Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, United Kingdom.
Biochemistry. 2013 Oct 15;52(41):7262-70. doi: 10.1021/bi400398t. Epub 2013 Oct 2.
Cytochromes c comprise a diverse and widespread family of proteins containing covalently bound heme that are central to the life of most organisms. In many bacteria and in certain mitochondria, the synthesis of cytochromes c is performed by a complex post-translational modification apparatus called System I (or cytochrome c maturation, Ccm, system). In Escherichia coli , there are eight maturation proteins, several of which are involved in heme handling, but the mechanism of heme transfer from one protein to the next is not known. Attachment of the heme to the apocytochrome occurs via a novel covalent bond to a histidine residue of the heme chaperone CcmE. The discovery of a variant maturation system (System I*) has provided a new tool for studying cytochrome c assembly because the variant CcmE functions via a cysteine residue in the place of the histidine of System I. In this work, we use site-directed mutagenesis on both maturation systems to probe the function of the individual component proteins as well as their concerted action in transferring heme to the cytochrome c substrate. The roles of CcmA, CcmC, CcmE, and CcmF in the heme delivery process are compared between Systems I and I*. We show that a previously proposed quinone-binding site on CcmF is not essential for either system. Significant differences in the heme chemistry involved in the formation of cytochromes c in the variant system add new pieces to the cytochrome c biogenesis puzzle.
细胞色素 c 组成了一个多样化和广泛分布的蛋白质家族，它们含有共价结合的血红素，这对于大多数生物体的生命活动至关重要。在许多细菌和某些线粒体中，细胞色素 c 的合成是由一个称为系统 I（或细胞色素 c 成熟、Ccm 系统）的复杂翻译后修饰装置完成的。在大肠杆菌中，有八个成熟蛋白，其中几个参与血红素处理，但血红素从一个蛋白转移到另一个蛋白的机制尚不清楚。血红素与脱辅基细胞色素的附着是通过与血红素伴侣蛋白 CcmE 的组氨酸残基形成新的共价键来实现的。变体成熟系统（系统 I*）的发现为研究细胞色素 c 组装提供了新的工具，因为变体 CcmE 通过在系统 I 中的组氨酸位置的半胱氨酸残基发挥作用。在这项工作中，我们使用定点突变两种成熟系统来探测单个组成蛋白的功能以及它们在将血红素转移到细胞色素 c 底物中的协同作用。比较了系统 I 和 I*中 CcmA、CcmC、CcmE 和 CcmF 在血红素传递过程中的作用。我们表明，先前在 CcmF 上提出的醌结合位点对于两个系统都不是必需的。变体系统中涉及细胞色素 c 形成的血红素化学的显著差异为细胞色素 c 生物发生之谜增添了新的内容。
