Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Biochemistry. 2010 Jul 13;49(27):5651-61. doi: 10.1021/bi1003876.
The Cu(I) chaperone Cox11 is required for the insertion of Cu(B) into cytochrome c oxidase (CcO) of mitochondria and many bacteria, including Rhodobacter sphaeroides. Exploration of the copper binding stoichiometry of R. sphaeroides Cox11 led to the finding that an apparent tetramer of both mitochondrial and bacterial Cox11 binds more copper than the sum of the dimers, providing another example of the flexibility of copper binding by Cu(I)-S clusters. Site-directed mutagenesis has been used to identify components of Cox11 that are not required for copper binding but are absolutely required for the assembly of Cu(B), including conserved Cys-35 and Lys-123. In contrast to earlier proposals, Cys-35 is not required for dimerization of Cox11 or for copper binding. These findings, and the location of Cys-35 at the C-terminus of the predicted transmembrane helix and thereby close to the surface of the membrane, allow a proposal that Cys-35 is involved in the transfer of copper from the Cu(I) cluster of Cox11 to the Cu(B) ligands His-333 and His-334 during the folding of CcO subunit I. Lys-123 is located near the Cu(I) cluster of Cox11, in an area otherwise devoid of charged residues. From the analysis of several Cox11 mutants, including K123E, -L, and -R, we conclude that a previous proposal that Lys-123 provides charge balance for the stabilization of the Cu(I) cluster is unlikely to account for its absolute requirement for Cox11 function. Rather, consideration of the properties of Lys-123 and the apparent specificity of Cox11 suggest that Lys-123 plays a role in the interaction of Cox11 with its target.
铜（I）伴侣蛋白 Cox11 对于将铜（B）插入线粒体和许多细菌（包括球形红杆菌）的细胞色素 c 氧化酶（CcO）中是必需的。对球形红杆菌 Cox11 的铜结合化学计量的探索导致发现，线粒体和细菌 Cox11 的明显四聚体比二聚体的总和结合更多的铜，为 Cu(I)-S 簇对铜结合的灵活性提供了另一个例子。定点突变已被用于鉴定 Cox11 中不参与铜结合但绝对需要组装 Cu(B)的组件，包括保守的 Cys-35 和 Lys-123。与早期的提议相反，Cys-35 对于 Cox11 的二聚化或铜结合不是必需的。这些发现，以及 Cys-35 位于预测跨膜螺旋的 C 末端的位置，因此靠近膜的表面，允许提出 Cys-35 参与 Cox11 的 Cu(I)簇与 His-333 和 His-334 的 Cu(B)配体之间的铜转移在 CcO 亚基 I 的折叠过程中。Lys-123 位于 Cox11 的 Cu(I)簇附近，在该区域没有带电残基。从对包括 K123E、-L 和 -R 在内的几种 Cox11 突变体的分析中，我们得出结论，以前的提议即 Lys-123 为稳定 Cu(I)簇提供电荷平衡不太可能解释其对 Cox11 功能的绝对要求。相反，考虑到 Lys-123 的性质和 Cox11 的明显特异性，Lys-123 可能在 Cox11 与其靶标的相互作用中发挥作用。
