Frey T G, Murray J M
Department of Biology, San Diego State University, CA 92182-0057.
J Mol Biol. 1994 Apr 1;237(3):275-97. doi: 10.1006/jmbi.1994.1231.
Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.
细胞色素c氧化酶是通过去污剂提取从牛心线粒体中分离出来的，得到了两种不同的晶体形式。用Triton去污剂提取产生了具有细胞色素c氧化酶二聚体二维晶体阵列的囊泡晶体，而用脱氧胆酸钠提取则产生了细胞色素c氧化酶单体的晶体薄片。通过对嵌入玻璃冰（冷冻水合）中的晶体进行冷冻电子显微镜观察，沿垂直于膜平面的轴在二维投影中确定了两种晶体形式的结构。未染色的冷冻水合单体和二聚体的投影结构与负染色晶体的结构相似。二聚体的分子轮廓可以近似为一个44埃×82埃的平行四边形，夹角为80度。单体不太规则，由两个大结构域组成，一端有一个较小的结构域，总长度约为82埃。两种结构的比较揭示了细胞色素c氧化酶单体在二聚体内的取向，这种取向与早期单体-单体相互作用模型不同，并表明单体在缔合形成二聚体时相互之间有非常紧密的相互作用。细胞色素c氧化酶单体的晶体薄片紧密结合小的外周膜蛋白底物细胞色素c，这种结合加剧了这些晶体相互堆叠的趋势。通过与单体晶体图像的互相关分析，分析了细胞色素c氧化酶/细胞色素c复合物晶体的图像。鉴定出了两种类型的双层晶体。两种类型都有一层相对于另一层旋转180度，但它们在两层沿晶体轴的原点位移上有所不同。通过从复合物晶体（细胞色素c氧化酶加细胞色素c）中减去模拟的多层晶体图像（没有结合细胞色素c）形成的差异图像，在一个晶胞内每个细胞色素氧化酶单体都有一个正差异峰。基于单层细胞色素氧化酶单体晶体图像中的共有细胞色素c结合位点，解释了不同晶体形式之间差异峰位点的比较。
