光诱导细胞色素b_(5)还原反应及机理的光谱学研究  

Spectroscopic Study on the Mechanism of Photoduction of Cytochrome b_(5) by Ultraviolet Light

作　　者：唐乾[1,3] 黄婷 宫婷婷[1,3] 曹洪玉[1,3] 王爱玲[2,3] 王立皓 郑学仿[1,2,3] TANG Qian;HUANG Ting;GONG Ting-ting;CAO Hong-yu;WANG Ai-ling;WANG Li-hao;ZHENG Xue-fang(College of Life Science and Technology,Dalian University,Dalian 116622,China;College of Environmental and Chemical Engineering,Dalian University,Dalian 116622,China;Liaoning Key Laboratory of Bio-organic Chemistry,Dalian University,Dalian 116622,China)

机构地区：[1]大连大学生命科学与技术学院,辽宁大连116622 [2]大连大学环境与化学工程学院,辽宁大连116622 [3]辽宁省生物有机化学重点实验室,大连大学,辽宁大连116622

出　　处：《光谱学与光谱分析》2022年第3期821-827,共7页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(21571025,21601024,21601025,21601023);大连大学科研平台重点项目(2021012D01)资助。

摘　　要：在生命体内有关电子转移的研究备受关注,其中蛋白质以及酶之间的电子转移研究已成为热点,目前有关光诱导血红素蛋白还原的详细机理尚不明确,电子转移也许是解释这一机理的有效途径。通过紫外-可见吸收光谱、稳态荧光光谱和圆二色等光谱系统地研究了体外近生理溶液环境中,不同的紫外定波长、pH、游离氨基酸、谷胱甘肽、咪唑对Cyt b_(5)光照还原的影响,以阐明Cyt b_(5)不能用传统提出的机制去解释的光还原机制。结果表明:在近紫外区通过直接的光激发高铁细胞色素b_(5)(Cyt b_(5))可以使其被还原成亚铁Cyt b_(5),经过分析发现了Cyt b_(5)被光还原的机制和促进条件。用280 nm单色光照射Cyt b_(5)溶液,发现随着光照时间的延长,Cyt b_(5)溶液紫外-可见光谱中Soret带由412 nm红移到421 nm,Q带556 nm处吸收峰逐渐增强。Cyt b_(5)被光照后发生了类似化学还原剂作用的还原反应,同时发现了不同的光照波长、不同的pH、氨基酸种类以及各种其他配体等存在下对Cyt b_(5)的还原程度影响不同。采用280 nm波长、在偏碱性条件下光照Cyt b_(5)时还原程度最强;溶液中加入谷胱甘肽和咪唑能通过提供电子和氢供体途径促进Cyt b_(5)的光照还原反应发生;溶液中游离Met的存在能以最大速率促进光照还原反应的发生。基于以上结果提出Cyt b_(5)光还原的机理是从卟啉环到三价铁的电子转移,通过280 nm激发形成卟啉π阳离子基团和亚铁。采用稳态荧光光谱和圆二色谱对Cyt b_(5)光照还原发生前后进行检测,发现光诱导蛋白发生还原后,Cyt b_(5)主链结构逐渐伸展,二级结构发生了一定改变,α-螺旋含量逐渐降低,β-折叠含量逐渐升高,但是整个Cyt b_(5)的二级结构仍以α-螺旋为主。该研究结果不仅对了解光对含血红素蛋白(酶)的结构和功能的影响有重要的理论和实践意义,而且对于生命体内的氧化还原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 b_(5) in vitro near-physiological environment to clarify the photoreduction mechanisms of Cyt b_(5) which wasn’t proposed by the traditional methods.The results show that ferric cytochrome b_(5) 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 b_(5)-FeⅢ in phosphate-buffered was photoreduced to Cyb_(5)-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 b_(5) had the strongest reduction degree.Under 280 nm alkaline conditions,Cyt b_(5) 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

关 键 词：细胞色素b_(5) 光诱导 氧化还原 光谱 

分 类 号：O657.32[理学—分析化学]