Spectroscopic observations of ferric enterobactin transport.
We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology. The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein. FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C. These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods. Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer. These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport. We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons. Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion.
Cao
Zhenghua
Z
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Warfel
Paul
P
Newton
Salete M C
SM
Klebba
Phillip E
PE
D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004758:Enterobactin / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature
D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D004758:Enterobactin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone