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J. Biol. Chem., Vol. 282, Issue 1, 397-406, January 5, 2007

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Evidence of Ball-and-chain Transport of Ferric Enterobactin through FepA^*

Li Ma, Wallace Kaserer, Rajasekeran Annamalai¹, Daniel C. Scott², Bo Jin³, Xiaoxu Jiang, Qiaobin Xiao, Hossein Maymani, Liliana Moura Massis⁴, Luiz C. S. Ferreira, Salete M. C. Newton, and Phillip E. Klebba⁵

From the Department of Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 and the Departamento de Microbiologia, Universidade de São Paulo, São Paulo 05508-900, Brazil

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane -barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the -barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal -barrel domain confirmed the feasibility of this mechanism.

Received for publication, June 5, 2006 , and in revised form, September 22, 2006.

^* This work was supported in part by National Science Foundation Grant MCB-0414694 and National Institutes of Health Grant GM53836 (to P. E. K. and S. M. C. N.). 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 Current address: University of Missouri Medical School, Columbia, MO 65211.

² Current address: Howard Hughes Medical Institute, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105.

³ Current address: Howard Hughes Medical Institute, Washington University, 660 S. Euclid Ave., Box 8022, St. Louis, MO 63110.

⁴ Supported by the FAPESP (Grant 02/04771-7).

⁵ To whom correspondence should be addressed: Dept. of Chemistry & Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019. Tel.: 405-325-4969; Fax: 405-325-6111; E-mail: peklebba{at}ou.edu.

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