Ultraviolet Resonance Raman Study on Protein Structure Dynamics of Cytochrome c Oxidase

研制了一台高性能的生物学用紫外共振拉曼光谱仪。将日盲增强电荷耦合器件附接到具有200 nm闪耀、3600凹槽/mm光栅的1260 mm单摄谱仪。对缺模氩离子激光器的488 nm输出进行倍频，获得244 nm紫外光，用于共振拉曼激发。这种拉曼光谱已被应用于细胞色素c氧化酶的探测酪氨酸和色氨酸残基的结构变化后，氧化还原和连接状态的变化。静息氧化，完全还原和混合价态形式的酶在一氧化碳或氰化物的存在和不存在下进行了测量。结果表明，血红素a和/或Cu_A的氧化还原变化对酪氨酸和色氨酸残基的影响最大，而血红素a_3的连接以及血红素a_3和/或Cu_B的氧化还原变化对蛋白质结构的影响较小。建立了分析细胞色素c氧化酶时间分辨紫外共振拉曼光谱的基础数据。

A high performance ultraviolet resonance Raman spectrophotometer for biological applications has been constructed. A solar-blind intensified charge-coupled device was attached to a 1260 mm single spectrograph with a 200 nm-blazed, 3600 grooves/mm grating. The 488 nm output of a mode-lacked argon ion laser was frequency doubled to obtain 244nm UV light for resonance Raman excitation. This Raman spectrophotometer has been applied to cytochrome c oxidase to probe structural changes of tyrosine and tryptophan residues upon redox and ligation-state changes. Resting oxidized, fully-reduced and mixed-valence forms of the enzyme in the presence and absence of carbonmonoxide or cyanide have been measured. It became evident that a redox change of heme a and / or Cu_A induced most prominent changes on tyrosine and tryptophan residues while ligation to heme a_3 and redox change of heme a_3 and / or Cu_B less affected the protein structures. Basic data to analyze time-resolved UV resonance Raman spectra of cytochrome c oxidase have been established.