【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 b₅光照还原的影响，以阐明Cyt b₅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b₅（Cyt b₅）可以使其被还原成亚铁Cyt b₅,经过分析发现了Cyt b₅被光还原的机制和促进条件。用280 nm单色光照射Cyt b₅溶液，发现随着光照时间的延长，Cyt b₅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 b₅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 b₅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 b₅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 b₅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 b₅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 b₅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 b₅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 b₅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反应和电子传递机制提供一定理论依据。更多还原

【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-Fe^Ⅲ in phosphate-buffered was photoreduced to Cyb₅-Fe^Ⅱ 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.更多还原