细胞色素C氧化酶亚基Ⅱ可溶性结构域的结构改变、转换和功能研究

【作者】 宋爱新；

【导师】 黄仲贤；

【作者基本信息】 复旦大学 ， 无机化学， 2004， 博士

【摘要】 细胞色素 c 氧化酶是哺乳动物线粒体或细菌呼吸链上的末端酶，催化电子从细胞色素 c 到 O2 的转移并使后者还原为水。该酶是一个复合的膜蛋白，包括多个亚基及不同的金属活性中心。其中，亚基II中的 CuA 中心被认为是电子从细胞色素 c 进入该酶的入口，在生理过程中起着重要作用。细胞色素 c 氧化酶的缺损或不足可导致许多疾病，在一些退行性疾病 (如：老年痴呆症) 或癌症研究中已发现了与其相关的基因突变。本工作以该酶的亚基 II 可溶区为研究对象，成功表达并获得了 Paracoccus versutus 的细胞色素 c 氧化酶亚基II可溶区重组蛋白 (128-280位的氨基酸序列)；通过基因工程方法构建突变体蛋白，研究了 Trp121 和 Val162氨基酸残基在该酶中的重要地位，报道了突变和其它物理化学因素引起的蛋白结构改变、转换，以及与此相关的性质和功能变化。主要工作和结果如下：(一) 细胞色素 c 氧化酶亚基II可溶区蛋白的体外表达、纯化及性质表征以大肠杆菌BL21 (DE3) 为表达载体，建立并优化了表达条件，获得了细菌Paracoccus versutus 的细胞色素 c 氧化酶亚基II可溶区重组蛋白。该蛋白大量存在于包涵体中，通过体外复性，金属中心重组和FPLC纯化，获得了水溶性的紫色蛋白，在SDS-PAGE中表现为单一条带。经电喷雾质谱测定分子量，蛋白的成功制备得到了进一步确证。采用紫外-可见和园二色谱表征了蛋白的金属活性中心和二级结构。紫外可见光谱显示出明显的 CuA 特征，360，480，530 和 810 nm 的吸收带是双核混价铜中心的特有贡献；在园二色谱中，215 nm 附近的负峰是 (-折叠的典型特征。这些光谱学特征与文献报导的同类蛋白质基本一致。稳态荧光谱研究提供了有关亚基II可溶区的三级结构信息，细胞色素 c 氧化酶亚基II可溶区的最大荧光发射波长为342±2 nm，主要反映了色氨酸的荧光特征，表明存在着Tyr → Trp的能量转移；Apo-WT 蛋白的荧光较Holo-WT 强，表明 CuA 中心对蛋白荧光有淬灭作用。采用还原态的细胞色素c，对蛋白的活性也作了检测。通过监测可见区 (520，530，550 nm) 的光谱变化，证明细胞色素 c 氧化酶亚基II可溶区可以氧化细胞色素c，具有酶活性。(二) 121位色氨酸突变对蛋白结构和功能的影响细胞色素 c 氧化酶亚基II中存在一个高度保守的芳香性残基簇 (Trp121，Tyr122，Trp123，Tyr125和Tyr127)，晶体结构研究表明：其中的Trp121距离 CuA <WP=6>中心仅有5 ?，且与 CuA 中心的多个配体有相互作用。同时，Trp121还被看作是电子从细胞色素 c 进入细胞色素 c 氧化酶的入口。然而，Trp121对蛋白结构所起的作用并不清楚，其影响电子传递的机制尚需进一步探讨。利用蛋白质工程的定点突变技术，我们构建了121位色氨酸残基的三个突变基因：W121Y、W121L 和 ?W121，经DNA测序证明其正确性。突变基因转入大肠杆菌中进行表达，三个突变蛋白的制备过程均与野生型类似。其中，只有W121Y突变体可以重组 CuA 中心，并具有与野生型一致的二级结构特征。突变体 W121L 和 ?W121 均丧失了形成 CuA 中心的能力，园二色谱研究显示它们的二级结构也发生了改变，在215 nm 处的负峰转化为 208 和 220 nm 的双负峰，暗示着蛋白中 (–螺旋和 (-折叠含量的相对变化。电子自旋共振 (EPR) 和拉曼光谱 (Raman) 测定表明：在这两个突变体中，特征的 CuA 配位结构已明显转换为II型 Cu2+ 配位。尤为重要的是，对Trp121残基的取代明显地影响了蛋白的电子传递功能。利用Stopped-flow 快速动力学技术研究 CuA 中心与细胞色素 c 的电子转移反应发现：与野生型相比，突变体W121Y与细胞色素 c 的电子传递速率减少了七倍；而在同样条件下，由于 CuA 活性中心的破坏和蛋白结构的转换，W121L 和 (W121 突变体与细胞色素 c 几乎不发生电子传递。研究表明，121位氨基酸残基的芳香性和氢键生成能力对于维系稳定的 CuA中心和天然的构象是必需的，同时也有利于高效的电子转移。用平衡滴定实验分别测得了野生型和W121Y突变体的电位，比较了可以部分保持电子传递功能的突变体W121Y与野生型的差异，进一步研究其活性减小的原因。结果表明：W121Y突变体的电位与野生型接近，并非导致其活性下降的主要因素；相应地，侧链体积的减小及氨基酸突变所引起的微环境的扰动可能起着不可忽略的作用。(三) 野生型及W121位突变体蛋白的稳定性及去折叠研究结合紫外-可见、园二色谱和稳态荧光光谱，考察了不同条件下细胞色素c氧化酶亚基II可溶区野生型及突变体的稳定性，并对变性过程中的去折叠机制进行了探讨。细胞色素c氧化酶亚基II可溶区的稳定性包括两个方面：CuA 中心的稳定性和蛋白骨架的稳定性。总的来讲，第121位色氨酸的突变使二者稳定性均有所下降。在蛋白变性过程中，我们再次观察到与 CuA 中心相关的构象转换，其CD光谱特征与W121L 和 (W121突变体类似。在热、碱或盐酸胍诱导的变性过程中，蛋白的去折叠不符合简单的二态机制，而是存在稳定的中间体。从实验结果看，中间体的构象应该是对应于蛋白发生构象转换后的状态。另外，蛋白酸变性<WP=7>的过程比较特殊，蛋白分子在等电点附近严重聚集，但是整个过程不发生构象转更多还原

