3-4 orders of magnitude. Thus, both carotenoids displayed rather weak protector properties. Moreover, phytofluene due to its high photosensitizing activity might be considered as a promoter of cell photodamage and a promising UVA photosensitizer for medical purposes.

Kinetic Measurements of Singlet Oxygen Phosphorescence in Hydrogen-Free Solvents by Time-Resolved Photon Counting.

Solvents lacking hydrogen atoms are very convenient models for elucidating the properties of singlet oxygen, since the lifetime of singlet oxygen in these solvents reaches tens milliseconds. Measuring intrinsic infrared (IR) phosphorescence of singlet oxygen at 1270 nm is the most reliable method of singlet oxygen detection. However, efficient application of the phosphorescence method to these models requires an equipment allowing reliable measurement of the phosphorescence kinetic parameters in the millisecond time range at low rates of singlet oxygen generation, which is a technically difficult problem. Here, we describe a highly sensitive LED (laser) spectrometer recently constructed in our laboratory for the steady-state and time-resolved measurements of the millisecond phosphorescence of singlet oxygen. In the steady-state mode, this spectrometer allows detection of singlet oxygen phosphorescence upon direct excitation of oxygen molecules in the region of dark-red absorption bands at 690 and 765 nm. For kinetic measurements, we used phenalenone as a photosensitizer, microsecond pulses of violet (405 nm) LED for excitation (irradiance intensity, ≤50 µW/cm2), a photomultiplier and a computer multichannel scaler for time-resolved photon counting. The decays of singlet oxygen in air-saturated CCl4, C6F6, and Freon 113 and quenching of singlet oxygen by phenalenone and dissolved molecules of triplet oxygen were measured. The relative values of the radiative rate constants of singlet oxygen in these media were determined. The results were compared with the absorption coefficients of oxygen measured by our group using the methods of laser photochemistry. Critical discussion of the obtained results and the data of other researchers is presented.