Harvat Edgar M, Stevens Julie M, Redfield Christina, Ferguson Stuart J
Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom.
J Biol Chem. 2005 Nov 4;280(44):36747-53. doi: 10.1074/jbc.M508355200. Epub 2005 Aug 29.
CcmE is a heme chaperone involved in the periplasmic maturation of c-type cytochromes in many bacteria and plant mitochondria. It binds heme covalently and subsequently transfers it to the apo form of cytochromes c. To examine the role of the C-terminal domain of CcmE in the binding of heme, in vitro heme binding to the apo form of a truncated (immediately before Pro-136) version of the periplasmic domain of the heme chaperone from Escherichia coli was studied. Removal of the C-terminal domain dramatically altered the ligation of non-covalently bound heme in CcmE' (the soluble form lacking the membrane anchor) but only slightly affected its affinity for protoporphyrin IX and 8-anilino-1-naphthalenesulfonate. This finding has significant mechanistic implications for in vivo holo-CcmE formation and indicates that the C-terminal region is not required for the recruitment and docking of heme into its binding site but is likely to contain amino acid(s) involved in heme iron axial coordination. Removal of the C-domain significantly impaired in vivo heme binding to CcmE and conversion of apocytochrome to holoprotein by a similar factor, suggesting that the C-terminal domain of the chaperone is primarily involved in heme binding to CcmE rather than in heme transfer to the apo cytochrome.
CcmE是一种血红素伴侣蛋白，参与许多细菌和植物线粒体中c型细胞色素的周质成熟过程。它与血红素共价结合，随后将其转移到细胞色素c的脱辅基形式上。为了研究CcmE C端结构域在血红素结合中的作用，我们研究了体外血红素与来自大肠杆菌的血红素伴侣蛋白周质结构域截短版本（紧接在Pro-136之前）的脱辅基形式的结合情况。去除C端结构域显著改变了CcmE'（缺乏膜锚定的可溶性形式）中非共价结合血红素的连接方式，但仅轻微影响其对原卟啉IX和8-苯胺基-1-萘磺酸盐的亲和力。这一发现对体内全CcmE的形成具有重要的机制意义，表明C端区域对于血红素募集到其结合位点并对接并非必需，但可能包含参与血红素铁轴向配位的氨基酸。去除C结构域显著损害了体内血红素与CcmE的结合以及脱辅基细胞色素向全蛋白的转化，且程度相似，这表明伴侣蛋白的C端结构域主要参与血红素与CcmE的结合，而非血红素向脱辅基细胞色素的转移。
