Stevens Julie M, Uchida Takeshi, Daltrop Oliver, Kitagawa Teizo, Ferguson Stuart J
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Biol Chem. 2006 Mar 10;281(10):6144-51. doi: 10.1074/jbc.M508765200. Epub 2005 Dec 22.
The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.
细胞色素c成熟蛋白CcmE是一种必需的膜锚定血红素伴侣蛋白，参与革兰氏阴性菌（如大肠杆菌）中血红素与c型细胞色素的翻译后共价连接。先前的体外研究表明，CcmE既能共价结合血红素（通过组氨酸残基），也能非共价结合血红素。在这项工作中，我们展示了关于血红素与可溶性CcmE结合的后一种形式的研究结果。通过共振拉曼光谱对大肠杆菌CcmE的多个定点突变体进行检测，确定了血红素铁的配体，并深入了解了CcmE在共价结合血红素之前血红素结合的初始步骤。血红素结合组氨酸（His-130）似乎在三价氧化态下与血红素铁配位，但另外两个残基在二价形式下与铁配位，从而使His-130能够与血红素乙烯基进行共价连接。非共价形式的血红素配位似乎与全酶形式不同，这表明配位的变化可能是共价键形成的触发因素，并显示了CcmE动态且对氧化态敏感的配位特性。
