Nett J H, Hunte C, Trumpower B L
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Eur J Biochem. 2000 Sep;267(18):5777-82. doi: 10.1046/j.1432-1327.2000.01650.x.
Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.
细胞色素bc1复合物的晶体结构表明，携带[2Fe-2S]簇的 Rieske 铁硫蛋白的催化结构域通过一个柔性连接区与跨膜锚定结构相连。这个柔性连接区允许催化结构域在靠近细胞色素b和细胞色素c1的两个位置之间移动。在 Rieske 蛋白的柔性连接区添加一个丙氨酸残基会使线粒体膜的泛醇-细胞色素c还原酶活性降低一半，并使泛醇的表观 Km 值从9.3微摩尔降至2.6微摩尔。添加两个丙氨酸残基会使活性降低90%，表观 Km 值降至1.9微摩尔。删除一个丙氨酸残基会使活性降低约40%，表观 Km 值降至5.0微摩尔。添加或删除一个丙氨酸残基还会导致抑霉唑对泛醇-细胞色素c还原酶活性的抑制效果显著降低，抑霉唑的结合类似于泛醇氧化的反应中间体。这些结果表明，柔性连接区的长度对于泛醇与bc1复合物的相互作用至关重要，这与泛醇必须同时与铁硫蛋白和细胞色素b相互作用的电子传递机制一致。
