Ferric enterobactin is a catecholate siderophore that binds with high affin
ity (K-d approximate to 10(-10) M) to the Escherichia coli outer membrane p
rotein FepA. We studied the involvement of aromatic amino acids in its upta
ke by determining the binding affinities, kinetics and transport properties
of site-directed mutants. We replaced seven aromatic residues (Y260, Y272,
Y285, Y289, W297, Y309 and F329) in the central part of FepA primary struc
ture with alanine, individually and in double combinations, and determined
the ability of the mutant proteins to interact with ferric enterobactin and
the protein toxins colicins B and D. All the constructs showed normal expr
ession and localization. Among single mutants, Y260A and F329A were most de
trimental, reducing the affinity between FepA and ferric enterobactin 100-
and 10-fold respectively. Double substitutions involving Y260, Y272 and F32
9 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly
. For Y280A and Y272A, the drop in adsorption affinity caused commensurate
decreases in transport efficiency, suggesting that the target residues prim
arily act in ligand binding. F329A, like R316A, showed greater impairment o
f transport than binding, intimating mechanistic involvement during ligand
internalization. Furthermore, immunochemical studies localized F329 in the
FepA ligand binding site. The mutagenesis results suggested the existence o
f dual ligand binding sites in the FepA vestibule, and measurements of the
rate of ferric enterobactin adsorption to fluoresceinated FepA mutant prote
ins confirmed this conclusion. The initial, outermost site contains aromati
c residues and probably functions through hydrophobic interactions, whereas
the secondary site exists deeper in the vestibule, contains both charged a
nd aromatic residues and probably acts through hydrophobic and electrostati
c bonds.