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Volume 272, Number 35, Issue of August 29, 1997 pp. 21950-21955
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Biphasic Binding Kinetics between FepA and Its Ligands

(Received for publication, April 4, 1997, and in revised form, June 26, 1997)

Marvin A. Payne , John D. Igo , Zhenghua Cao , Samuel B. Foster , Salete M. C. Newton ^§ and Phillip E. Klebba

From the  Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 and ^§ Departamento de Microbiologia, Universidade de São Paulo, São Paulo 08805-900, Brazil

The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced K_d values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B·FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.

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