【Abstract】 Cytochrome c Oxidase (CcO) is the terminal enzyme of the redox respiratory chain in mitochondria and many aerobic bacteria. It catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water. The enzyme is a complex metalloprotein embedded in the membrane and comprises several subunits and metal active sites. The CuA center in subunit II is essential to the physiological function of the enzyme because the electron from cytochrome c enters the CcO firstly via this site. Defects in cytochrome c oxidase can lead to many diseases, and several mutations in gene encoding the enzyme have been found in Alzheimers’disease and Cancers. In this work, we have expressed and isolated the native subunit II soluble CuA domain of CcO from Paracoccus versutus and its mutant proteins at sites Trp121 and Val162. Using spectral and kinetic techniques, we studied the effects of different mutation on structure, property and electron transfer function of the protein. The conformation transitions caused by mutation or other physical and chemical factors have also been reported. The followings are the main results.(I) Expression, purification and characterization of the soluble CuA domain of CcO The native soluble CuA domain of Paracoccus versutus has been expressed in E.coli BL21 (DE3). Almost the entire subunit II fragment was in inclusion bodies. After refolding in vitro, recombination of metal center and purification with Mono Q column running with a Pharmacia FPLC instrument, the soluble purple sample was obtained. The purified protein showed a single band on SDS-PAGE analysis, and the molecular mass of protein was measured accurately with electrospray mass spectrometry. The UV-visible and CD spectra were used to characterize the active site and secondary structure of the protein. The absorption peaks at 360, 480, 530 and 810 nm show a typical purple CuA center feature, and the far-UV CD spectra shows a negative peak at 215 nm, which presents a typical (-sheet structure. These characteristics are similar to that of other native and engineered CuA protein. The maximum of 342 nm in fluorescence emission spectrum of the protein shows that its fluorescence can be mainly assigned to the tryptophan residues. The efficient energy transfers were observed in the soluble CuA domain, including the energy transfer from Tyr to Trp <WP=9>residue and that from Trp to the CuA center.The activity of protein was demonstrated by monitoring the oxidation process of the reduced cytochrome c (Cyt c) by the soluble CuA domain.(II) The effects of mutation at Trp-121 on protein’s structure and functionTrp121 is one member of the highly conserved aromatic cluster (Trp121, Tyr122, Trp123, Tyr125 and Tyr127) in CcO sdII. It locates at the surface of subunit II about 5 ? above the CuA center and interacts with several different CuA ligands. Functionally, the Trp121 has been considered as the electron entry site from Cyt c to CcO. However, the contribution of Trp121 to protein’s structure and property had never been explored. The mechanism of transfer electron is also not clear.Using the site-directed technique of protein engineering, we have constructed and expressed the mutant proteins of W121Y, W121L and (W121 in soluble domain of subunit II of CcO from Paracoccus versutus. The W121Y mutant can refold properly and reform the CuA center almost completely. However, the (W121 and W121L mutant proteins lost the ability to reconstitute CuA center and mainly refold as another conformation. Their characteristics in CD spectrum are double negative peaks of 208 nm and 220 nm, which suggest the relative change in abundance of (-helices and (-sheets in those proteins. The conversion of CuA center to type II Cu2+ coordination is corroborated by EPR and Raman spectroscopy. Importantly, the mutations at Trp121 affect the electron transfer (ET) rate of the protein with Cyt c. The results from stopped-flow rapid kinetic studies show that the ET rate between the W121Y mutant and Cyt c decreases about seven folds. At the same更多还原