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J. Biol. Chem., Vol. 281, Issue 10, 6144-6151, March 10, 2006

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Dynamic Ligation Properties of the Escherichia coli Heme Chaperone CcmE to Non-covalently Bound Heme^*

Julie M. Stevens¹², Takeshi Uchida¹, Oliver Daltrop³, Teizo Kitagawa⁴, and Stuart J. Ferguson⁵

From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and Okazaki Institute for Integrative Bioscience, National Institutes for Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan

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.

Received for publication, August 9, 2005 , and in revised form, December 21, 2005.

^* This work was supported in part by Grant C20071 [GenBank] from the British Biotechnology and Biological Sciences Research Council (to S. J. F.) and Grant-in-aid 14001004 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ Both authors contributed equally to this work.

² Supported by the British Biotechnology and Biological Sciences Research Council.

³ A Junior Research Fellow of Christ Church, Oxford.

⁴ To whom correspondence may be addressed. Tel.: 81-564-59-5225; Fax: 81-564-59-5229; E-mail: teizo{at}ims.ac.jp. ⁵ To whom correspondence may be addressed. Tel.: 44-1865-275240; Fax: 44-1865-275259; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.

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