Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, United States.
Biochim Biophys Acta Bioenerg. 2018 Sep;1859(9):725-733. doi: 10.1016/j.bbabio.2018.03.017. Epub 2018 Apr 4.
Three mutant forms of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) were created to test for multiple K-path entry sites (E101W), the existence of an "upper ligand site" (M350 W), and the nature and binding specificity of the "lower ligand site" (P315W/E101A) in the region of a crystallographically-defined deoxycholate at the K-path entrance. The effects of inhibitory and stimulatory detergents (dodecyl maltoside and Tween20) on these mutants are presented, as well as competition with other ligands, including the potentially physiologically relevant ligands cholesterol and retinoic acid. Ligands are shown to be able to compete with natural lipids to affect the activity of membrane-bound RsCcO. Results point to a single K-path entrance site at E101, with a single ligand binding pocket proximal to the entrance. The affinity of this pocket for amphipathic ligands is enhanced by removal of the E101 carboxyl and blocked by substituting a tryptophan in this area. A new crystal structure of the E101A mutant of RsCcO is presented that illustrates the structural basis of these results, showing that the loss of the E101 carboxyl creates a more hydrophobic groove consistent with altered ligand affinities.
三种突变体形式的红假单胞菌细胞色素 c 氧化酶（RsCcO）被创建来测试多个 K 通道入口位点（E101W）、是否存在“上配体位点”（M350W）以及“下配体位点”的性质和结合特异性（P315W/E101A）在 K 通道入口处的结晶定义去氧胆酸盐区域。本文介绍了这些突变体对抑制性和刺激性去污剂（十二烷基麦芽糖和 Tween20）的影响，以及与其他配体（包括潜在生理相关的配体胆固醇和视黄酸）的竞争情况。结果表明，配体能够与天然脂质竞争，从而影响膜结合的 RsCcO 的活性。结果表明，E101 处只有一个 K 通道入口位点，入口附近有一个单一的配体结合口袋。这个口袋对两亲性配体的亲和力通过去除 E101 羧基得到增强，而在该区域取代色氨酸则会被阻断。本文还呈现了 RsCcO 的 E101A 突变体的新晶体结构，说明了这些结果的结构基础，表明 E101 羧基的缺失会产生更疏水的凹槽，与改变的配体亲和力一致。
