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J. Biol. Chem., Vol. 280, Issue 44, 36747-36753, November 4, 2005

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Functional Characterization of the C-terminal Domain of the Cytochrome c Maturation Protein CcmE^*

Edgar M. Harvat1, Julie M. Stevens, Supported by Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom and EMBO2, Christina Redfield, and Stuart J. Ferguson3

From the Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom and Department of Biochemistry and Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, Mansfield Rd., Oxford OX1 3TA, United Kingdom

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.

Received for publication, July 29, 2005 , and in revised form, August 18, 2005.

^* This work was supported in part by Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom Grant C20071 [GenBank] (to S. J. F.). 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.

¹ Supported by an Abraham Newton Scholarship in Biological Sciences from Oxford University.

² To whom correspondence may be addressed. Tel.: 44-1865-275242; Fax: 44-1865-275259; E-mail: julie.stevens{at}bioch.ox.ac.uk ³ To whom correspondence may be addressed. Tel.: 44-1865-275240; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.

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This article has been cited by other articles:

				J. M. Stevens, T. Uchida, O. Daltrop, T. Kitagawa, and S. J. Ferguson Dynamic Ligation Properties of the Escherichia coli Heme Chaperone CcmE to Non-covalently Bound Heme J. Biol. Chem., March 10, 2006; 281(10): 6144 - 6151. [Abstract] [Full Text] [PDF]