From the Department of Molecular Genetics and Department of Molecular Cellular Biochemistry and.
J Biol Chem. 2010 Sep 24;285(39):29738-49. doi: 10.1074/jbc.M109.099069. Epub 2010 Jul 13.
The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c(6) in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b(6)f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c(6) in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.
C 型细胞色素是一种金属蛋白,其血红素分子与半胱氨酸残基共价连接到 CXXCH 血红素结合位点。在质体中,至少需要六个组装因子将血红素附着到类囊体腔中的细胞色素 f 和细胞色素 c(6)的脱辅基形式上。CCS5 通过在单细胞绿藻衣藻中进行插入诱变来控制质体细胞色素 c 的组装而被鉴定。互补基因编码的蛋白与拟南芥 HCF164 具有相似性,HCF164 是一种定位于类囊体膜的蛋白,具有面向腔的硫氧还蛋白样结构域。HCF164 是细胞色素 b(6)f 生物发生所必需的,但它在组装过程中的活性和作用部位迄今仍未破译。我们表明,CCS5 是跨类囊体氧化还原途径的一个组成部分,并且在血红素连接反应之前通过还原脱辅基细胞色素 c 的 CXXCH 血红素结合位点来发挥作用。这一观点基于以下发现:1)外源硫醇可拯救 ccs5 突变体;2)CCS5 在酵母双杂交测定中与脱辅基细胞色素 f 和 c(6)相互作用;3)重组 CCS5 能够还原脱辅基细胞色素 f 的 CXXCH 血红素结合位点中的二硫键。