Spectroscopic Study on the Mechanism of Photoduction of Cytochrome b5 by Ultraviolet Light
TANG Qian1, 3, HUANG Ting2, 3, GONG Ting-ting1, 3, CAO Hong-yu1, 3, WANG Ai-ling2, 3, WANG Li-hao2, 3, ZHENG Xue-fang1, 2, 3*
1. College of Life Science and Technology，Dalian University，Dalian 116622，China
2. College of Environmental and Chemical Engineering，Dalian University，Dalian 116622，China
3. Liaoning Key Laboratory of Bio-organic Chemistry，Dalian University，Dalian 116622，China
Abstract：The study of electron transfer in life has attracted much attention, and the research on the electron transfer of proteins and enzymes has become a hot spot. However, electron transfer may be an effective way to explain this mechanism. The detailed mechanism of photoinduced heme protein reduction is still unclear. In this paper, UV-visible absorption spectroscopy, steady-state fluorescence spectroscopy and circular dichroism spectroscopy were used to study the effects of different UV wavelengths systematically, pH, amino acids,Glutathione and Imidazoleon the photoreduction of Cyt b5 in vitro near-physiological environment to clarify the photoreduction mechanisms of Cyt b5 which wasn’t proposed by the traditional methods. The results show that ferric cytochrome b5 can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this study, we studied the mechanism and facilitating conditions for photoreduction. Based on the sort band blue-shifted of 412 nm and absorbance intensity increase of Q band 556 nm, Cyt b5-FeⅢ in phosphate-buffered was photoreduced to Cyb5-FeⅡ similar to the action of a chemical reducing agent occurs. Considering that the fixed wavelength, pH values, amino acids and ligands of photoreduction were irradiated by 280 nm light, Cyt b5 had the strongest reduction degree. Under 280 nm alkaline conditions, Cyt b5 had the strongest reduction degree; glutathione and imidazole promoted the photoreduction reaction by providing electron and hydrogen donors; free Met in solution promoted the photoreduction reaction at the maximum rate happened. The photoreduction mechanism of Cyt c was intramolecular electron transfer, including the formation of porphyrin cation radical as an active intermediate excited by 280 nm light. In addition, results of fluorescence and CD spectra indicated that the protein-peptide chain structure, while the secondary structure of the protein changes, α-helix content decreased, β-sheet content increased.However, the secondary structure of Cyt b5 is still dominated by α-helix in the photoreduction process. Moreover, it provides a theoretical basis for the redox reaction and electron transfer mechanism in life.
唐 乾，黄 婷，宫婷婷，曹洪玉，王爱玲，王立皓，郑学仿. 光诱导细胞色素b5还原反应及机理的光谱学研究[J]. 光谱学与光谱分析, 2022, 42(03): 821-827.
TANG Qian, HUANG Ting, GONG Ting-ting, CAO Hong-yu, WANG Ai-ling, WANG Li-hao, ZHENG Xue-fang. Spectroscopic Study on the Mechanism of Photoduction of Cytochrome b5 by Ultraviolet Light. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2022, 42(03): 821-827.
[1] Johnson M P. Essays Biochemistry，2016，60(3)：255.
[2] Wood W H J, MacGregor-Chatwin C, Barnett S F H, et al. Nature Plants, 2018, 4(2): 116.
[3] Zheng T W，Li J，Zhang Y L，et al. Front Bioeng Biotechol, 2020, 8: 10.
[4] Ferapontova E E. Chem. Asian J, 2019, 14(21): 3773.
[5] Duggal R，Denisov I G. FEBS Letters，2018, 592(13): 2282.
[6] Holien J K，Parker M V，Conley A J，et al. Current Protein & Peptide Science，2017, 18(5): 515.
[7] Amdahl M B，Petersen E B，Bocian K, et al. Biochemistry，2019, 58(29): 3212.
[8] Hilda M U，Mariana A M，Juan Horacio E R，et al. PLOS ONE, 2020, 15(4): 130. 232408.
[9] Peter Guengerich F，Wilkey C J, Phan T T N. J Biol Chem，2019，294(28)：10928.
[10] Lmdahl M B，Sparacino-Watkins C E，Corti P，et al. J. Biochemistry, 2017, 56(30): 3993.
[11] ZHOU Hua-wei，CAO Hong-yu，TANG Qian，et al（周华伟，曹洪玉，唐 乾，等）. Acta Chim. Sinica(化学学报)，2011，69(13)：1559.
[12] AN Liang-mei，CAO Hong-yu，TANG Qian，et al（安良梅，曹洪玉，唐 乾，等）. Chinese Journal of Inorganic Chemistry（无机化学学报），2012, 28(7)：1461.
[13] CAO Hong-yu, SHI Fei, TANG Qian, et al(曹洪玉，史 飞，唐 乾，等). Chinese Journal of Inoganic Chemistry(无机化学学报)，2017, 33（8）：1339.
[14] Tang Q，Peng X J，Cao H Y，et al. Asian Journal of Chemistry，2013，25(4)：2054.
[15] Consani C，Auböck G，Van M F，et al. Science，2013，339(6127): 1586.
[16] Cao H Y, Liu Y W，Tang Q，et al. Protein & Peptide Letters，2015，22：853.
[17] Lakowica J R. Principles of Fluorescence Spectroscopy，3rd Edition. Science Press, 2008，546.
[18] Tang Q, Peng X J, Cao H Y, et al. The Journal of Spectroscopy，2013, (9): 1.
[19] Villar-Guerra R D, Trent J O, Chaires J B. Angew. Chem. Int. Ed. Engl., 2018, 57: 7171.
